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Fertilisation

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Specific adhesion of spermatozoa to the oocyte's extracellular coat, the zona pellucida (ZP) is an essential process for mammalian fertilization. Therefore, the molecular nature of sperm-ZP interactions (components of the ZP as well as in the sperm plasma membrane fractions) has been extensively studied in various species of eutherian mammals. In mice, the ZP sialoglycoprotein ZP3 is the sperm receptor. Contrary to this relatively simple situation, there are more than two dozen candidates that have been claimed to be ZP (ZP3) receptors in various mammalian species.

One paradigm considers the ZP receptor(s) to be localized on the plasma membrane of acrosome-intact spermatozoa, which derived from observations that acrosome-intact spermatozoa adhere to the ZP during in vitro fertilization (IVF). Subsequently, the sperm acrosome reaction (AR) is believed to occur on the ZP surface. Underpinning with this view, a large number of reports demonstrate that ZP3 is the inducer of the sperm AR. Thus, the theoretical model of spermegg interactions has been formed based on discontinuous observations of some key events during IVF. However, using transgenic mice and retrospective videoimaging microscopy, we recently reported that fertilizing spermatozoa initiated the acrosome reaction (as defined by a loss of acrosomal EGFP) before their contact with the ZP. These observations raise the question of whether fertilizing spermatozoa, prior to contact with the ZP, have begun or completed acrosomal exocytosis. In this report, we performed real-time assessment of the acrosome status of living spermatozoa approaching and thereafter contacting the ZP of the cumulus-oocyte complex (COC).

Recent studies highlighted that the AR is not a binary event (acrosome being either completely intact or totally reacted), but rather constitutes a continuum of different states. We investigated cell surface staining for acrosomal makers, i.e., sp56/ZP3R and peanut agglutinin lectin (PNA)-reactive carbohydrates in sperm populations under capacitating conditions where spermatozoa trigger the spontaneous AR. PNA-positive (PNA⁺) spermatozoa are regarded as having undergone the AR as defined by externalization of the acrosomal constituents,, whereas sp56/ZP3R is exposed to the cell surface before soluble EGFP is diffused away from the acrosome and released in progressive stages (). Analyses with fluorescence microscopy () and flow cytometry () revealed progressive changes in acrosomal antigens on the sperm surface, confirming the results previously presented. Notably, at any time point following capacitation, spermatozoa that exhibit both EGFP − and PNA −, representing an intermediate state of the AR, were rarely observed (), suggesting a rapid transition from EGFP⁺/PNA − to EGFP −/PNA⁺ during the spontaneous AR.

We next developed assays for visualizing the acrosome status of living spermatozoa during passage through the cumulus cell layer (CCL). First, anti-sp56/ZP3R antibody or PNA was coupled to fluorescent polystyrene microspheres by which the acrosomal status of sperm population entering in the CCL could be analyzed statistically by counting the number of beads carried by spermatozoa (). In this assay, we found that large populations of sp56/ZP3R-exposed and/or PNA antigen-exposed spermatozoa could enter the CCL, suggesting that acrosome-reacted or acrosome-reacting spermatozoa were capable of entering and passing through the cumulus matrix (). Second, microscopic IVF (MIVF) was performed in the presence of Alexa 594-labeled PNA, by which a real-time imaging of the acrosomal status and even its transition are enabled at the zona periphery (). Transition from acrosome-intact (EGFP⁺) to acrosome-reacted (PNA⁺) was rarely seen in the vicinity of the ZP (3 cases in 17 experiments) and the time required for such transition was approximately 2 min (). In this assay, among EGFP − sperm that had bound to the ZP, PNA⁺ spermatozoa were most abundant (91.2%), whereas there were small numbers of double-negative (i.e., transitional state of EGFP −/PNA −) spermatozoa (). Among PNA⁺, ZP-bound spermatozoa, only 4% initiated penetration through the ZP (5/125, ).

The long-standing conceptual view that the soluble acrosomal lysins create a path in the ZP, which facilitates sperm penetration through the egg extracellular matrix, imposes that the AR must be triggered in the close vicinity of the target for lytic action (i.e., the ZP). However, recent investigations call into a question whether soluble components housed in the acrosome play a role in egg-coat lysis. More importantly, sperm take a considerable period of time to complete the sequential processes of membrane fusions, time-dependent exposure and release of intraacrosomal proteins and post-translational modifications of cell surface antigens., The relationship of the progression of the AR to the penetration of the ZP and fusion with the oocyte remains unknown. Several laboratories reached the same conclusion that sperm binding to the ZP is not sufficient for the AR induction. We recently found that most fertilizing spermatozoa initiate the AR before they reach the ZP. Therefore, it is important to determine acrosomal status at the moment of sperm contact with the ZP. To address these questions, we performed live-cell imaging with transgenic sperm and chemical probes to detect cell surface antigens. Acrosome staining with PNA would represent sperm that have undergone the AR and proceeded to the advanced stages following point fusions between the outer acrosomal membrane and the plasma membrane. Early stages of the AR can be defined by absence of both GFP and PNA fluorescence, which was observed in a minor population of sperm during MIVF (). The majority of GFP-spermatozoa exhibited PNA fluorescence when they made contact with the ZP. Although the extent of original membrane loss remains to be identified, our studies provide the first blueprint of physiological changes associated with the acrosomal exocytosis in fertilizing spermatozoa. In a perspective view, electron microscopy analysis in conjunction with live-cell imaging would be useful to identify the precise status of the acrosome at the moment of gamete interaction. This would give new insights into the cellular and molecular bases of mammalian fertilization.

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Video imaging of the sperm acrosome reaction during in vitro fertilization

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Specific adhesion of spermatozoa to the oocyte's extracellular coat, the zona pellucida (ZP) is an essential process for mammalian fertilization. Therefore, the molecular nature of sperm-ZP interactions (components of the ZP as well as in the sperm plasma membrane fractions) has been extensively studied in various species of eutherian mammals.

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The Cell: An Image Library - Image CIL:11962

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"The time lapse movie spans fertilization, ooplasmic segregation, and early divisions in the egg of the tunicate Phalusia mammilata. Contrast is provided by both polarizing microscopy and differential interference microscopy.\nThe ent...

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länt lab mais j'ai pas regardé le contenu!

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Browse data

The Furlong lab maintains several browsing instance of genome browsers (using JBrowse). Click on the thumbnails to access one.

Our browser instances runs with our in-house Dynamix plugin, for automated genome browsing. Learn more about it here.

The Furlong lab maintains and serves a genome browser instance where all of our public data is made available for browsing (with complementary datasets from other labs). Access the Furlong Browser.

Non-coding RNA expression, function and variation during Drosophila embryogenesis

Schor IE, Bussotti G, Males M, Forneris M, Viales RR, Enright AJ, Furlong EEM (2018)

Developmental enhancers and chromosome topology.

Furlong EEM ‡, Levine M ‡ (2018)

‡ Co-corresponding authors

The Insulator Protein CTCF Is Required for Correct Hox Gene Expression, but Not for Embryonic Development in Drosophila.

Gambetta MC ‡, Furlong EEM ‡ (2018)

‡ Co-corresponding authors

The cis-regulatory dynamics of embryonic development at single-cell resolution

Cusanovich DA*, Reddington JP*, Garfield DA*, Daza RM, Aghamirzaie D, Marco-Ferreres R, Pliner HA, Christiansen L, Qiu X, Steemers FJ, Trapnell C, Shendure J ‡, Furlong EE ‡ (2018)

* These authors contributed equally to this work.

‡ Co-corresponding authors

Opbp is a new architectural/insulator protein required for ribosomal gene expression.

Zolotarev N, Maksimenko O, Kyrchanova O, Sokolinskaya E, Osadchiy I, Girardot C, Bonchuk A, Ciglar L, Furlong EE ‡, Georgiev P ‡ (2017)

‡ Co-corresponding authors

Dual functionality of cis-regulatory elements as developmental enhancers and Polycomb response elements

Erceg J*, Pakozdi T*, Marco-Ferreres R*, Ghavi-Helm Y, Girardot C, Bracken AP, Furlong EE (2017)

* These authors contributed equally to this work.

See also genesdev.org/multitasking-by-polycomb-response-elements

Dynamix: dynamic visualization by automatic selection of informative tracks from hundreds of genomic datasets

Monfort M, Furlong EE, Girardot C (2017)

Promoter shape varies across populations and impacts promoter evolution and expression noise

Schor IE, Degner JF, Harnett D, Cannavo E, Casale FP, Shim H, Garfield D, Birney E, Stephens M, Stegle O ‡, Furlong EE ‡ (2017)

‡ Co-corresponding authors

Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers.

Girardot C, Scholtalbers J, Sauer S, Su SY, Furlong EE (2016)

Qualitative Dynamical Modelling Can Formally Explain Mesoderm Specification and Predict Novel Developmental Phenotypes.

Mbodj A, Gustafson EH, Ciglar L, Junion G, Gonzalez A, Girardot C, Perrin L, Furlong EE ‡, Thieffry D ‡ (2016)

‡ Co-corresponding authors

FourCSeq: Analysis of 4C sequencing data

Klein FA, Pakozdi T, Anders S, Ghavi-Helm Y, Furlong EE, Huber W (2015)

easyRNASeq: a bioconductor package for processing RNA-Seq data

Delhomme N, Padioleau I, Furlong EE, Steinmetz LM (2012)

Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development

Bonn S*, Zinzen RP*, Girardot C*, Gustafson EH, Perez-Gonzalez A, Delhomme N, Ghavi-Helm Y, Wilczyński B, Riddell A, Furlong EE (2012)

* These authors contributed equally to this work.

A transcription factor collective defines cardiac cell fate and reflects lineage history

Junion G*, Spivakov M*, Girardot C, Braun M, Gustafson EH, Birney E, Furlong EE (2012)

* These authors contributed equally to this work.

Molecular biology: A fly in the face of genomics

Furlong EE (2011)

The importance of being specified: cell fate decisions and their role in cell biology

Furlong EE (2010)

Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity

Cunha PM*, Sandmann T*, Gustafson EH, Ciglar L, Eichenlaub MP, Furlong EE (2010)

* These authors contributed equally to this work.

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The Furlong lab maintains several browsing instance of genome browsers (using JBrowse). Click on the thumbnails to access one. Our browser instances runs with our in-house Dynamix plugin, for automated genome browsing. Learn more about it here.

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Trés complet

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Welcome to the Embryology education and research website now in the 22nd year online! There are many ways to find what you are looking for. Click on the left image term, or use the menu items at the page top, or the search window.

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whole embryos etc...

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Acknowledgements

We thank the mechanical and electronics workshop of the European Molecular Biology Laboratory (EMBL) for customized hardware; D. Holzer, T. Schneidt and H. Gustafson for help with the flies and G. Heuvelman and M. Wachsmuth for discussions on optics. We thank S. DeRenzis and the Wieschaus laboratory for the Gap43-mCherry flies and E. Lemke for hardware support. We thank J. Ellenberg for his helpful comments on the manuscript and anonymous reviewers for their constructive comments. We acknowledge the advanced light microscopy facility of EMBL for its kind support. We are grateful for financial support from EMBL, the EMBL Interdisciplinary Postdocs Program (EIPOD) and the Center for Modelling and Simulation in the Biosciences (BIOMS).

Author information

Affiliations

Contributions

U.K. and L.H. designed and built the microscope software and hardware. U.K., S.G. and T.E.S. performed the experiments. U.K., S.G., T.E.S. and S.J.S. performed data analysis, quantification and visualization. All authors contributed to the writing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lars Hufnagel.

Supplementary information

PDF files

Videos

1. 5.

Color ranges from blue to yellow, where the former corresponds to the time T, and the latter to T - 250 s.

2. 8.

The embryo was imaged for 15 h every 30 s, and for a further 5 h every 10 min to confirm that the high frame rate of imaging didn't alter the embryonic development. Only one image every 10 min is shown in the video after the initial 5 h. Every 3D image was background-corrected and maximum-projected from a middle plane towards the ventral and dorsal side, respectively, using Fiji ( www.fiji.sc).

3. 10.

Rotation of a Drosophila melanogaster embryo expressing the Gap43-mCherry membrane marker in cycle 14, corresponding to Figure 2e.

4. 12.

View along the long-axis of a Drosophila melanogaster embryo expressing the Gap43-mCherry membrane marker at a fixed time point, 10 min prior to ventral furrow formation.

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Multiview light-sheet microscope for rapid in toto imaging

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A selective-plane illumination microscope with two illumination and two detection objectives rapidly records four three-dimensional images of an entire developing fly embryo and processes them into a single high-content image in real time. This allows for cell tracking and quantification of cell shape changes across the embryo.

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animal model

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Summary

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Cite this Article
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Range, R. C., Martinez-Bartolomé, M., Burr, S. D. The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions. J. Vis. Exp. (120), e55113, doi:10.3791/55113 (2017).

Abstract

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

Introduction

Gene regulatory networks (GRNs) and signal transduction pathways establish the spatial and temporal expression of genes during embryonic development that are used to build the adult animal body plan. Cell-to-cell signal transduction pathways are essential components of these regulatory networks, providing the means by which cells communicate. These cellular interactions establish and refine the expression of regulatory and differentiation genes in and among the various territories during embryogenesis ¹,². Interactions among secreted extracellular modulators (ligands, antagonists), receptors, and co-receptors control the activities of signal transduction pathways. An assortment of intracellular molecules transduces these inputs resulting in altered gene expression, division, and/or shape of a cell. While many of the key molecules used at the extracellular and intracellular levels in the major pathways are known, it is an incomplete knowledge due in large part to the complexity of individual signaling pathways. In addition, different signaling pathways often interact with one another either positively or negatively at the extracellular, intracellular, and transcriptional levels ³,⁴,⁵,⁶. Importantly, the core components of signal transduction pathways are highly conserved in all metazoan species, and, remarkably, most of the major signaling pathways often perform similar developmental functions in many species when comparing organisms from closely related phyla in particular ⁷,⁸,⁹,¹⁰,¹¹.

The study of signaling during development is a daunting task in any organism, and there are several significant challenges to studying signaling pathways in most deuterostome models (vertebrates, invertebrate chordates, hemichordates, and echinoderms): 1) In vertebrates there are large numbers of possible ligand and receptor/co-modulator interactions, intracellular transduction molecules, as well as potential interactions among different signaling pathways due to the complexity of the genome ¹²,¹³,¹⁴; 2) The complex morphology and morphogenetic movements in vertebrates often make it more difficult to interpret functional interactions in and among signal transduction pathways; 3) Analyses in most non-echinoderm invertebrate deuterostome model species are limited by short windows of gravidity with the exception of some tunicate species ¹⁵,¹⁶.

The sea urchin embryo has few of the above-mentioned limitations and offers many unique qualities for performing a detailed analysis of signal transduction pathways in vivo. These include the following: 1) The relative simplicity of the sea urchin genome significantly reduces the number of possible ligand, receptor/co-receptor and intracellular transduction molecule interactions ¹⁷; 2) The GRNs controlling the specification and patterning of the germ layers and major embryonic axes are well established in sea urchin embryos, aiding in the understanding of the regulatory context of the cell/territory receiving the signals ¹⁸,¹⁹; 3) Many signal transduction pathways can be studied between early cleavage and gastrula stages when embryos consist of a single layered epithelium whose morphology is easier to analyze; 4) The molecules involved in signaling pathways in the sea urchins are easily manipulated; 5) Many sea urchins are gravid for 10 to 11 months a year ( e.g. Strongylocentrotus purpuratus and Lytechinus variegatus).

Here, we present a method to systematically and efficiently characterize components of the signaling pathways that specify and pattern territories in sea urchin embryos to illustrate the advantages that several invertebrate model systems offer in the study of complex molecular mechanisms.

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Protocol

1. High Throughput Morpholino Design Strategy

1. Identify a gene(s) of interest (e.g. candidate gene approach, cis-regulatory analysis, RNAseq and/or proteomic differential screens).

2. Use genomic, transcriptomic, and gene expression data available on frequently updated websites (e.g. SpBase http://www.echinobase.org​²⁰ and S. purpuratus Genome Search http:///urchin.nidcr.nih.gov/blast/index.html) to determine that the spatiotemporal expression profile overlaps with the developmental mechanism in question. If no expression data is available, then generate qPCR primers and/or an antisense in situ probe to assess the gene expression pattern.

3. After determining the spatial and temporal expression pattern, obtain the DNA sequence from the genomic web sites.

1. For generating translation-blocking morpholino oligonucleotides, obtain sequence of the 5' un-translated region (5' UTR) directly upstream from the initiation codon from expressed sequence tag (EST) databases available on SpBase or S. purpuratus Genome Search websites. If this information is unavailable for the gene of interest, then perform 5' RACE.

2. To design a splice-blocking morpholino, search for the genomic sequence including the exons and introns using the scaffold blastn tool in SpBase or S. purpuratus Genome Search tool.

4. Use the oligonucleotide designing website http://oligodesign.gene-tools.com in order to design the desired 25 base pair morpholino sequence.

1. For a translational-blocking morpholino, use the mRNA sequence from 5' to 3' as the source of translational target sequence. Include the 5' UTR sequence (approximately 70 nucleotides upstream of the transcription start site) plus 25 bases of coding region (downstream of the start site) with the start codon marked with parenthesis (ATG).

2. For a splice-blocking morpholino, select intron-exon or exon-intron boundary sequence and include 50 bases (25 bases of exon sequence and 25 bases of intron sequence) around the boundary regions. Scan the genome with the morpholino sequence to verify that the sequence is unique.

2. Microinjection of Morpholino Oligonucleotides

1. Prepare 3 mM stock solutions of the morpholino oligonucleotides by adding 100 µL of nuclease-free water into the 300 nmol morpholino vial.
NOTE: Do not use DEPC treated water for the re-suspension because diethyl pyrocarbonate can damage the morpholino.

2. For the first reconstitution spin down the vial containing the stock oligonucleotide solution for 30 s at full speed (14,000 - 16,000 x g), briefly vortex, heat at 65 °C for 5 to 10 min, briefly vortex, and keep the morpholino stock at room temperature for at least 1 h. Morpholino stock solutions are stored from -20 °C to +4 °C.

3. Prepare injection solution containing morpholino oligonucleotides at the desired concentration. This solution usually contains 20% glycerol or 125 mM KCl as a carrier and 15% FITC-Dextran (fluorescein isothiocyanate dextran 10,000 MW 2.5 mg/μL stock solution). FITC-Dextran and other fluorescent dextran conjugates are routinely used to identify injected embryos by epifluorescence microscopy. Store injection solutions at -20 °C.

4. Heat the morpholino solution at 65 °C in a heat block or water bath for at least 2 - 5 min.

5. Briefly spin the morpholino solution for 30 s at full speed (14,000 - 16,000 x g), vortex for 1 min and centrifuge at full speed (14,000 - 16,000 x g) for at least 10 min.

6. Load the injection needles with the morpholino solution. For a detailed microinjection protocol please see Stepicheva and Song, 2014²¹ and Cheers and Ettensohn, 2004²².

3. Fixation and In Situ Protocol at 24 h Post-fertilization (hpf) in S. purpuratus Embryos

NOTE: This protocol is modified from Arenas-Mena et al., 2000 ²³ and Sethi et al., 2014 ²⁴.

1. Fixation

1. Add several drops of fixative (see below) to wells containing embryos, mix gently by pipetting, and allow them to settle. Remove the fixative solution, and then mix embryos with two additional 180 µL washes of fixative.
NOTE: It is important to thoroughly mix the embryos during the washes by gentle pipetting up and down several times.Failure to do so can lower the signal to noise ratio.

2. Leave the embryos to fix in the second fixative wash for 50 min to 1 h at room temperature (RT) in 4% electron microscopy grade paraformaldehyde consisting of 10 mM MOPS pH 7.0, 0.1% Tween-20, and artificial seawater (ASW). Make this solution fresh each time for best results. In addition, for ease and practicality embryos are fixed in 96-well plates.
For 20 mL fixative use 5 mL 16% paraformaldehyde, 15 mL ASW, 200 µL 1 M MOPS pH 7.0, and 20 µL Tween-20.

3. Wash 5 times at RT with 180 µL of MOPS wash buffer consisting of 0.1 M MOPS pH 7.0, 0.5 M NaCl and 0.1% Tween-20 for at least 5 min or until embryos drop to the bottom of the well. Again, It is important to thoroughly mix the embryos into the wash buffer by gently pipetting up and down several times. MOPS wash buffer can be used for 2 days if stored at 4°C.

1. For 40 mL MOPS wash buffer use 4 mL 1 M MOPS pH 7.0, 4 mL 5 M NaCl, 32 mL dH₂O, and 40 µL Tween-20.

2. Fixed embryos can be stored at 4 oC for 2 days.
NOTE: If storing fixed embryos longer, then add 0.2% sodium azide in MOPS wash buffer to prevent bacterial growth.

2. Pre-hybridization

1. Aspirate the MOPS wash buffer and add 180 μL of a 2:1 ratio of MOPS wash buffer to hybridization buffer, mix embryos into the solution by gentle pipetting several times, and incubate for at least 20 min at RT. Hybridization buffer consists of 70% formamide, 0.1 M MOPS pH 7.0, 0.5 M NaCl, 1 mg/mL BSA and 0.1% Tween-20.
For 40 mL hybridization buffer use 4 mL 1 M MOPS pH 7.0, 4 mL 5 M NaCl, 4 mL dH₂O, 0.04 g BSA, and 40 µL Tween-20. Mix well by vortexing, add 28 mL formamide, and vortex again.

2. Remove the 2:1 ratio of MOPS wash and hybridization buffers and add 180 µL of a 1:2 ratio of MOPS wash buffer to hybridization buffer, mix embryos into the solution, and incubate for at least 20 min at RT.

3. Before probe hybridization gently mix embryos into 100 - 150 µL of hybridization buffer. Incubate embryos at 50 °C for at least 1 h.
NOTE: Incubating embryos overnight is acceptable. Before incubating, seal the wells with an adhesive sheet in order to prevent evaporation.

3. Hybridization

1. In a separate tube add 0.1 - 0.3 ng/µL of probe and 500 µg/mL yeast tRNA into hybridization buffer, then vortex gently to create probe solution. Yeast tRNA is added to the probe solution to decrease non-specific binding of the anti-sense probe. Preheat this solution to 50 °C, and aspirate hybridization buffer.

2. Mix pre-hybridized embryos into 100 µL of the probe solution, seal with an adhesive sheet, and hybridize at 50 °C for 2 to 7 days depending on the probe (the foxq2 probe can be incubated for 2-days).
NOTE: Incubation times will vary based off the probe. Some genes expressed at low levels require up to a 7-day incubation. 96-well plates can be placed in a humidity box as insurance against evaporation if the adhesive sheet fails to seal properly.

3. Wash 5 times at 50 °C with 180 μL of freshly made MOPS wash buffer for a total of 3 h. Then, wash 3 times (15 min) with 180 µL of MOPS wash buffer at RT. Remember to mix embryos into the wash buffer each time by gently pipetting several times.

4. Antibody Incubation

1. Aspirate the MOPS wash buffer and mix embryos in 180 µL of blocking buffer consisting of 10% Normal Sheep Serum and 5 mg/mL BSA in MOPS wash buffer and incubate for at least 45 min at RT or 4 °C overnight.

2. Remove blocking buffer and then mix embryos into blocking buffer containing a 1:1,500 dilution of alkaline phosphatase-conjugated anti-digoxigenin antibody diluted in blocking buffer. Incubate overnight at RT in a sealed plate to avoid evaporation. NOTE: Do not leave antibody on longer than overnight.

3. Wash embryos 6 times (5 min or until embryos drop) in MOPS wash buffer at RT. Embryos can be stored overnight at 4 °C.

5. In situ development

1. Wash embryos 3 times (10 min) at RT with freshly made pH 9.5 buffer consisting of 0.1 M Tris pH 9.5, 100 mM NaCl, 50 mM MgCl₂, 1 mM Levamisole, and 0.1% Tween-20.
For 20 mL pH 9.5 buffer use 2 mL 1 M Tris pH 9.5, 400 µL 5M NaCl, 1 mL 1 M MgCl₂, 20 µL Tween-20, 16.6 mL dH₂O, and 0.0048 g Levamisole.

2. Incubate embryos at 37 °C in staining buffer protected from light. Staining buffer consists of 10% Dimethyl Formamide, 4.5 µL/mL 4-Nitro blue tetrazolium chloride (NBT), and 3.5 µL/mL 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) in freshly made pH 9.5 buffer.
For 1 mL of staining buffer use 100 µL Dimethyl Formamide, 4.5 µL NBT, and 3.5 µL BCIP.

3. Stop the alkaline phosphatase reaction by washing 3 to 5 times in MOPS wash buffer. The reaction with the foxq2 probe typically takes 30 min to 1 h. Some probes may need overnight incubation at either RT or 4 °C.

4. Mix embryos into a solution of 70% MOPS wash and 30% glycerol. The glycerol provides a refractive index necessary for microscopy. Embryos can be stored in this solution for several weeks. Seal the plates with plastic paraffin to prevent evaporation.

6. Slide preparation and image capturing

1. Prepare a slide by arranging three small strips of double-sided tape into a triangle with small gaps between strips onto a slide.

2. Transfer the embryos in the 70% MOPS wash and 30% glycerol solution to the center of the triangle and cover with a coverslip.

3. Seal the coverslip by applying a layer of nail polish around the edges of the coverslip.

4. Capture images using a compound light microscope with a 20X objective and an attached camera.

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Representative Results

In the sea urchin embryo we have shown that 3 different Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) ⁴,²⁵ interact to form a Wnt signaling network that governs anterior-posterior (AP) patterning. One of the most important consequences of these signaling events is that the initial broadly expressed anterior neuroectoderm (ANE) GRN becomes restricted to a small territory around the anterior pole by the beginning of gastrulation (24 hpf in S. purpuratus). These results indicate that Wnt/β-catenin signaling prevents ANE gene activation in the posterior half of the embryo by the 32-cell stage. Then, this pathway relays a signal to the non-canonical Wnt/JNK signaling pathway that progressively down regulates the ANE GRN in the anterior half of the embryo between the 60-cell stage and early gastrulation. Finally, a third non-canonical Wnt pathway, Wnt/PKC, antagonizes the Wnt/JNK signaling pathway and prevents it from eliminating ANE specification around the anterior pole ( Figure 1A) ⁴.

We use the spatiotemporal expression of foxq2 to assay for the activity of each of the Wnt signaling branches during AP patterning because it is one of the first two genes activated in the ANE GRN and it is easily assessed by in situ hybridization because of its robust expression ²⁶. If any individual Wnt signaling branch is perturbed, then there are clear expression phenotypes that indicate which pathway is involved: 1) In the absence of Wnt/β-catenin signaling a broad maternal regulatory mechanism ( Figure 1A) activates foxq2 expression throughout the entire embryo ( Figure 1Bb); 2) In the absence of Wnt/JNK signaling foxq2 is expressed throughout the anterior half of the embryo ( Figure 1Bc), but it is still down regulated in the posterior half due to the activity of Wnt/β-catenin signaling ( Figure 1A); In the absence of Wnt/PKC signaling foxq2 expression is completely down regulated throughout the embryo ( Figure 1Bd), because the Wnt/β-catenin and Wnt/JNK signaling pathways are up regulated ⁴,²⁵. Thus, we have developed an assay that we have termed the foxq2 transcriptional readout system which, when combined with our systematic workflow ( Figure 2), allows us to efficiently identify and test whether a gene of interest is involved in one or more of the Wnt signaling branches.

Using the methodology and foxq2 readout system presented here, we have identified several putative extracellular or intracellular molecules likely involved in the Wnt network governing AP axis specification, four of which are presented in Figure 3. Embryos injected with morpholinos designed to knockdown the expression of either the transcription factor, ATF2, or the secreted extracellular Wnt modulator, Wif-1, showed an expansion of foxq2 expression toward the posterior pole of the embryo at early gastrulation stages ( Figure 3A - C). These results mimic the phenotypes observed when members of the Wnt/JNK signaling pathway are knocked down ⁴,²⁵, suggesting that they are members of this signaling branch that is necessary to down regulate ANE GRN expression in the anterior half of the embryo. In contrast, foxq2 expression was eliminated when we knocked down the expression of the transcription factor, NFAT ( Figure 3D), and the secreted extracellular modulator, sFRP3/4 ( Figure 3E), suggesting that these molecules are involved in the Wnt/PKC pathway that is necessary to antagonize the down regulation of the ANE GRN by Wnt/JNK signaling. Based on these rapidly obtained results we are now able to perform more detailed functional analyses that will place these factors within the evolving Wnt signaling network involved in the GRN that governs AP patterning in the sea urchin embryo.

Figure 1. The Model for AP Specification and Patterning in the Sea Urchin and the foxq2 Transcriptional Readout System. (A) The progressive down regulation of the ANE GRN to a territory around the anterior pole by the Wnt signaling network detailed in the text and in ⁴,⁹. (B) A transcriptional readout system showing foxq2 expression in the indicated Wnt pathway knockdowns (KO). Scale bar = 20 μm. Please click here to view a larger version of this figure.

Figure 2. An Experimental Flow Diagram for Efficient Wnt Network Analysis in the Sea Urchin Embryo. Please click here to view a larger version of this figure.

Figure 3. Signal Transduction Molecules Involved in the Wnt Network Governing AP Axis Specification and Patterning Identified Using the foxq2 Transcriptional Readout System. (A, B, C) Morpholino knockdowns suggest that an intracellular signaling modulator, ATF2, and secreted extracellular modulator, Wif-1, are potential players of the Wnt/JNK signaling pathway. (A, D, E) Knockdown experiments indicated that NFAT, an intracellular signaling modulator, and sFRP3/4, a secreted Wnt signaling modulator, are involved in Fzl1/2/7-PKC signaling. Scale bar = 20 μm. Please click here to view a larger version of this figure.

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Discussion

The methodology presented here is an example that illustrates the power of using embryos with less genomic and morphological complexity than vertebrates to understand the signaling transduction pathways and GRNs governing fundamental developmental mechanisms.. Many labs are using similar assays during early sea urchin development to dissect the signaling pathways involved in other cell fate specification events ( e.g. Notch, Hedgehog, TGF- β, and FGF signaling) ²⁷,²⁸,²⁹,³⁰,³¹. These sea urchin studies have revealed many interesting and novel signaling mechanisms, and many aspects of these signaling mechanisms governing early sea urchin development appear to be conserved among deuterostomes ⁹,²⁷,³⁰,³¹,³². Importantly, the exploration of the developmental biology of non-traditional metazoan embryos has seen an increase in recent years, and many of these share characteristics with sea urchin embryos during early development ⁹,¹⁵,¹⁶,³³. Thus, the methodology presented here could be widely applied to the study of the signal transduction pathways in many of these organisms during early development.

All techniques used to knock down gene expression can potentially produce off-target knock down effects. Morpholinos have proven to be a remarkably effective gene perturbation method in sea urchin embryos in large part because the community performs rigorous controls to alleviate concerns that the knockdown phenotype is non-specific. These basic control assays can be modified and applied to other knockdown techniques ( i.e. CRISPER/Cas9). The controls are: 1) Careful dose response assays are performed with each morpholino until ≥ 80% of injected embryos show defective phenotype and/or marker gene expression; 2) Morpholinos are discarded if they cause severe developmental delays or even death at moderate concentrations. These phenotypes are likely due toxic off-target effects; 3) At least two morpholinos that are designed to target different binding sites are used to confirm knockdown phenotypes; 3) Missense morpholinos are injected; 4) Morpholino phenotypes are rescued by introducing mRNA for the target gene into morpholino knockdown embryos; 5) When available, whole mount antibody staining assays are used to show that morpholino knockdowns prevent translation of the gene of interest. It is important to note that the sea urchin community uses at least four species for functional studies, depending on where scientists are located. In most cases, to our knowledge, the various morpholinos used to knock down the orthologs have generated similar phenotypes in each species (for example see these functional studies on Nodal signaling ³⁴,³⁵,³⁶,³⁷). These results argue convincingly that our rigorous control experiments strongly select for those morpholinos that produce on-target knockdown effects.

Another important aspect of this methodology is to carefully identify the transcriptional targets that are most likely directly activated by the signaling pathway under consideration. The example that we provide here is fortuitous, because the spatiotemporal activation of the signaling pathways and the down regulation of a robustly expressed gene, foxq2, occur early in development and the GRNs governing germ layer and axis specification in the sea urchin embryo are well-established. Thus, an obvious limitation of this methodology is that in many cases it may be necessary to knockdown the activity of a particular signaling pathway during later stages of development and within specific territories. There are several technologies now available that can be used to overcome these limitations ( e.g. photo-morpholinos, Crisper/Cas9 ³⁸, FACseq) even in non-traditional model embryos that are not amenable to genetic manipulation. In many cases it may not be possible to identify a candidate gene down stream of a signaling pathway of interest whose spatiotemporal expression is as easily assessed as foxq2, which has a remarkably high signal-to-noise ratio. It is common to find genes with much lower signal-to-noise ratios. If another gene is unavailable for a particular assay, then the in situ probe generated for the gene of interest should be as long as possible. In general, using probes longer than 1,000 base pairs significantly increases the signal and reduces the noise in colorimetric and fluorescent in situ assays.

Once the transcriptional assay that indicates where and when the signal transduction pathway of interest is signaling is established, the next important step is to determine both the spatial and the temporal expression of the putative regulatory factors involved in the developmental mechanism under study. Differential screen and/or QPCR data are important tools, but they can be misleading. Confirming with whole mount in situ hybridization that the gene of interest is expressed within the territory where the signaling pathway is active prevents a waste of time and resources. In addition, these data allow predictions to be made about the gene regulatory architecture, which will inform the more detailed analyses that will follow once the players involved are identified with this initial assay ( Figure 2). We present a simple colorimetric assay in this protocol; however, fluorescent in situ hybridization (FISH) can also be used to visualize several fluorescent probes at the same time, allowing for enhanced spatial resolution within the territory of interest as well as confocal microscopy ²⁴. In addition, FISH can be paired with antibody staining to identify post-translational modifications and/or where the signaling pathway of interest is active ²⁴.

There are two critical steps in the protocol that are often overlooked. One is the preparation of the morpholino oligonucleotide solution. It is important to heat the morpholino solution at 65 °C for 2 min to 5 min and to centrifuge it at full speed for at least 10 min before loading the injection needles. These steps are essential when the morpholino stock solution is stored at -20 °C because morpholino oligonucleotides can precipitate from the solution at low temperatures and the spinning of the solution prevents the injection needles from being clogged by particulates. Another critical aspect to consider is that embryos should be thoroughly mixed into the various solutions during every step of the fixation and in situ hybridization protocols. In our hands, the signal is reduced and/or the background is increased if this simple step is not performed.

Perhaps the most important aspect of the methodology presented here is that it allows for efficient functional analyses of large sets of potential signal transduction molecules generated by next generation transcriptomics and proteomics. Once these initial functional analyses confirm a group of regulatory factors are involved in a pathway of interest, the next challenge is to use established assays ( e.g. functional knockdowns; detailed multi-colored whole mount in situ; biochemical interactions) to assess how they fit into the extracellular, intracellular, and transcriptional levels of the signal transduction pathways involved in a particular developmental process.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

Materials

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References

1. Stepicheva, N. A., Song, J. L. High throughput microinjections of sea urchin zygotes. Journal of visualized experiments : JoVE. e50841 (2014).

2. Arenas-Mena, C., Cameron, A. R., Davidson, E. H. Spatial expression of Hox cluster genes in the ontogeny of a sea urchin. Development. 4631-4643 (2000).

3. Sethi, A. J., Angerer, R. C., Angerer, L. M. Multicolor labeling in developmental gene regulatory network analysis. Methods in molecular biology. 249-262 (2014).

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions | Protocol

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This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos. Copy Citation |Download Citations Range, R. C., Martinez-Bartolomé, M., Burr, S. D.

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Abstract

Embryogenesis is a dynamic process with an intrinsic variability whose understanding requires the integration of molecular, genetic, and cellular dynamics. Biological circuits function over time at the level of single cells and require a precise analysis of the topology, temporality, and probability of events. Integrative developmental biology is currently looking for the appropriate strategies to capture the intrinsic properties of biological systems. The "-omic" approaches require disruption of the function of the biological circuit; they provide static information, with low temporal resolution and usually with population averaging that masks fast or variable features at the cellular scale and in a single individual. This data should be correlated with cell behavior as cells are the integrators of biological activity. Cellular dynamics are captured by the in vivo microscopy observation of live organisms. This can be used to reconstruct the 3D + time cell lineage tree to serve as the basis for modeling the organism's multiscale dynamics. We discuss here the progress that has been made in this direction, starting with the reconstruction over time of three-dimensional digital embryos from in toto time-lapse imaging. Digital specimens provide the means for a quantitative description of the development of model organisms that can be stored, shared, and compared. They open the way to in silico experimentation and to a more theoretical approach to biological processes. We show, with some unpublished results, how the proposed methodology can be applied to sea urchin species that have been model organisms in the field of classical embryology and modern developmental biology for over a century.

Keywords: Morphogenesis, Sea urchin, Cellular dynamics, In vivo imaging, Multiscale digital specimens, In silico modeling

The phenomenological and theoretical reconstruction of multiscale dynamics in animal embryogenesis: a general perspective

In an ideal scenario, morphogenetic events could be understood through a circular path integrating quantitative reconstructions, data analysis, and modeling (depicted in Fig. ). The bottom-up digital reconstruction and analysis from the sub-cellular scale to the whole embryo level should be combined with a top-down approach. The latter includes the effect of high-level phenotypic features, such as tissue biomechanics on biochemical processes and on the regulation of gene expression. Events at the subcellular level modify the properties of cells and extracellular matrices, and the latter, in turn, modulates the biochemical activity of the cell [ 13, 74].

Among all possible genetically and epigenetically encoded processes, cells divide, differentiate, and migrate in the context of their self-produced external environment [ 67, 102]. Cell features over time can be described with a set of parameters including the cell position, cell lineage, volume, surface, shape indexes, local convexity or concavity, nuclear/cytoplasmic ratio, cell neighborhood and surface of contact, cell polarization assessed by the asymmetric distribution of sub-cellular structures, intrinsic motility, and directionality. At the mesoscopic level, tissue patterning depends on cell displacements, cell-cell adhesion, and cell division characteristics. The emergence of the most macroscopic features shapes the whole embryo and provides global mechanical constraints. Changes in cell and tissue properties can be described by biomechanical models whose behavior depends on initial and boundary conditions given by the cellular environment [ 135]. A number of recent publications on different model organisms have focused on the mechanical properties of cells and their emergence from the interaction of cells with their environment [ 41, 46, 65, 66] and the interplay between mechanical constraints and gene expression patterning [ 13, 74, 114]. The biomechanical approach readily leads to the integration of the genetic and molecular basis of cell activities into the macroscopic deformation of tissues that shape the embryo [ 20].

Building multiscale models, integrating the biomechanics, and the molecular and genetic dynamics of embryonic development requires the quantitative description of cell behaviors. We review here how the state of the art reflects this paradigm, focusing on the reconstruction of digital specimens from in vivo and in toto imaging.

From in vivo imaging to modeling to understand animal embryogenesis: the premises

State of the art imaging techniques combined with the expression of fluorescent proteins provide time lapse recordings of the embryonic development of small transparent animal model organisms with resolution at the cellular level [ 69, 89]. The tridimensional images can be processed to reconstruct digital specimens composed of labeled volumes or surfaces corresponding to subcellular structures such as cell nuclei and membranes over time, and encompassing the clonal history of each cell. The digital reconstruction provides many advantages compared with raw 3D + time data. The visualization of biological structures (e.g., nuclei and membranes) in the digital specimen is simpler and can be combined with different color maps to highlight various features, select cells, and assess their clonal history (examples are shown for the sea urchin in Figs. and ). Even more importantly, digital embryos can be analyzed to quantify features at all scales.

A number of studies in different model organisms have more or less explicitly tackled the reconstruction of digital specimens from live observation. Pioneering work in the late 1970s [ 23, 128] led to the full reconstruction of the cell lineage tree of Caenorhabditis elegans [ 129]. This was obtained from the manual annotation of different embryos observed through Nomarski optics and was made possible thanks to the lineage invariance in Caenorhabditis elegans. Then, in the late 1990s, advances in microscopy and computation to handle high-dimensional data brought the first computer-aided cell tracking [ 124]. The authors used a new 3D multifocal time lapse system [ 55] to observe the early embryogenesis of Caenorhabditis elegans and the software Simi BioCell provided means for annotating and comparing the lineage trees of different embryos. The synergistic efforts of different disciplines brought further major advances. Developmental biology benefited from breakthroughs in fluorescent protein engineering [ 14, 16, 84, 85], microscopy imaging systems [ 34, 60, 107, 127, 130], image processing methods for cell segmentation and cell tracking [ 69, 90- 92], the development of software for computer-aided data processing and visualization [ 26], and computer hardware for processing high dimensional datasets on computing grids [ 115].

From in vivo imaging to modeling to understand animal embryogenesis: reconstructing multiscale digital specimens

However, the reconstruction of animal embryogenesis from 3D + time imaging data remains a challenging approach, suffering from intrinsic limitations mainly related to the quality of the original data and the simultaneous requirement of automation and precision. The field of image processing has only recently turned to biological images, first dealing with 2D images, then 3D data, and then addressing one more level of complexity with 3D + time processing [ 93]. But even the most sophisticated algorithms are currently unable to distinguish objects or track them where the human eye cannot provide an accurate benchmark. So, the next breakthroughs in the field will have to originate from the biological side. In vivo 3D + time imaging has to deal with the trade-off between conflicting requirements including spatial and temporal resolution, signal-to-noise ratio, and photo damage. High spatial resolution and signal-to-noise ratio are needed to capture cell position and shape, but high temporal resolution is necessary to achieve single cell tracking. Further constraints are imposed by photo bleaching and photo damage, which limit the acquisition rate and overall imaging duration. In addition, fluorescent staining of subcellular structures in live model organisms is often non-homogeneous and adds artifacts in data acquisition, limiting the quality of the reconstruction, despite the development of sophisticated pre-processing methods [ 72, 134].

We can expect further improvements in fluorescent staining with more stable and brighter protein variants. Revealing contrasts intrinsic to the tissues such as the generation of harmonics by multiphoton illumination, has also proved to be useful [ 101]. When the cell density becomes too high, e.g., at late developmental stages, strategies to decrease the image complexity by providing color combinations and mosaic staining should be further developed. On the side of microscopy techniques, there is still room for improvement in depth imaging, spatial and temporal resolution, and signal-to-noise ratio. The recently developed single-plane illumination microscopy seems a valuable alternative to laser point-scanning microscopy, as it definitely improves the temporal resolution and the signal-to-noise ratio. But various imaging artifacts have not been solved yet and single-plane 2-photon excitation, which looks very promising, is still under development by physicists [ 62].

Except with ideal image data sets, e.g., highly contrasted objects at low density and moving slowly enough, state of the art image processing strategies are still far from providing error-free, fully automated processing of large 3D + time images. Every cell tracking error propagates to its progeny and impairs the reconstruction of cell clonal history. Similarly, shape segmentation inaccuracy adversely affects further analysis of the cells' characteristic features. Consequently, although protocols for 3D + time imaging of development based either on 2-photon or confocal laser scanning or on selective plane illumination microscopy have been described for several model organisms- Caenorhabditis elegans [ 44, 97], Ascidians [ 116- 118], Drosophila melanogaster [ 105, 131], zebrafish [ 88], zebrafish and Drosophila [ 64], mouse [ 37, 100], and avian [ 73]-the most reliable results still come from manual reconstructions or extensively supervised and corrected data. In this context, the best results have been obtained for Nematoda and Ascidiacea embryos, thanks to their small cell number and largely invariant cell lineage that allows the matching of different specimens with precision at the single cell level (Table ). Drosophila [ 35, 36, 68, 71, 80, 87, 109, 131] and zebrafish [ 63, 82, 101, 137] are more difficult to handle and cell tracking results obtained from state of the art 3D + time imaging data of developing embryos are suitable for average analysis but lack precision for cell clonal analysis. We review below the main results obtained for Nematodes which provides an example of error-free reconstruction of the cell lineage, and for Ascidians which gives an example of the digital representation of cell shapes combined with a quantitative description of cell interactions.

Table 1

The digital reconstruction of Nematoda

The reconstruction of the cell lineage tree of Caenorhabditis elegans led to the development of dedicated software applications. The first available tools were designed to perform manual tracking and annotations. Worth mentioning, as it was pioneering in the field, is the development of 3D-DIASemb [ 50] for performing manual tracking, nuclear and membrane segmentation, and cell motion analysis from Nomarski video microscopy imaging of Caenorhabditis elegans up to the 28-cell stage. Simi BioCell, first mentioned in 1997, was used for manual tracking and annotation in Nematoda [ 58, 59], for combining the lineage tree with gene expression [ 96], and even lately for investigating cell lineages in the early mouse embryo [ 94]. Semiautomatic tracking with StarryNite [ 11] and AceTree [ 97] (available at http://waterston.gs.washington.edu/) provided the cell lineage tree of the Caenorhabditis elegans embryo up to 350 cells [ 8]. A digital atlas of the first larval stage (L1) with single-cell resolution was obtained from confocal images of 15 individual worms [ 78]. Gene expression and cell fate were then investigated [ 77] with the volume-object annotation system [ 108]. More recently, NucleiTracker4D (available at http://sourceforge.net/projects/nucleitracker4d/) was used for the semiautomatic tracking of nuclei imaged by laser scanning confocal microscopy of Caenorhabditis elegans development [ 39]. Further details concerning the cell clonal analysis in Caenorhabditis elegans can be found in [ 38, 44] and all the available data has been collected by the Nematode community on http://www.wormatlas.org/. It should be noted that the digital reconstruction performed so far on Nematoda has focused on displaying the gene regulatory network data according to cell position, but little integration has been achieved with cell behavior, and to our best knowledge, the assessment of cell shape through the segmentation of membrane images has not been addressed.

The digital reconstruction of Ascidiacea

As for the Nematodes, Ascidians exhibit a largely invariant cell lineage and digital embryos have been reconstructed from confocal images of fixed specimens at different stages of development. The most remarkable work has been collected in the Ascidian Network for In Situ Expression and Embryological Data (ANISEED) database [ 133] and the 3D Virtual Embryo software for visualization and interaction [ 132], both available through http://aniseed-ibdm.univ-mrs.fr/virtual_embryo.php. The 3D Virtual Embryo has been introduced as a tool for quantifying the geometry of cells and cell-cell contacts in an interactive three-dimensional environment and provides cell volumes, mathematical descriptors of cell shape, and cell-cell contacts. Initially used to describe the first developmental stages of Ciona intestinalis and a late 32-cell stage of Halocynthia roretzi, the concept has been extended to other Ascidian species. The ANISEED database integrating molecular, embryological, and anatomical data in the virtual embryo provides a new paradigm in the field of developmental biology. It should, however, be noted that so far, ANISEED has been based on the segmentation of fixed embryos. Further extending the concept will require the segmentation of live specimens. The Ascidian community has also contributed another interesting initiative. At first, dealing with raw data only, the Four-dimensional Ascidian Body Atlas, with interactive visualization of the development of Ciona intestinalis [ 57] is available online through http://chordate.bpni.bio.keio.ac.jp/faba/1.4/top.html. More recently, Hotta et al. achieved the manual segmentation of Ciona intestinalis tailbud embryos, and the interactive visualization of the data is available through 3D pdf files [ 98].

Reconstructing the multiscale dynamics of the sea urchin embryogenesis

The sea urchin as an animal model in experimental biology: historical perspective

The importance of the sea urchin as a model organism in biological investigations dates back to the nineteenth century and its characteristic features led to cornerstone discoveries in the history of embryology and cell biology. The transparency of the embryo allowed for the first time to observe the fusion between oocyte and sperm during the process of fertilization [ 52]. In 1892, blastomeres isolated at the 2-cell stage were shown to produce complete sea urchin larvae [ 24]. Such experimental possibilities made the sea urchin a very valuable model for investigating the components of individual variation. Pioneering studies on teratogenesis carried out by changing the chemical content of the sea urchin embryo helped to found the concept of embryonic induction [ 51]. The manipulation of double fertilized eggs [ 9] inspired the chromosome theory of inheritance and opened the way to the understanding of cancer as a genetic disease (English translation in [ 10]). Further work opened the way to modern Developmental Biology with the exploration of early development including gastrulation [ 56], cell mechanics underlying morphogenetic movements [ 18], and the mechanical properties of the sea urchin embryo at early stages of development [ 53, 54]. During the same period, Gustafson and Kinnander developed a technique allowing the gastrulation of the sea urchin Psammechinus miliaris to be analyzed for the first time, through time-lapse cinematography [ 42]. A few years later, the sea urchin was selected as a suitable animal model for large-scale analysis of gene expression regulation during early development [ 12]. More recently (2001), the discovery of cyclin and its role in cell cycle progression in the sea urchin [ 33] resulted in the award of the Nobel Prize in Physiology and Medicine to Tim Hunt (jointly with Leland H. Hartwell and Paul M. Nurse). Further information about the contribution of the sea urchin animal model to the foundation of paradigms in embryology and developmental biology can be found in [ 27].

The sea urchin in modern developmental biology

The sea urchin remains a leading model organism in developmental biology [ 4- 6, 32], and the sea urchin community made a major breakthrough in building the architecture of the gene regulatory networks (GRN) underlying the embryo patterning and functional differentiation [ 86, 110]. The corresponding resources including protocols, database, and data annotation are publicly available (Table ). However, cell lineage and biomechanics have only been scarcely addressed. The fate of embryonic cells was identified by means of fluorescent dyes [ 113, 120, 121] and uncaging methods [ 112], but very little work has been done to achieve in vivo and in toto 3D digital reconstructions from time-lapse imaging, and the resulting data have not yet permitted 3D + time quantitative analysis of cell behavior. Regarding the early development of Paracentrotus lividus, Luengo et al. [ 81] modeled cell geometries with a Voronoi diagram and compared results with membrane shapes obtained with a viscous watershed segmentation method [ 136]. Pastor et al. [ 106] designed a cell tracking method based on the introduction of a 4D tube-structuring element. Rubio et al. [ 119] merged lineage trees encompassing different periods of the sea urchin development using an error-tolerant graph matching technique. So far, however, these methods and the corresponding data have not led to new biological insights or any quantitative assessment of cell behavior. A quantitative 2D analysis of the mitotic gradient observed in different sea urchin species [ 1, 2, 103, 104] has been performed in the sand dollar Dendraster excentricus [ 25]. The quantitative assessment of cell behavior from time-lapse video microscopy imaging suggested a possible dependency of cell division timing and position on the cell volume between the fourth and the tenth cell cleavage. In this work, cell volumes were calculated indirectly from the measurement of cell diameter under the assumption of a spherical cell shape. Conclusions derived from 2D analysis might, however, be revisited at some point with more precise 3D data. This paradigm has been explored-although the corresponding data did not have single cell resolution-for the 3D reconstruction of adult sea urchin soft tissue from magnetic resonance imaging [ 138]. The method proposed for the inference of phylogenetic relationships was described together with a simple and attractive way to explore 3D structures interactively in digital specimens embedded in pdf files.

Reconstructing the digital sea urchin from 3D + time in toto imaging

The sea urchin has a number of desirable characteristics, making it a model of choice for in toto time-lapse microscopy imaging of the embryonic and larval stages. The embryo is easily accessible, relatively transparent and small, develops fast, grows to a fairly small number of cells and can be engineered to express fluorescent proteins. After overcoming the problem of the immobilization of the swimming blastula, the embryo can be imaged in toto for extended periods of time by two-photon laser scanning or selective plane illumination microscopy. The sea urchin is the ideal model for making a proof of concept for the reconstruction of the multiscale dynamics of a developing model organism from Deuterostomia, exploiting the unique data available in terms of the GRN architecture and dynamics.

We should, however, emphasize here that the task of long-term 3D + time imaging of the developing sea urchin and automated image processing to achieve cell tracking and shape segmentation is still subject to a number of limitations. These limitations are mainly on the biological side: highlighting structures without ectopic staining, e.g., staining membranes or nuclei without spurious cytoplasmic staining, immobilizing the specimen without mechanical deformation, compensating for light absorption and dispersion and thus loss of signal at depth, resolving individual nuclei throughout the entire volume including at late developmental stages, keeping a temporal resolution high enough to optimize the cell tracking outcome, e.g., ideally keeping the time step smaller than 2 min. Both 2-photon laser scanning and selective plane microscopy imaging are suitable, although they are based on quite different compromises, for the long-term 3D + time imaging of sea urchin embryos. It may soon be demonstrated that for specimens as small as sea urchin embryos, SPIM/DSLM offers the best conditions, provided that specimen mounting is improved compared with what is currently achieved [ 62].

Over the past few years, the BioEmergences platform ( http://www.bioemergences.eu) and its interdisciplinary team have been exploring this paradigm, starting with the systematic reconstruction of the cell lineage tree of Paracentrotus lividus from in toto imaging and automated algorithmic processing. The reconstructed digital embryo consists of cell positions and shapes over time and cell clonal history. The corresponding data can be displayed and interactively explored, validated, and annotated with the visualization interface, Mov-IT, [ 101] (Figs. and ). The lineages of the different cell populations described in the early embryo according to [ 19] were highlighted from the 32-cell stage until blastula stages. These reconstructions provide precise measurements hitherto unavailable to model the dynamics at the microscopic scale and reconstruct the overall dynamics of the morphogenetic process at the macroscopic scale as well as possible. This is a most promising approach for the systematic identification of symmetry breaking or intra-individual and inter-individual variation. It would be a major paradigm change in the field of developmental biology, as it uses new theoretical frameworks to revisit classical embryology concepts.

When completed up to late developmental stages, the precise reconstruction of the cell's clonal history with associated shape, volume, and contact changes will contribute to our understanding of morphogenesis and differentiation processes [ 47- 49] and answer a number of open questions. Digital specimens and the corresponding raw data should be made available to the entire community and scientists should all work to enrich a common database and contribute to the validation and correction of cell trajectories and cell shape. By sharing this 3D + time quantitative data for cohorts of individuals in defined genetic and environmental conditions, scientists will have the tools to bring a new perspective to classical questions of embryology.

First, a series of questions would be settled with the analysis of cell clonal history and cell fate such as for ectoderm/endoderm boundaries in the veg1 territory (reviewed by [ 32]) or the clonal origin of larval structures such as coelomic pouches. A second series of questions concerns the biomechanics of generic morphogenetic movements. The thickening of the vegetal plate during the ingression of the primary mesenchymal cells [ 28, 29] is described as a case of epithelial-mesenchymal transition, that should be compared to other similar processes [ 126]. The elongation and narrowing of the archenteron and the flattening of the epithelial cells in the wall gut rudiment [ 30], described in terms of the convergent extension movements [ 45], can be related to processes described in other model organisms [ 66, 67]. The Drosophila germ band extension [ 61], and the gastrulation and axis elongation in Ascidians [ 95], zebrafish [ 40, 70], avians [ 76, 122, 125], and mice [ 123] should, at some point, be quantified and compared. Achieving such a goal in the sea urchin will be insightful for a comparative study of morphogenetic processes and their evolutionary relationship.

Modeling morphogenetic processes in sea urchin development

The long-term goal of integrated modeling of the multiscale dynamics of sea urchin development will take advantage of the partial models established at different scales. The tentative modeling strategies available so far are scarce and much remains to be done in terms of comparing the models with quantitative data. A careful bibliographic search has brought to light four papers, all addressing early developmental steps and dealing respectively with: the biomechanical modeling of the primary invagination [ 21], the mechanical modeling of division asymmetry [ 3], the molecular modeling of cell proliferation [ 17] and the Boolean modeling of GRN dynamics [ 111].

Finite element modeling was used to discriminate between the possible mechanisms accounting for the primary invagination leading to the formation of the archenteron [ 21]. Hypothetical mechanisms driving invagination included at the time: apical constriction [ 7, 31], annular ring contraction [ 15, 31], cell tractoring [ 15], gel swelling [ 75], and apicobasal contraction [ 43]. But each of these mechanisms required different properties of the composite epithelial sheet. Further studies on Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage [ 22] revealed that the extracellular matrix of the blastula wall was considerably stiffer than the cell layer. The authors then suggested that neither apical constriction nor annular ring contraction could drive invagination while all other hypotheses remained plausible.

Akiyama et al. developed an ad hoc mathematical model to reproduce the first four cleavages of the sea urchin egg, including the asymmetric divisions giving rise to micromeres [ 3]. The model established the planes of mitosis by assuming a chemotactic motion of centrosomes based on the diffusion of a chemical substance with repellent effects at the animal pole and attractive activity at the vegetal pole.

Ciliberto and Tyson [ 17] modified a previously developed mathematical model [ 99] based on molecular interactions underlying early embryonic cell-cycle control to investigate the hypothesis of a mitotic gradient along the animal-vegetal axis. Their results exhibited a good match with experimental data from [ 83] on Temnopleurus toreumaticus. They concluded that the mitotic wave was primarily attributable to differences in interdivision times in different parts of the embryo, although some subtle details of the wave were probably due to diffusive coupling between neighboring cells.

The transformation of the well-established GRN architecture into a predictive, dynamic Boolean computational model was a major recent breakthrough by the team led by Eric Davidson. This Boolean model computes spatial and temporal gene expression according to the GRN structure established for the embryonic development in the sea urchin. Direct comparison with experimental observations showed that the model predictively computed known gene expression patterns with remarkable spatial and temporal accuracy, thus validating the GRN architecture established so far.

All these models are intended to be both predictive and explanatory and to provide a formal basis to support or refute the intuition. They also tackle specific and limited scales and components of the system. Their integration in a multiscale modeling of morphogenesis will require a huge interdisciplinary and synergistic effort.

Conclusions

We have depicted an ideal scenario for a complex systems approach to morphogenetic processes underlying early animal development. The proposed strategy, based on the evidence that morphogenesis is a multiscale dynamic process, requires the integration of quantitative data across spatio-temporal scales into a yet-to-be-defined theoretical framework. A new interdisciplinary community coming from different perspectives and sharing the same goals is progressing fast. However, the overall conceptual and experimental landscape is still disparate, resembling a huge puzzle whose pieces do not yet fit together. Synergistic efforts by the scientific community are producing public databases of genome and GRN components for many model organisms. State-of-the-art microscopy imaging has made it possible to observe embryo development in small transparent organisms in toto, in vivo, with resolution at the cellular level. Software tools are being developed for visualizing, reconstructing and analyzing 3D + time image data. Computer hardware enables the processing of high-dimensional datasets on computing grids. Digital embryos can be directly manipulated and visualized in 3D to inspect and analyze cell clonal history and cell shape changes and cell-cell interactions over time. Much effort is being dedicated to the development of mathematical models of morphogenesis, based on either GRN architecture or the mechanical properties of tissue, for testing biological hypotheses and predicting the system's behavior. A new generation of digital resources anticipates the need to integrate multimodal and multiscale data and to provide tools for in silico experimentation. The availability of multimodal and multiscale quantitative data from the in vivo observation and reconstruction of cohorts of individuals should revolutionize practices in the field of developmental biology. Some major bottlenecks remain, related to the current limitations of long-term in vivo imaging and their consequences in terms of automated image processing. Imaging artifacts make 3D + time data from developing organisms difficult to process automatically. And the need for human supervision and time-consuming manual validation and correction is a major issue. The next breakthroughs may come from imaging techniques and may also rely on crowd sourcing for validating and correcting data. We also need new concepts in data analysis and theoretical modeling based on new representations of biological systems, faithful to biological observations but derived from formal descriptions to be shared by a new interdisciplinary community. In this context, the sea urchin is a model organism of choice for unprecedented achievements.

Acknowledgments

We thank Louise Duloquin for the time-lapse imaging of live sea urchin embryos and our collaborators at the BioEmergences platform for their contribution. This work was supported by the Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche, the European Commission and the Region Ile de France. We thank Paul Bourgine for helpful discussions and Yannick Kergosien for critical reading of the manuscript. We apologize to the many authors whose work we were unable to cite owing to space constraints.

References

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# data[].attributes.attachment_link.title
Towards 3D in silico modeling of the sea urchin embryonic development

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Embryogenesis is a dynamic process with an intrinsic variability whose understanding requires the integration of molecular, genetic, and cellular dynamics. Biological circuits function over time at the level of single cells and require a precise analysis of the topology, temporality, and probability of events.

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MOl Biol as a method (transfection)

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Summary

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

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Hirsinger, E., Carvalho, J. E., Chevalier, C., Lutfalla, G., Nicolas, J. F., Peyriéras, N., Schubert, M. Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes. J. Vis. Exp. (95), e52042, doi:10.3791/52042 (2015).

Abstract

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Introduction

During development, a single cell gives rise to an entire organism in a highly complex process that involves both cell divisions and movements. To better understand the biological principles underlying the dynamics of cell behavior, developmental biologists have started to use fluorescence-based in vivo imaging techniques. Specific compartments of cells, such as cell membranes, can either be labeled by treatments with fluorescent dyes, an approach hampered by a lack of specificity and of tissue penetration ¹, or by the specific introduction into the embryo of exogenous mRNAs encoding fluorescent proteins ². Different techniques can be used for the efficient delivery of exogenous compounds, such as mRNAs. These include, but are not limited to, microinjection, electroporation, bombardment with microparticles, lipofection and transduction 3,4. Although all of these approaches can be used to introduce exogenous compounds into a developing embryo, only microinjection allows the application of predefined and precise quantities into each cell ³. Microinjection techniques have been described for all major developmental model systems ⁴ ( e.g., fruit flies, nematode worms, zebrafish, frogs, mice) as well as for some alternative models ⁴, including those used for comparative studies aimed at understanding the evolution of developmental mechanisms ( e.g., sea anemones, annelid worms, sea urchins, ascidian tunicates, the cephalochordate amphioxus).

Cephalochordates, which together with tunicates and vertebrates establish the chordate phylum, are particularly well-suited models to study the evolution of chordates and the diversification of vertebrates from an invertebrate ancestor ⁵⁻⁸. The cephalochordate lineage diverged very early during chordate evolution; and extant cephalochordates, which are subdivided into three genera ( Branchiostoma, Asymmetron and Epigonichthys), resemble vertebrates both in terms of overall anatomy and genome architecture ⁵⁻⁸. Of the about 30 species of cephalochordates that have been described so far, five are available for embryological and developmental studies 6,9: Asymmetron lucayanum (the Bahama lancelet), Branchiostoma floridae (the Florida amphioxus), Branchiostoma lanceolatum (the European amphioxus), Branchiostoma belcheri (the Chinese amphioxus) and Branchiostoma japonicum (the Japanese amphioxus). Ripe adults of three of these species ( B. lanceolatum, B. belcheri and B. japonicum) can be induced to spawn on-demand during the breeding season 10,11. In addition, at least for B. lanceolatum, efficient spawning can also be induced in artificial sea water ¹², thereby making this particular cephalochordate species accessible for laboratories that do not have access to natural seawater. The combination, in B. lanceolatum, of a convenient and reliable access to embryos with an efficient delivery method, such as microinjection, so far the only delivery technique developed in amphioxus (in both B. floridae and B. belcheri) ¹³⁻¹⁵, will enable the development of a novel suite of manipulative techniques, including lineage tracing- and dynamic cell behavior-based approaches.

A protocol for the efficient microinjection of mRNAs to express fluorescent proteins in the B. lanceolatum embryo was hence developed. Furthermore, to provide a basic toolkit for live imaging of B. lanceolatum embryos, vector systems were developed that allow membrane-associated and nuclear expression of fluorescent proteins. For membrane targeting, enhanced green fluorescent protein (eGFP) was fused to the human HRAS CAAX box and nuclear localization of mCherry and eGFP was obtained by fusion to the zebrafish histone 2B (H2B) exon ( Figure 1, Supplementary File 1). Furthermore, with the goal to optimize protein translation, the Kozak sequences and codons of the constructs have been modified and adapted to usage in B. lanceolatum. Taken together, the injection method and expression vectors presented here will serve as a basis for the generation of new experimental approaches for cephalochordates, notably analyses using the latest fluorescence-based in vivo imaging techniques.

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Protocol

1. Preparation of Instruments and Reagents

1. Transfer Pasteur pipettes

1. Generate a series of transfer Pasteur pipettes with different tip diameters by pulling 230 mm long Pasteur pipettes above a flame at different speeds. Ensure that the taper is as long as possible for smooth and fine control of aspiration.

2. With a diamond scribe, scratch the pipette along a line perpendicular to the length of the pipette. With both hands, pull the pipette parallel to its length to generate a blunt cut. Swiftly flame-polish the pipette without sealing the tip.

3. Vary the diameter of the tip with the stage of the oocytes/embryos to be pipetted: 300-400 µm for unfertilized oocytes (around 150 µm in diameter), 600 µm for fertilized eggs (around 500 µm in diameter), 200-400 µm for hatched neurulae. Use pipettes with a mouth-monitored aspiration tube to transfer oocytes/embryos from one dish to another.

2. Injection needles

1. Manipulate the capillaries with gloves to ensure RNase-free conditions. Use borosilicate glass with filament capillaries with dimensions of OD 1.20 mm, ID 0.94 mm, length 10 mm.

2. If capillaries are pulled on the type of heating-filament needle puller described in the Materials List, use the following settings: Heat 600, Pull 50, Velocity 80, Time 60, Pressure 200 or 300. Otherwise, ensure that the shape, which is crucial for successful injections, is as shown in Figure 2 with the following properties: 4-8 µm outer tip diameter, 2 cm taper length.
NOTE: Needles can be pulled before the spawning season and used throughout the season.

3. 5% Phenol Red stock solution (4x)

1. Prepare fresh before each spawning season.

2. In a 0.22 µm-filtration tube, weigh out 25 mg of Phenol Red powder.

3. Add 0.5 ml of DNase- and RNase-free water to the tube containing the powder.

4. Spin for 3-5 min at 18,000 x g at room temperature to filter-sterilize the solution and remove crystals that could clog the injection needle.

5. Store at 4 °C or store 250 µl aliquots at -20 °C.

4. 0.25 mg/ml poly-lysine solution

1. Prepare fresh before each spawning season.

2. Dissolve 5 mg of poly-L-lysine in 20 ml distilled water. Store 5 ml aliquots at -20 °C.

3. Use a defrosted aliquot immediately and only once to ensure reproducible and robust adhesion of the oocytes to the poly-lysine-coated dish.

5. mRNA synthesis

1. Prepare fresh before each spawning season.

2. Linearize 5 µg of DNA of interest with the adequate enzyme (usually for 2 hr, at 37 °C). To check the completeness of the digestion, run 2% in volume of the digestion mix on a 1% agarose-TBE gel in TBE buffer at 150 W for 20 min.

3. Extract the linearized DNA with 25:24:1 phenol (pH 8.0):chloroform:isoamyl alcohol. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous (upper) phase.

4. Extract the aqueous phase again with 24:1 chloroform:isoamyl alcohol. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.

5. Precipitate the linearized DNA with 100:10:300 linearized DNA:3 M sodium acetate (pH 5.2):100% ethanol overnight at -20 °C.

6. Centrifuge 20 min at 18,000 x g at 4 °C. Rinse in 70% ethanol.

7. Centrifuge 10 min at 18,000 x g at 4 °C. Let dry and resuspend in RNase-free water at a final concentration of 0.5 µg/µl.

8. Transcribe 1 µg of linearized DNA using an mRNA synthesis kit with the appropriate polymerase, according to manufacturer's instructions.

9. Extract the mRNA with 5:1 phenol (pH 4.7):chloroform and ammonium acetate stop solution provided with the kit.

10. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.

11. Extract the aqueous phase again with 24:1 chloroform:isoamyl alcohol. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.

12. Precipitate the mRNA with 100% isopropanol overnight at -20 °C.

13. Centrifuge 20 min at 18,000 x g at 4 °C. Rinse in 80% ethanol.

14. Centrifuge 10 min at 18,000 x g at 4 °C. Let the pellet dry at room temperature for no longer than 5-10 min as it will then be difficult to resuspend. Resuspend in DNase- and RNase-free water to a final concentration of at least 2 µg/µl to ensure a decent final mRNA concentration in the injection mix.

15. To check the quality and size of the transcription product, run 0.5 µl of the mRNA on a RNase-free 1% agarose-TBE gel in TBE buffer at 150 W for 20 min. Store 2 µl aliquots at -80 °C.

6. Poly-lysine-coated dishes
NOTE: Poly-lysine coated dishes are used to immobilize the oocytes during injection.

1. For each 35 mm cell-culture Petri dish (5 in total), cover the bottom of the Petri dish with 1 ml of the thawed 0.25 mg/ml poly-lysine solution. Incubate at room temperature for 5 min.

2. For each 35 mm cell-culture Petri dish (5 in total), transfer the 0.25 mg/ml poly-lysine solution into another 35 mm cell-culture Petri dish. Incubate at room temperature for 5 min.

3. Discard the 0.25 mg/ml poly-lysine solution.

4. Let the Petri dishes dry, upside-down at room temperature for 2 hr.

5. Store the poly-lysine-coated dishes wrapped in a plastic wrap at 4 °C to avoid contamination for one week maximum.

7. Agarose-coated dishes
NOTE: They are used to culture injected embryos. The agarose provides a cushion for the injected embryos and prevents them from sticking to the bottom of the dish.

1. Make artificial seawater (ASW) using 37-38 g/L commercial salts + 0.25 mM NaHCO₃ in reverse osmosis water.

2. Dissolve agarose to a 1% concentration in 0.22 µm-filtered ASW by heating the solution in a microwave.

3. Swiftly pour the warm agarose solution from one 35 mm Petri dish into another one in order to ensure a very thin agarose coating of the dish.

4. Store the agarose-coated dishes wrapped in Saran wrap at 4 °C to avoid contamination for one week maximum.

8. Injection mix and loading of the injection needles

1. About 2 hr before starting the injections, make a 2 µl injection mix in RNase- and DNase-free water with final concentrations of 1-1.8 µg/µl of mRNA, 15% glycerol, 1.25% Phenol Red.
NOTE: The Phenol Red colors the solution, which allows monitoring of the injection efficiency and the identification of successfully injected embryos. Glycerol favors mRNA diffusion within the oocyte.

2. Centrifuge 4 min at 18,000 x g to pellet crystals. Keep on ice until use.

3. With a 10 µl pipette, collect 0.5 µl of the injection mix, avoiding the bottom of the tube, where the crystals have been pelleted.

4. Backfill at least two injection needles (in case one breaks during the injection) by pipetting the 0.5 µl drop of injection mix at the large opening of the needle.

5. Install the needles in a storage jar with liquid at the bottom at 4 °C to prevent evaporation of the injection mix. Let the injection mix slowly travel to the tip of the injection needle for at least 1 hr to minimize the creation of bubbles.

6. Store additional injection mix at -80 °C for maximum of three additional uses, after which the mRNA quality deteriorates (data not shown).

2. Collection of Biological Material, Microinjection and Embryo Culture

1. Oocyte and sperm collection
NOTE: See Theodosiouet al. ¹² for a detailed protocol for inducing spawning and for gamete collection.

1. Shock males and females in ASW at 23 °C for 24 hr.

2. One to two hours before sunset, transfer the adults into individual cups in ASW at 19 °C because most adults will spawn 1-2 hr after sunset.

3. Rinse 35 mm Petri dishes in filtered ASW and let them dry upside-down to prevent the oocytes from sticking to the bottom of the dish.

4. Upon spawning, immediately collect sperm and oocytes with a 1,000 µl pipette.

1. Keep sperm and oocytes separate from adult amphioxus because the contact is detrimental for gamete health (data not shown).

2. Furthermore, avoid startling the adults, which leads to movements that dissipate, and hence dilute, both sperm and oocytes. To keep the sperm active as long as possible and to optimize fertilization rate, collect the sperm as concentrated as possible.

5. Keep sperm on ice in a 1.5 ml tube.

6. Transfer oocytes in filtered ASW into the pre-rinsed 35 mm Petri dishes.

7. Transfer 100-500 oocytes with a previously pulled 300-400 µm transfer Pasteur pipette to another 35 mm Petri dish to perform the injections.

8. Fertilize the remainder of the clutch as a control for sperm and oocyte quality or for other experiments.

2. Oocyte injection

1. Install the injection needle on the micromanipulator at a 50° angle relative to the horizontal plane.
NOTE: Angles of less than 50° will push the oocytes around on the dish, while angles of more than 50° will not allow an appropriate monitoring of the needle position relative to the oocyte.

2. Under a fluorescent dissecting scope with 25X oculars, transfer 30 oocytes with the 300-400 µm transfer Pasteur pipette on a poly-lysine-coated dish containing filtered ASW.

3. Deposit the oocytes along a line to carry out injections in an ordered way and to distinguish injected from non-injected oocytes. Inject small numbers (30 oocytes) to minimize the exposure time of oocytes to poly-lysine, which tends to deform developing embryos (data not shown).

4. Use the dark field illumination to render the oocytes as translucent as possible.

5. With the coarse movement knob of the micromanipulator, bring the injection needle close to an oocyte.

6. With fine forceps, cut open the needle at the level where the tip starts to be curved. By pulsing with the injector, verify that red injection mix is actually flowing out of the needle.

7. At 200X magnification and with the fine movement knob of the micromanipulator, gently move the injection needle inside the core of the oocyte.
NOTE: If inserted too superficially, the injected solution will not remain inside the oocyte. If inserted too far, the oocyte will be destroyed.

8. Inject with 1-3 pulses of 120 msec duration and 1-10 psi pressure. If the needle is fine enough, inject with continuous flow at constant pressure. Ensure that the injection volume corresponds to 1/5 to 1/3 of the volume of a single oocyte.

9. Following injection, pull the needle out swiftly to avoid leakage of the oocyte.

10. Verify that the injected solution remains within the oocyte and that after a few sec, the injected solution spreads throughout the oocyte.

11. Move on to the next oocyte in line.

12. Keep some uninjected embryos of each series as negative control to estimate the background fluorescence when scanning for injected embryos.

3. Fertilization, selection of injected embryos and embryo culture

1. Fertilize the oocytes as soon as a series has been injected. As oocyte quality declines with time, inject and fertilize oocytes within 1 hr after spawning¹².

2. Depending on the sperm concentration, add 1-5 drops of sperm to the oocytes and swirl the dish.
NOTE: The fertilization envelope should become apparent on the embryos after about 1 min.

3. Allow the embryos to detach from the poly-lysine-coated dish, while injecting another series of oocytes.

4. Transfer the embryos with the 600 µm transfer Pasteur pipette into an agarose-coated Petri dish. Remove the embryos from the poly-lysine-coated dish as soon as possible, if at all possible before the 2-cell stage.
NOTE: In case of prolonged exposure to poly-lysine, the embryos tend to become densely-packed blastulae, flattened on the side touching the bottom of the dish.

5. At the 2-cell to 4-cell stage, select with a fluorescent dissecting scope with DSR filter the successfully-injected embryos, i.e., those with a normal morphology that exhibit a Phenol Red-derived red fluorescent signal.

6. Keep the embryos in culture in filtered ASW in agarose-coated Petri dishes at 19 °C until the desired stage for in vivo imaging.

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Representative Results

The protocol detailed above provides the basis for the microinjection of B. lanceolatum oocytes and hence for the introduction into developing B. lanceolatum embryos of mRNA encoding fluorescent proteins for in vivo imaging. Although the technique is certainly robust and reliable, the rate of successful injections using this protocol remains variable ( Table 1). The very likely explanation for this intriguing fact is the extreme variability of oocyte clutches: different egg batches do indeed behave very differently, when subjected to the injection pressure. Some oocytes are rather elastic and tend to flatten at the bottom of the dish, while others are quite stiff and remain round upon injection. Furthermore, some oocyte batches tend to inflate their chorion membranes upon injection, while others simply lyse (data not shown). No obvious correlation could be established between oocyte behavior upon injection and embryonic development after fertilization. It is thus difficult to anticipate which category of oocytes is best suited for microinjection. However after fertilization, embryos undergoing normal development can be identified as early as cleavage stages: 2-cell to 4-cell stage embryos can be considered normal when the contact surface between blastomeres is small, while a compacted cleavage-stage embryo, where individual cells are difficult to discern, is definitely abnormal.

In our hands, about half of the oocytes do not survive the trauma caused by the injection and about half of the embryos that do survive the injection exhibit a specific fluorescent label. Thus, we estimate that with this injection protocol, between 50 and 120 embryos can be successfully injected on a given spawning day yielding between 15 and 60 labeled embryos.

The red fluorescence of the Phenol Red in 2-cell to 4-cell stage embryos very reliably marks embryos that have been properly injected, as 100% of embryos selected in this manner subsequently produce the fluorescent proteins encoded by the injected mRNA. Additionally, there is a very clear positive correlation between the intensity of Phenol Red fluorescence at the 2-cell to 4-cell stage and the time of onset of the fluorescence produced by the proteins encoded by the injected mRNA. This correlation thus allows the identification of those embryos that have received the highest quantity of mRNA during the injection process.

If the signal of the fluorescent protein encoded by the injected mRNA is not detected after selection of Phenol Red-positive embryos, we recommend to run the injection mix on an RNAse-free gel in order to check for mRNA degradation or trapping ( Figure 3). In lane 1, an intact mRNA band is detected. Injection of this mix led to strong fluorescent signal in the embryo. On the contrary, in lane 2 which corresponds to another experiment where Phenol Red was replaced with Dextran dye in the mix (see discussion for further details), the mRNA seems to have been trapped by the Dextran dye and injection of this mix never led to fluorescent signal in the embryo.

Using this microinjection technique and our constructs ( Tables 2 and 3, Supplementary File 1) (see discussion), we can thus reproducibly produce homogenous fluorescent labeling throughout the embryo with mCherry or eGFP in the nucleus and with eGFP at the membrane ( Figure 4). The imaging protocol used to generate Figure 4 will be described elsewhere (Faure et al., currently in preparation). Depending on the amount of mRNA injected, fluorescent protein expression typically becomes detectable between 16-cell ( Figure 4A) and 64-cell stages and stays detectable at least up to the late neurula stage (data not shown). Later stages have not been tested. Nuclear signal typically appears earlier than membrane signal, potentially because of the intrinsically diffuse nature of the membrane compared to the compact nature of the nucleus (data not shown). Although it has not been monitored systematically, embryos injected with both a nuclear and a membrane label appear to be less healthy than embryos injected exclusively with a nuclear eGFP label. Intriguingly, this effect seems to be independent of the total amount of mRNA injected as the total amount of injected mRNA is the same, whether one or two mRNA species are included to the mix (data not shown).

Figure 1: Construct details. Sketches of (A) the membrane-targeted eGFP (eGFP:CAAX box), (B) the nuclear-targeted eGFP (H2B:eGFP) and (C) the nuclear-targeted mCherry (H2B:mCherry) constructs are shown.

Figure 2: Shape of a representative injection needle. (A) The taper is about 2 cm in length and the needle curves at the tip. Scale bar = 2 mm. (B) Magnification of the boxed area in A. The position for clipping the needle (arrow) is in the curve of needle. The tip of the needle is indicated by the arrowhead. Scale bar = 500 µm. Please click here to view a larger version of this figure.

Figure 3: Interactions of mRNA and selected fluorescent dyes. (A) Result of the migration of a mix containing mRNA and Phenol Red in lane 1 and of mRNA and Texas Red dextran (at a final concentration of 3.3 mg/ml) in lane 2. (B) Result of the migration of a mix containing mRNA and Oregon Green dextran (at a final concentration of 3.3 mg/ml) in lane 3, of mRNA and FITC dextran (at a final concentration of 3.3 mg/ml) in lane 4 and of mRNA and Rhodamine dextran (at a final concentration of 3.3 mg/ml) in lane 5. Gel migration time in A and B was different. For the sake of clarity, the black rectangle in A masks a light reflection and the dashed lines delineate the migration lanes. Please click here to view a larger version of this figure.

Figure 4: 3D rendering (Amira software) of amphioxus embryos injected with mRNAs coding for fluorescent proteins. Images are extracted from 3D time-lapses acquired on an optimized Leica SP5 2-photon laser-scanning microscope. (A) 16-cell stage embryo expressing nuclear H2B:eGFP. (B) Gastrula stage embryo expressing nuclear H2B:mCherry and the membrane-targeted eGFP:CAAX box. (C) Gastrula stage embryo expressing nuclear H2B:mCherry and the membrane-targeted eGFP:CAAX box. The optical section shows internal cells with nuclear mCherry and membrane-associated eGFP signals. Scale bar = 50 µm.

Experiment number Injected construct mRNA Number of injected embryos Number of labeled embryos

H2B:mCherry + eGFP:CAAX box

H2B:mCherry + eGFP:CAAX box

H2B:mCherry + eGFP:CAAX box

Total 906 448

Success rate 49%

Table 1: Injection success rates. The injected constructs, the number of injected embryos that survived the injection and the number of labeled (or successfully injected) embryos are indicated, as is the overall percentage of successful injections.

Construct Position in the pCS2+ vector Original sequence Mutated sequence

pCS2+ eGFP:CAAX box

79..87

GGA TCC ACC

ACC GTC A A C

pCS2+ H2B:eGFP

82..90

TCC GAC ACG

ACC G TC A AC

pCS2+ H2B:mCherry

82..90

TCC GAC ACG

ACC G TC A AC

Table 2: Tentative optimization of Kozak sequences. Original and mutated Kozak sequences are indicated for each construct. The introduced mutations recover the Kozak sequence of the B. lanceolatum actin gene, which very closely resembles that of the inferred theoretical preferred Kozak sequence of amphioxus (A/CGA/C G/TTC A/GAC atg TG/CT).

pCS2+ eGFP:CAAX box

Original codon Mutated codon Position in the pCS2+ vector

(usage level)(usage level)

(0.08)

(0.43)

(0.09)

(0.27)

pCS2+ H2B:eGFP

Original codon Mutated codon Position in the pCS2+ vector

(usage level)(usage level)

(0.08)

(0.43)

(0.06)

(0.51)

pCS2+ H2B:mCherry

Original codon Mutated codon Position in the pCS2+ vector

(usage level)(usage level)

(0.08)

(0.43)

(0.06)

(0.51)

Table 3: Tentative optimization of codon usage. Original and mutated codons are indicated for each construct. While this has not been experimentally tested, the introduced mutations are meant to optimize mRNA translation in amphioxus.

Supplementary File 1: Construct sequences. The nucleotide sequences of (A) the membrane-targeted eGFP (eGFP:CAAX box), (B) the nuclear-targeted eGFP (H2B:eGFP) and (C) the nuclear-targeted mCherry (H2B:mCherry) constructs are given in the context of the pCS2+ vector backbone. The main domains encoded by each of the constructs are indicated in color: eGFP (green), mCherry (red), membrane localization signal from human HRAS (CAAX box) (cyan blue), zebrafish histone 2B exon (H2B) (yellow).

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Discussion

In this article, we present, for the first time, a detailed and reproducible protocol for the injection of B. lanceolatum oocytes, which, after B. floridae 13,14 and B. belcheri ¹⁵, is thus the third amphioxus species, for which such a technique has been described. Importantly, the protocol described here also includes the description of vector systems suited for the production of fluorescent proteins in B. lanceolatum from injected mRNA produced in vitro (described below). Together, these new tools allow in vivo imaging of the early development of amphioxus and the analysis of dynamic cell behaviors that underlie the earliest morphogenetic events in the embryo, such as cleavage and gastrulation.

In general, the microinjection technique has the benefit of allowing the delivery, into a target cell, of a pre-defined volume of a specific compound. With this being said, one major limitation of this technique is the limited number of cells that can be injected during a given experiment. For example, the protocol described here for B. lanceolatum allows the injection of 100 to 120 eggs per injection session, of which about half will be labeled ( Table 3). For B. floridae, the numbers are very similar with 500 or more eggs that can be injected per day, of which more than 50% will survive the injection 13,14. While the protocols for injecting B. lanceolatum and B. floridae eggs closely resemble each other, the B. belcheri technique is slightly different: the eggs are placed on poly-lysine-coated coverslips and injections are performed under an inverted microscope using a microinjector of a different brand. This approach apparently allows the injection of between 200 and 300 B. belcheri eggs per injection session with a survival rate, after hatching at the neurula stage, of 89.39% to 95.83% ¹⁵. Considering the differences in the protocols, it is difficult to know whether the differences in success rates are due to species-related or protocol-related differences. One would have to test the different species in parallel with the different protocols to answer this question. Nonetheless, to further increase the number of oocytes targeted for the delivery of exogenous compounds, other protocols will have to be developed for amphioxus. Candidate techniques include electroporation, bombardment with microparticles, lipofection, and transduction ⁴. Of note, electroporation has already been established as a standard technique for the introduction of exogenous material into fertilized ascidian tunicate eggs, another invertebrate chordate model used for studying the evolution of developmental mechanisms ⁸.

For successful microinjection and subsequent in vivo imaging of fluorescent proteins, suitable expression vectors are a mandatory prerequisite. Towards this end, three plasmids have been developed and experimentally validated ( Figure 1, Supplementary File 1): for membrane targeting, the eGFP gene was fused at its 3' end to the human HRAS CAAX box (resulting in a eGFP:CAAX box construct) ¹⁶ and, for nuclear targeting, both the eGFP and the mCherry genes were fused at their 5' ends to the zebrafish histone 2B (H2B) exon (resulting, respectively, in a H2B:eGFP and a H2B:mCherry construct) ¹⁷. Each one of these constructs has been cloned into the pCS2+ plasmid containing a SV40 late polyadenylation site and a SP6 promoter allowing in vitro capped mRNA synthesis 18,19. Furthermore, with the goal to optimize translation of the constructs in B. lanceolatum, both the Kozak sequences and the codons have been adapted using single nucleotide mutagenesis ( Tables 2 and 3, Supplementary File 1). Based on the approach by Nakagawa ²⁰, a small pool of B. lanceolatum genes was analyzed to identify a preferred Kozak sequence in this species. The closest known naturally-occurring sequence to this theoretical preferred sequence is that of the B. lanceolatum actin gene. The Kozak sequences of the three constructs were hence modified to match this actin gene sequence. Furthermore, the codon usage of B. lanceolatum was analyzed using the codon usage database ²¹ and codons in the constructs with a low usage probability in B. lanceolatum (<10%) were replaced, wherever possible, by equivalent codons with a higher usage probability.

The results of the injections show that the level of protein expression from these vectors is suitable for in vivo imaging analyses. Given that the expression output of the modified vectors has not been compared with that of unmodified constructs, we cannot conclude on the efficiency of the modifications that were introduced in the Kozak consensus and the coding sequences. It would certainly be interesting to test whether these modifications indeed have an impact on protein expression levels. Along these lines, a recent analysis based on microinjections into B. belcheri suggests that other, unmodified vectors can also be used for the synthesis of mRNA for fluorescent protein production in amphioxus ¹⁵.

Amphioxus contains endogenous green fluorescent proteins ²². The embryos therefore exhibit low levels of green as well as red fluorescence (our unpublished observations). However, these endogenous levels are negligible with respect to the fluorescence levels resulting from the injection of our mRNA constructs. Therefore, the endogenous fluorescence of amphioxus embryos does not disturb experimental observations using fluorescent binoculars or multi-laser scanning microscopes. In addition, the injection experiments reveal that the intensity of the exogenous fluorescence generated by mRNA injection depends on the actual amount of injected material, which is directly correlated with the final concentration of mRNA used in the injection mix. B. lanceolatum embryos tend to tolerate a concentration of up to 1.8 µg/µl mRNA, although, independent of the actual mRNA concentration, some mRNA combinations seem to be less tolerated by B. lanceolatum embryos than others. Thus, the combination of nuclear mCherry and membrane eGFP mRNAs appeared to be more toxic than the injection of nuclear eGFP alone.

Texas Red dextran has previously been used as a tracer for successful injections into amphioxus oocytes ¹³⁻¹⁵. Intriguingly, a fluorescent signal derived from the injected mRNA was never obtained after injection of solutions containing Texas Red dextran and this in both amphioxus oocytes (n = 4 experiments) and zebrafish embryos (n = 3 experiments). When an injection mix containing in vitro transcribed mRNA and Texas Red dextran is loaded on a RNase-free agarose gel ( Figure 3, lane 2), the band corresponding to the mRNA is absent. In contrast, in vitro transcribed mRNA is detectable on an RNase-free agarose gel in the presence of Phenol Red ( Figure 3, lane 1). Given that there is no evidence for mRNA degradation on the gel, these results suggest that Texas Red dextran tends to trap mRNA. When used in an injection solution, Texas Red dextran might thus prevent the translation of the injected mRNA. Following this observation, the interactions of other dyes (FITC dextran, Rhodamine dextran and Oregon Green dextran) with mRNA were tested, both on RNase-free agarose gels and by injection into B. lanceolatum or zebrafish ( Figure 3). The analyses indicate that FITC dextran does not trap mRNA on gel ( Figure 3, lane 4) and leads to fluorescent protein expression in zebrafish (n = 1 experiment), but not in embryos of B. lanceolatum (n=1 experiment) (data not shown). Rhodamine dextran does not trap mRNA on gel ( Figure 3, lane 5) and leads to fluorescent protein expression in zebrafish embryos (n = 1 experiment) (data not shown), but its activity could unfortunately not be tested in B. lanceolatum embryos. Finally, as the Oregon Green dextran migrates at the same level as the mRNA, mRNA trapping could not be assessed by agarose gel ( Figure 3, lane 3). This latter dye prevented fluorescent protein expression in amphioxus (n = 1 experiment), but not zebrafish embryos (n=1 experiment) (data not shown). In sum, these data suggest that some dextrans can efficiently inhibit the translation of injected mRNA. Given that dose-response experiments were not carried out, these data are qualitative. Intriguingly, this inhibitory effect seems to be dependent on the animal species (zebrafish versus amphioxus), which highlights the need for further studies to understand the mechanisms underlying this effect. Furthermore, these results call for preparatory analyses, if dextrans are to be used as color tracers for injections.

The development of the microinjection technique for a given model opens the door for gene-specific manipulations. In amphioxus, for example, the first description of microinjections (in B. floridae) was followed by the successful knockdown of a gene, hox1, using morpholino oligonucleotide injections 23,24. The number of B. lanceolatum embryos that can successfully be injected every day using the protocol presented here is certainly compatible with this type of study. Furthermore, with these methods and the novel constructs at hand, one can now also design and perform overexpression and knockdown studies by injection of mRNAs of interest (native or dominant-negative forms) or use other strategies for disrupting gene expression (for example microRNA- or shRNA-based strategies). So far, amphioxus injections can only be performed at the oocyte stage, simply because this is the only stage that can efficiently be immobilized. After injection at the oocyte stage, the molecule of interest is ubiquitously expressed in the embryo. Thus, if gene expression needs to be altered or monitored only in a subset of cells, DNA constructs need to be injected, as these are expressed mosaically in the developing amphioxus embryo 15,25-27. DNA construct injections can also be used to test the activity of regulatory regions during amphioxus development in transient transgenic assays, with the aforementioned drawback of mosaic expression 15,25-27.

Ultimately, the amphioxus microinjection technique also opens the door for stable transgenesis, including the targeted knock-out or knock-in of specific genetic loci. Systems for stable insertion of exogenous DNA already exist, for example with the fish transposon-based Tol2 system that seems to function in both insects and amniote vertebrates ²⁸. Furthermore, approaches based on modified zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) or RNA-guided genome editing tools (CRISPR/Cas system) could be used to generate point mutations and knock-in modifications in specific regions of the genome ²⁹. These efforts will require the year-round breeding of amphioxus in captivity, which has already been set up for B. belcheri 11,30 and B. japonicum 30,31 and is currently being developed for A. lucayanum, B. floridae and the amphioxus species featured here, B. lanceolatum ³².

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Disclosures

The authors have nothing to disclose.

Acknowledgments

The authors would like to acknowledge the support by the "Animalerie Centrale de Gif-sur-Yvette" for animal husbandry. This work was supported by funds from ANR (ANR-09-BLAN-0262-02 and ANR-11-JSV2-002-01) to Michael Schubert, by the European Union FP6 grant "Embryomics" and by the ANR grant "ANR-10-BLAN-121801 Dev-Process" to Jean-François Nicolas and Nadine Peyriéras. João Emanuel Carvalho is financed by a FCT doctoral fellowship (SFRH/BD/86878/2012).

Requests for the vectors described here can be addressed directly to the authors.

Materials

Name Company Catalog Number Comments

Consumables

Reagents

H ₂ O, DNase/RNase-free

Gibco

10977-035

NaHCO ₃

Sigma

S6014

Equipment

DOWNLOAD MATERIALS LIST

References

Stepicheva, N. A., Song, J. L. High throughput microinjections of sea urchin zygotes. J. Vis. Exp. e50841 (2014).

Usage Statistics

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in vivo imaging

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Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes | Protocol

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We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

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Mais bcp de dvpt: le YT channel du MBL

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biology ecology "marine biology" "woods hole" "university of chicago" research education science

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery and improving the human condition through research and education in biology, biom...

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First trigger for orientation of first division
Paper by Sardet team  (old)

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Paper Sardet team 2005

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Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo. - Semantic Scholar

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The mature ascidian oocyte is a large cell containing cytoplasmic and cortical domains polarized along a primary animal-vegetal (a-v) axis. The oocyte cortex is characterized by a gradient distribution of a submembrane monolayer of cortical rough endoplasmic reticulum (cER) and associated maternal postplasmic/PEM mRNAs (cER-mRNA domain).

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different lab

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Cell polarity emerges at first cleavage in sea urchin embryos

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In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We no...

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This is where the first embryonic cell begins! Fertilization is the fusion of haploid gametes, egg (oocyte) and sperm (spermatozoa), to form the diploid zygote. Note though there can be subtle differences in the fertilization process which occurs naturally within the body or through reproductive technologies outside the body, the overall product in both cases is a diplod zygote.

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drosophila (check explanations, in other movie). In droso, the

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Fruitfly Drosophila complete embryonic development at high resolution

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Drosophila

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Fruitfly Drosophila complete embryonic development at high resolution

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Drosophila

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Early Drosophila embryo development - Eric Wieschaus (Princeton/HHMI)

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droso

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Maximum-intensity projections of a DSLM time-lapse recording of a membrane- and nuclei-labeled zebrafish embryo. Membranes were imaged using structured illumination (SI-25, top row), nuclei using standard light sheet illumination (bottom left). The top-most cell layer (the enveloping layer, EVL) was removed computationally in the ras-eGFP channel and is shown on the right, separate from the deeper cell layers (left).

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extraction of  nuclei for quantification by image segmentation

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Dynamic 3D visualization of nuclei during early embryogenesis

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This section contains a collection of audio-visual resources that can be browsed to learn more about development and biology. This page is part of the redeveloped resources section on the Node, you can find out more about the update here.

Links to some interesting videos teaching about biology.

Posters can be a great tool to convey developmental biology, both in terms of practical information and concepts.

Audio resources including podcasts to further interest and understanding in biology.

This section contains links to some examples of the pairing of art and science. Development is such a beautiful part of biology, and turning experimental images into art is an easy way to tell a story to the public.

Videos

Seminars on development and stem cells from .

Stated Clearly videos explaining biological concepts to lay audiences.

Suggested YouYube channels from the popular IFLS.

The Company of Biologists YouTube channel, including a Development playlist.

Acapella Science explaining science in pop songs.

Minute lectures on a range of biology topics.

YouTube video on the importance of funding basic science.

Movie about Antonie van Leeuwenhoek and bacteria.

Video to illustrate the problem of image resolution.

Learn about blood stem cell development.

Video explaining the cytoskeleton.

Videos explaining the process of transcription.

SDB CoRE: database of reference images, movies and diagrams.

EuroStemCell highlights animated videos from the Institut Pasteur explaining Muscle and Stem Cells

Nicole Theodosiou shares her experience of teaching evo-devo to, and promoting creativity in, university students

The UNSW Embryology page has collated hundreds of embryology movies, including human and model organisms as well as computer models

If you want to introduce students (or anyone for that matter) to the beauty of developmental biology, this movie of newt development by Jan van IJken is a good place to start.

Our own YouTube channel includes playlists devoted to animal and development, specific model organisms, history of the field, talks and interviews, education and science art, and advice for scientists. Get in touch if you see anything we've missed!

Posters

Model organism staging and phenotyping. Developmental Dynamics has put together a series of staging posters for some of the main model organisms of developmental biology (plus humans). You can find them .

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How to fish and how to fly. The journal genesis also has two wonderful posters on zebrafish developmental stages and phenotypic markers in Drosophila (which adorns the walls of many a fly lab). You can find them .

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Drosophila phenotyping. Flybase and Thomas Kaufman's lab have put together a comprehensive, high resolution collections of commonly used fly mutants. Check them out .

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Development's 'Development at a glance' series. Since 2012 Development has run a series of posters and accompanying articles visually exploring some of the topical aspects of the field. You can find the whole series Disease Models and Mechanisms' here. (Also check out Journal of Cell Science's Cell Science at a Glance andAt a Glance series, which both contain lots of relevance to developmental biologists.)

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Nature Reviews Molecular Cell Biology has since 2002 produced posters on various topics relevant to developmental biology. Check them out .

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Abcam's poster library mainly focuses on pathways but also some processes and concepts (some posters require registration to view). Check it out .

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Microscopy aides. Zeiss has a useful collection of microscopy posters - check them out .

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Levin lab posters. Michael Levin's lab at Tufts has had two poster made for them by the graphic designer Ben Oldroyd (with support of the Templeton World Charity Foundation) covering interesting concepts in developmental biology.

Audio

CellPress podcast webpage.

A wide range of science podcasts for many levels.

Plantae's The Taproot is a podcast devoted to the stories behind papers in plant science

Genetics Unzipped is a podcast from The Genetics Society hosted by Kat Arney.

En Fase Experimental is the science podcast of the Society of Spanish Researchers in the United Kingdom, covering current advances in science but also the role and challenges that STEM research faces in the current world, in Spanish.

SciArt
Have we missed a useful link? Get in touch!
Resources section map

# data[].attributes.attachment_link.description
This section contains a collection of audio-visual resources that can be browsed to learn more about development and biology. This page is part of the redeveloped resources section on the Node, you can find out more about the update here . Links to some interesting videos teaching about biology.

# data[].attributes.attachment_link.description
Development is a dynamic process with structures changing (shape, name and relationships) over time and the best way to show this is with dynamic images, such as movies. This current page introduces the various movies and animations in UNSW Embryology that illustrate many different aspects of development.

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The Company of Biologists is a not for profit publishing organisation dedicated to supporting and inspiring the biological community. The Company publishes five specialist peer-reviewed journals: Development, Journal of Cell Science, Journal of Experimental Biology, Disease Models & Mechanisms and Biology Open.

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cell lineage study

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Allen Discovery Center for Lineage Tracing

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During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula.

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3D visualisation early embryonic development

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Open science resources for the discovery and analysis of Tara Oceans data - Semantic Scholar

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The Tara Oceans expedition (2009-2013) sampled contrasting ecosystems of the world oceans, collecting environmental data and plankton, from viruses to metazoans, for later analysis using modern sequencing and state-of-the-art imaging technologies. It surveyed 210 ecosystems in 20 biogeographic provinces, collecting over 35,000 samples of seawater and plankton.

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rien à voir

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Tiny claws and single-celled algae are among the top images in the 2012 Olympus BioScapes Microscopic Life Photo Contest.

Ralph Grimm, a teacher from Australia, filmed the tiny animals' madly beating cilia, or small hairs, to win $5,000 worth of Olympus equipment. The rotifers use their cilia to create water currents that draw in food.

A psychedelic image captures the reproductive structures, or spores, of a common U.S. East Coast fern, Polypodium virginianum. This photo took third place, adding to the record number of prizes won by photographer Igor Siwanowicz of Howard Hughes Medical Institute. His four other entries are honourable mentions, for a total of five awards. (Related: "Ferns of Rocky Mountain National Park." )

Despite its fearsome appearance, this wicked-looking claw belongs to a group of tiny crustaceans called amphipods. Female members of the genus Phronima attack gelatinous ocean creatures called salps, eating their flesh and leaving a barrel-shaped outer structure. The female amphipods then crawl inside and lay their eggs.

This single-celled green diatom won Rogelio Moreno Gill of Panama fifth place in the BioScapes Imaging Competition. Although many diatoms are found in the oceans, they inhabit freshwater too. Specimens for this composite image came from a lake.

Edwin Lee of Texas took eighth place with an image of the male reproductive organs - stamens, anthers, and filaments - of an annual plant called ( henbit deadnettle Lamium amplexicaule). This member of the mint family is native to Europe and the Mediterranean and is considered an invasive species in the U.S.

This composite image of a Delphinium seed won Sahar Khodaverdi of Iran ninth place in the BioScapes Imaging Competition. Otherwise known as , these plants are highly toxic and are responsible for many livestock losses in the U.S.

The scales covering the wings of this prola beauty butterfly ( Panacea prola) reflect the light, producing this dazzling display of colour. Charles Krebs of Washington State won tenth place in the BioScapes Imaging Competition with this image. (See more pictures of butterflies. )

Top image: Rotifers feeding. Screen capture from video.

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10 JAW-DROPPING IMAGES CAPTURING THE BEAUTY OF LIFE THROUGH A MICROSCOPE

# data[].attributes.attachment_link.description
Tiny claws and single-celled algae are among the top images in the 2012 Olympus BioScapes Microscopic Life Photo Contest. Ralph Grimm, a teacher from Australia, filmed the tiny animals' madly beating cilia, or small hairs, to win $5,000 worth of Olympus equipment. The rotifers use their cilia to create water currents that draw in food.

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2.1 Introduction

The acrosome is a single exocytotic vesicle overlying the nucleus of most animal sperm. While the sperm of some taxa (e.g., teleost fish) do not possess an acrosome, it is generally thought that mammalian sperm acrosomes may perform two principal exocytosis-dependent functions: (1) to serve as a site for the sequestration and release of proteins required for binding to and penetration of the zona pellucida and (2) to enable the sperm to fuse with the oolemma. For the past several decades, research has been largely guided by the concept that mammalian sperm acrosomes must be intact to bind to the zona pellucida prior to undergoing exocytosis, enabling the sperm to penetrate this vestment and fertilize the egg ( Wassarman et al. 2004). Although this widely accepted dogma has been challenged on many levels, recent experiments utilizing modern, highly sophisticated imaging techniques have enabled investigators to track the acrosomal status of individual fertilizing spermatozoa. Using genetically engineered mouse strains that express green fluorescent protein in their acrosomes, these experiments have shown that almost all fertilizing mouse sperm have undergone acrosomal exocytosis prior to the time they penetrate the zona pellucida ( Jin et al. 2011).

These contemporary experiments raise new questions of how the acrosome forms during spermatogenesis and functions in the fertilization process. Specific issues that need to be addressed include (1) the contribution of acrosomal biogenesis to the fertility of individual sperm, (2) the timing of acrosomal exocytosis in vivo, (3) the role of acrosomal compartmentalization (including structurally distinct domains within the vesicle and biochemical segregation of overlapping soluble materials and insoluble acrosomal matrix compartments), and (4) the functions of specific proteins in fertilization. While recent results challenge or even contradict experiments that were based upon previous reductionist paradigms, many of the older studies were carefully designed and yielded a solid body of evidence that should be reevaluated from a more holistic standpoint to see whether a reinterpretation sheds new light on the question of the physiological role of the acrosome in fertilization.

In a previous review, we focused on the structure and function of the sperm acrosomal matrix in fertilization, where we espoused three principal points: (1) the acrosome is not a "bag of enzymes," simply comprised of soluble substances that are rapidly released when the outer acrosomal membrane fuses with the plasma membrane, (2) acrosomal exocytosis is not an "all-or-none" event, where the acrosome is either "intact" or "reacted" but instead incorporates intermediate steps involving the progressive loss of acrosomal matrix material, and (3) the acrosomal matrix is a molecular scaffold assembly that is dismantled by a self-regulated mechanism driven, in part, by proteolysis ( Buffone et al. 2008). We will not recapitulate these points in this review, but we will discuss them in light of the many new findings from studies using genetically modified mice.

It is abundantly clear that fertilization is a complicated process that requires models to extend from a cluster of hundreds or thousands of sperm to the single-cell level. Given the heterogeneous nature in the structure and physiology in a population of sperm, it is not surprising that aspects of acrosomal structure, including the matrix, could depend on events during spermatogenesis and could impact function at several steps in the female reproductive tract or the cumulus-oocyte complex. Furthermore, the heterogeneity in the timing and location of acrosomal exocytosis in individual cells present within a population of spermatozoa is not yet fully understood. Thus, it is a challenge to sort out real population cell behaviors (e.g., disruption of cumulus cells) from irrelevant or deleterious cell behaviors (spontaneous exocytosis, cell demise). Perhaps the most important question is, what is the location and timing of these events in the one sperm that successfully fertilizes the egg? We must also recognize that there might be multiple means (pathways) to an end (fertilization). In light of the contemporary imaging studies indicating that most mouse sperm that fertilize mouse oocytes enclosed by cumulus masses have lost the green fluorescent protein marker of intact acrosomes ( Jin et al. 2011; Hirohashi et al. 2011), we recognize that work in this area is still very active so it would be premature to attempt to develop a consensus model. Nevertheless, it is useful at this time to consider results utilizing another modern approach: gene knockouts in mice. Toward this goal, recently developed, genetically modified mouse models provide valuable new insights into the molecular mechanisms of mammalian fertilization involving the sperm acrosome.

2.2 Gene Knockout Studies in Mice: What They Do and Do Not Tell Us About Acrosomal Matrix Function

The knockout of a specific reproductive gene in the mouse is usually engineered with an explicit outcome (hypothesis) in mind: to demonstrate the importance of a particular protein by interfering with fertility. However, the resulting phenotypes are often different and/or are more complicated than expected. In many cases, knockouts of individual genes encoding acrosomal matrix proteins have not caused sterility; this outcome can be explained away with the understanding that sperm function and fertilization are critical for survival of a species, so it makes sense to have built-in redundancy. Nevertheless, closer inspection of some of these "fertile" knockouts has revealed important insights into the functions of acrosomal matrix and other fertilization proteins. Also, knockouts of multiple proteins can address questions of complementary proteins in a complex system. As the body of literature involving genetically engineered mice has grown, there have been many surprising new observations that force us to think more broadly and to ask different questions than had previously been considered. For example, we now know not to assume that acrosomal exocytosis will only occur on the ZP, and we also understand proteins thought to be involved in sperm-egg interactions can have other functions such as acrosome formation, transiting the uterus and oviduct, etc. Thus, we feel the literature is rich with data from gene knockouts, and this review is an attempt to bring both widely known and understated results to the reader's attention with hope that new avenues for research will come to light ().

Table 2.1

Mouse gene name

Common protein names

Proposed function

Observed KO phenotype

References

Acrosome formation/globozoospermia

Zona interacting proteins

Oocyte cumulus complex and zona pellucida transit

Proprotein convertase subtilisin 4/Kexin type 4

Processing/activation of sperm/acrosomal proteins, capacitation, acrosomal exocytosis, ZP binding

Infertile males; abnormal acrosomal morphology

Mbikay et al. (1997), Gyamera-Acheampong et al. (2006) and Tardif et al. (2012)

Inner acrosomal membrane proteins

Equatorial segment proteins

2.3 Acrosome Formation/Globozoospermia

The function of the sperm acrosome is dependent upon its structure. The sperm of each species has a characteristic configuration with defined acrosomal shapes that are determined, in part, by the underlying nuclear morphologies. However, each acrosome shares the common features of an inner acrosomal membrane (IAM) that is laminated to the nuclear foundation, a fusogenic outer acrosomal membrane (OAM) that is closely apposed to the plasma membrane over the acrosome, and an equatorial segment (ES), which forms the posterior acrosomal margin where the IAM and OAM meet. Acrosomal formation begins during late meiosis when small, proacrosomal vesicles form in pachytene spermatocytes ( Bloom et al. 1975; Holstein and Roosen-Runge 1981; Anakwe and Gerton 1990). After the meiotic divisions, the resulting haploid spermatids inherit the proacrosomal vesicles and continue acrosome biogenesis in earnest. Proacrosomal vesicles eventually coalesce into a single acrosomal vesicle that then attaches to and begins to spread over the future anterior segment of the nucleus. The points of contact between the developing acrosome and spermatid nucleus define the prospective IAM. The acrosome and the nucleus continue developing throughout spermiogenesis until they coordinately achieve structures similar to those found in ejaculated sperm. Final steps of metamorphosis occur during epididymal transit.

In recent years, the molecular mechanisms supporting the transmogrification of a round spermatid acrosome to the form seen in mature testicular spermatozoa have been elucidated by many studies utilizing mutations or gene knockouts in the mouse. One of the earliest examples of the effect of acrosomal dysplasia is the blind-sterile mouse, whose round spermatids exhibit disorganized proacrosomal granules with no further development beyond step 3 of spermiogenesis ( Sotomayor and Handel 1986). This phenotype is caused by mutations in the Tbc1d20 gene, which encodes a GTPase-activating protein specific for Rab1 and Rab2 small GTPase families ( Liegel et al. 2013). Another GTPase-activating protein, AGFG1 (also known as Hrb), specific in this case for ARF proteins, shows a similar phenotype ( Kang-Decker et al. 2001; Kierszenbaum et al. 2004). Vesicle trafficking is key to formation of the acrosome since mutations affecting PICK1, the Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), the casein kinase II α′ catalytic subunit (CSNK2A2), and islet cell autoantigen 1-like protein (ICA1L) cause sterility or subfertility in males ( Xu et al. 1999; Yao et al. 2002; Xiao et al. 2009; He et al. 2015). Mutations affecting SEC23IP, a phospholipase A1-like protein that interacts with Sec23, also cause male subfertility by interfering with acrosome biogenesis and leading to round-headed spermatozoa ( Arimitsu et al. 2011). Spermatids of infertile male mice lacking GalNAc transferase 3 (GALNT3) produce proacrosomal vesicles of various sizes, which attach to the nuclear envelope but do not coalesce to form a single acrosomal vesicle, apparently causing oligoasthenoteratozoospermia through a severe reduction of mucin-type O-glycans, impaired acrosome formation, and increased apoptosis ( Miyazaki et al. 2012). Acrosomal dysplasia does occur in cases where certain acrosomal matrix components such as zona pellucida-binding protein 1 (ZPBP) or 2 (ZPBP2) are missing ( Lin et al. 2007).

Abnormalities in nuclear envelope structure can also interfere with the ability of the acrosome to form properly. Disruption of the mouse homologue ( Gmcl1) of the Drosophila melanogaster germ cell-less gene causes insufficient chromatin condensation and abnormal acrosome structures in mutant sperm ( Kimura et al. 2003). When the inner nuclear membrane protein DPY19L2 is absent, the nuclear dense lamina as well as the junction between the acroplaxome and the nuclear envelope are destabilized. As a result, the linking of the acrosome and the manchette to the nucleus is blocked; this disrupts vesicular trafficking, causes sperm nuclear shaping to fail, and leads to the elimination of the unbound acrosomal vesicle ( Pierre et al. 2012). In elongating spermatids, abnormalities of anterior head shaping involving CSNK2A2 or the sperm acrosome-associated 1 protein (SPACA1) also lead to globozoospermia ( Xu et al. 1999; Fujihara et al. 2012). Spermatids with mutant PAFAH1B1 protein (the regulatory subunit of platelet-activating factor acetylhydrolase 1b) are sterile because the spermatids fail to form acrosomes properly and exhibit nuclei distorted in size and shape ( Nayernia et al. 2003).

Final maturation of the sperm acrosome occurs within the epididymis. The classic case is the morphological alteration in the acrosome of guinea pig sperm where the head changes drastically from a planar, tennis racket-shaped structure to a form reminiscent of a cupped hand or xistera, the handheld basket used in jai alai, as the sperm transit from the testis to the cauda epididymis ( Fawcett and Hollenberg 1963). Another example is seen in the post-testicular development of spermatozoa of the tammar wallaby where the immature, uncondensed acrosome has a distinctive "thumbtack" appearance that is remodeled into a streamlined, condensed structure on the sperm head ( Setiadi et al. 1997). Phospholipase A2, group III (PLA2G3), which is secreted in the proximal (caput) region of the mouse epididymis, is reported to have a role in epididymal maturation of sperm ( Sato et al. 2010); however, the Pla2g3 gene is also expressed in the testis, so it is difficult to attribute the subfertility of males to the lack of the Pla2g3 protein in the epididymis rather than the testis. The molecular mechanisms for these structural alterations are not fully understood but could be due, in part, to changes in protein disulfide cross-links, oligosaccharide modifications, and/or repackaging of the acrosomal components ( Anakwe et al. 1991; Setiadi et al. 1997; Fléchon 2015).

2.4 Zona Interacting Proteins

For many years, the prevailing notion was that sperm bound to the zona pellucida of unfertilized eggs through cell surface receptors or binding proteins present on the exterior of acrosome-intact sperm. Biochemical studies in the mouse led to the identification of ZP3R (originally called "sp56") as the putative ZP3 receptor protein ( Bleil and Wassarman 1990; Cheng et al. 1994; Bookbinder et al. 1995). Subsequent experiments established that ZP3R and its guinea pig homologue are actually components of the acrosomal matrix ( Foster et al. 1997; Kim et al. 2001). ZP3R monomers are assembled into a multimeric complex in the particulate acrosomal matrix. During the course of spontaneous acrosomal exocytosis and coincident with release of ZP3R from the acrosome under capacitating conditions, the monomer is proteolytically processed from 67,000 to 43,000 ( Buffone et al. 2009). The processing of ZP3R is dramatically reduced when sperm are incubated under non-capacitating conditions. The cleavage probably takes place in complement control protein domain (CCP) 6 or the bridge region between CCP6 and CCP7, which is not present in the guinea pig orthologue AM67. This cleavage has functional consequences since the cleaved form of ZP3R does not bind to unfertilized eggs. However, although ZP3R binds the zona pellucida, the elimination of ZP3R in the mouse by homologous recombination did not affect fertility in the nullizygous mice ( Muro et al. 2012).

Other candidates proposed as proteins responsible for zona pellucida binding also turned out to be components of the acrosomal matrix. Zona-binding proteins include the homologues known as sp38 [boar ( Mori et al. 1993)], IAM38 [bull ( Yu et al. 2006)], ZPBP and ZPBP2 [mouse ( Lin et al. 2007)]. In the pig, this protein competes with proacrosin for binding to the zona pellucida ( Mori et al. 1993). As discussed previously, elimination of the Zpbp and/or Zpbp2 genes by homologous recombination results in infertility or subfertility in male mice due to multiple effects on acrosome biogenesis and other spermatogenic defects leading to dysfunctional spermatozoa ( Lin et al. 2007). The serine zymogen proacrosin is another zona-binding protein ( Howes et al. 2001; Furlong et al. 2005), but, as described below, elimination of the Acr gene by homologous recombination does not cause infertility in mice ( Baba et al. 1994; Adham et al. 1997). Zonadhesin (ZAN) was originally identified biochemically as a binding protein of the porcine zona pellucida ( Hardy and Garbers 1995) and then demonstrated to be associated with the acrosomal matrix and membranes ( Bi et al. 2003). Homologues in other species were identified and a gene knockout of Zan was performed in the mouse, which does not affect fertility but, instead, enables the mouse sperm to bind more effectively to pig, cow, and rabbit ZP without enhancing attachment to the mouse ZP, essentially eliminating the filter for the species-specific interactions between the mutant mouse sperm and oocytes of other animals ( Tardif et al. 2010). From the studies performed to date, it is clear that there are several zona pellucida-binding proteins present within the acrosomal matrix of sperm. Some of these are involved in essential steps of acrosome biogenesis such as ZPBP and ZPBP2. However, the elimination of other zona-binding proteins by homologous recombination does not affect fertility per se but may yield a subtler phenotype such as a loss of species-specific sperm-zona binding or the slower dissipation of the acrosomal matrix when exocytosis is initiated, leading to a delayed rate of fertilization. These traits appear to be redundant, yet they likely impart a competitive advantage to sperm from wild-type mice when one or more males mate with the same female.

2.5 Oocyte Cumulus Complex and Zona Pellucida Transit: Proteases, Regulatory Proteins, and Binding Proteins

Mammalian oocytes are surrounded by two formidable barriers to spermatozoa: the oocyte cumulus complex (OCC) that consists of multiple layers of cumulus cells with an extracellular matrix rich in hyaluronic acid and the ZP that is comprised of three or four glycoproteins ( Litscher and Wassarman 2014). The sperm surface and acrosome contain several proteases that are purported to have a role in at least some limited digestion of the OCC extracellular matrix and ZP, but it remains unclear whether these proteases are essential for sperm to transit these barriers or whether mechanical forces produced by the sperm tail are sufficient ( Bedford 1998; Litscher and Wassarman 2014). In addition, the acrosomal matrix contains numerous proteins that can bind to the ZP and presumably enable a spermatozoon to burrow into the ZP by the combined mechanical forces of the tail and limited enzymatic digestion by proteases. Gene knockouts of individual and multiple proteases have provided some important and unforeseen insights into this process. Indeed, the pattern of surprising knockout results was set by perhaps the first knockout in this field-the proacrosin (ACR) gene. For many years, proacrosin was purported to be the main sperm protease for ZP penetration, but, as discussed previously, gene knockout experiments showed that proacrosin was not essential for zona penetration as mice were fertile and proacrosin-null sperm were able to transit the ZP ( Baba et al. 1994). Further study showed that proacrosin-null sperm dispersed acrosomal contents more slowly than wild-type sperm, indicating that the role of acrosin was to disperse the acrosomal matrix ( Yamagata et al. 1998). This also inspired research to identify novel acrosomal proteases involved in OCC and ZP transit.

PRSS21 (TESP5, testisin) is a candidate serine protease to complement acrosin; it is a GPI-linked protein on the surface of the sperm head, cytoplasmic droplet, and midpiece of the tail ( Honda et al. 2002). PRSS21 knockout mice were fertile in long-term mating trials over several weeks, but in vitro analysis showed that the PRSS21-null sperm had decreased motility, abnormal morphology, and poor performance in fertilization assays ( Netzel-Arnett et al. 2009).

Double knockout ACR/PRSS21 male mice were able to produce offspring in vivo but with significantly reduced litter size ( Kawano et al. 2010). In vitro, ACR/PRSS21-null sperm failed to fertilize both cumulus-free and cumulus-intact oocytes, which suggests a role for these proteases in both cumulus penetration and ZP transit. Sperm penetration through the OCC was slower for ACR/PRSS21 sperm than wild-type sperm, and OCCs incubated with extracts of these sperm showed slower dispersal times than wild-type sperm extracts. Analysis of in vitro fertilization experiments showed numerous effects: the acrosomal contents of ACR/PRSS21 knockout sperm failed to disperse, the number of sperm bound to the ZP and the percentage of oocytes with fused sperm were very low, and the ratio of sperm bound to the ZP, percentage of ZP penetrated by sperm, number of sperm in the perivitelline space, and percent fertilized oocytes were zero or negligible. Scanning electron microscopy showed that oocytes incubated with ACR/PRSS21-null sperm did not have any sperm tracks, the putative paths of sperm through the ZP caused by limited enzymatic digestion. It is notable that the ACR/PRSS21 sperm appear to remain acrosome intact in these images, or perhaps they are acrosome reacted but retain a significant portion of undispersed acrosomal matrix material. The authors hypothesize that this may be due to the lack of PRSS21 activity rather than dispersal of the acrosomal matrix since ACR is known to activate the PRSS21 protease. They propose that ACR-null mice may have compensatory PRSS21 enzymatic activity for penetrating the ZP while PRSS21-null mice may have compensatory acrosomal matrix dispersal activity. ACR/PRSS21 double knockout mice lack both mechanisms and are subfertile due to the failure to fertilize all ovulated oocytes ( Kawano et al. 2010). The authors propose that sperm lacking these two proteases are able to fertilize oocytes due to a compensatory mechanism in the uterine fluid; it is also tempting to speculate that at least some sperm lacking these proteases are able to fertilize oocytes due to simple mechanical forces from sperm motility. The authors suggest a role in cumulus dispersal and penetration, which is interesting since sperm lacking known hyaluronidases SPAM1 ( Baba et al. 2002) and HYAL5 ( Kimura et al. 2009) were able to fertilize oocytes. Together with double knockout experiments of SPAM1 and ACR or SPAM1 and PRSS21 ( Zhou et al. 2012), these results suggest that ACR and PRSS21 may work in concert with hyaluronidases for cumulus penetration. These results are also consistent with the notion that acrosomal exocytosis occurs before or during interaction with the cumulus ( Jin et al. 2011).

A likely substrate of acrosin in the guinea pig acrosomal matrix is AM50, the orthologue of NPTX2 (neuronal pentraxin 2 or Narp) in mice. AM50, like other pentraxins, forms large disulfide-bonded pentameric or decameric complexes and is a major structural component of the acrosomal matrix in guinea pigs ( Noland et al. 1994; Reid and Blobel 1994). When acrosin is activated during acrosomal exocytosis in guinea pig sperm or in isolated acrosomal matrices, AM50 is processed at acrosin-specific sites coincident with acrosomal matrix disassembly ( Westbrook-Case et al. 1994; Kim et al. 2011). NPTX2 is present in the mouse and knockout of this protein yields fertile males ( Bjartmar et al. 2006), although analysis of sperm performance has not been done. Curiously, pentraxin 3 (PTX3) is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix; in vivo, female mice have been reported to be infertile or subfertile due to severe abnormalities of the cumulus oophorus, although oocytes can be successfully fertilized in vitro ( Varani et al. 2002; Salustri et al. 2004).

Protein C Inhibitor (PCI), a widely expressed serine protease inhibitor in seminal plasma, is present in the acrosome, has been shown to inhibit human sperm acrosin activity, and is detected on the surface of damaged or abnormal human sperm where it is assumed to prevent inappropriate acrosin activity ( Moore et al. 1993; Hermans et al. 1994; Zheng et al. 1994; Uhrin et al. 2007). PCI has also been linked to fertility in humans as infertile patients had inactive PCI ( He et al. 1999). PCI-null female mice are fertile but males are infertile and the primary defect appears to be with the Sertoli cell-spermatogenic cell junctions in the testis since spermatogenic cells were prematurely released into the seminiferous tubule lumen and immature and abnormal germ cells were found in the epididymis ( Uhrin et al. 2000; Uhrin et al. 2007). Further analysis of PCI-null epididymal sperm showed 95 % abnormal sperm morphology (many with separated heads and tails and abnormally shaped heads), significantly reduced motility, and failure to bind either cumulus-intact or cumulus-free eggs ( Uhrin et al. 2000; Uhrin et al. 2007). Whether the observed lack of cumulus and ZP binding and penetration in absence of PCI is due to problems of unregulated acrosin activity is a question to be investigated, but it is interesting to note that ACR/PRSS21-null mice also had this phenotype. Perhaps unregulated acrosin produces premature spontaneous acrosomal exocytosis and proteolysis of the acrosomal matrix such that sperm-ZP binding sites of the acrosomal matrix and/or ZP are prematurely processed and thus nonfunctional.

The pro-protein convertase PCSK4 is reported to be localized in the developing acrosome during spermatogenesis and, in mature sperm, it is present on the plasma membrane over the acrosome ( Gyamera-Acheampong et al. 2006); whether there are two PCSK4 pools (acrosomal and cell surface) or it migrates from the acrosome to the sperm surface is unclear. PCSK4-null mice are infertile ( Mbikay et al. 1997; Gyamera-Acheampong et al. 2006) and have abnormal acrosomal shapes ( Tardif et al. 2012) that are likely caused by reduced ACRBP processing during spermiogenesis that leads to improper packaging of proacrosin and perhaps other acrosomal matrix proteins into the acrosome ( Kanemori et al. 2013). PCSK4-null mature sperm had accelerated tyrosine phosphorylation during capacitation, improper processing of the egg-binding protein ADAM2, increased spontaneous acrosomal exocytosis, and reduced ZP binding. Thus, PCSK4 has multiple functions in developing and mature sperm and identification of substrate proteins will help elucidate the mechanisms through which this protein functions.

2.6 Inner Acrosomal Membrane Proteins

As acrosomal exocytosis proceeds and acrosomal matrix proteins are released, some components of the IAM remain associated with the sperm and are in position to interact with egg vestments. The term "secondary binding" has been used to describe the interactions of these proteins with the ZP, particularly ZP2, but we envision these interactions to be less well defined and to involve multiple binding and release events. In this model, sperm would adhere to the ZP via multiple proteins that are then released as a result of limited proteolysis to produce repeated binding and release at the molecular level; the continual force of the tail would drive the head progressively further into the ZP. Numerous proteins have the ZP-binding and/or enzymatic properties that are consistent with this model.

The cellular differentiation marker CD46 is ubiquitously expressed on the surface of most mammalian cells and is involved in complement protection in many species, including humans, but not in rats, mice, and guinea pigs. In mice, it appears to be expressed solely in the testis, and, in sperm, CD46 is not on the PM as would be expected, but is located inside the acrosome on the IAM. It was thought that this might be to compensate for loss of sperm surface CD proteins that protect the cell from attack by complement, but sperm from CD46 knockout mice are not more susceptible to complement than control sperm, so the function of CD46 is not likely to be complement protection. Interestingly, CD46 knockout mouse sperm have enhanced fertility; the sperm show accelerated spontaneous acrosomal exocytosis and have an elevated litter size in one mouse strain (DBA/2N), and CD46-null sperm fertilized superovulated oocytes at a significantly higher rate than controls ( Inoue et al. 2003). Thus, CD46 appears to be important in stabilizing the acrosomal membranes. How it does this from the inner acrosomal membrane is a mystery, but it is possible that, during acrosome formation in spermatogenesis, CD46 may interact with other proteins destined for the periacrosomal membranes. Consequently, CD46 might direct or influence sorting of proteins that stabilize these membranes so that, in the absence of CD46, these proteins are misdirected or do not function properly to stabilize the acrosomal membranes.

Several IAM proteins are known to have a role in sperm-ZP binding but do not have knockout models yet. Yu et al. (2006) also have extracted proteasomal activity from the IAM, but, again, there is no gene knockout data available with regard to its role in fertilization. SPACA4 (SAMP14) is present in the IAM, OAM, and ES of human sperm; it is GPI-linked and is exposed after acrosomal exocytosis ( Shetty et al. 2003). Antibodies to recombinant SPACA4 inhibit binding/fusion of human sperm to zona-free hamster eggs, but a knockout mouse line has not been generated.

2.7 Equatorial Segment Proteins

Equatorin (EQTN) localization is limited to the equatorial segment and the protein is resistant to fairly strong extraction conditions (high salt, 0.1 % Triton X-100, sonication), persists following acrosomal exocytosis on sperm in the perivitelline space, and is even present on sperm inside the egg ( Manandhar and Toshimori 2001). Following acrosomal exocytosis, some EQTN is redistributed onto the PM over the ES (and some on the IAM), but the significance of this is not clear. Male EQTN-null mice are subfertile (~50 % of controls) and have reduced litter sizes (5.8 vs. 12.3 in control). EQTN-null sperm undergo normal acrosomal biogenesis (acrosomes are normal by immunofluorescence and by transmission electron microscopy), but do not undergo acrosomal exocytosis above control levels. Co-immunoprecipitation with SNAP25 and syntaxin 1a suggests a role in vesicle fusion during acrosomal exocytosis, but since the protein does not have a canonical transmembrane domain, it must do this via a membrane protein that interacts with the vesicle fusion proteins ( Wolkowicz et al. 2003; Hao et al. 2014). Also, since the outer acrosomal and plasma membranes don't fuse in the ES, EQTN is not likely to be involved in fusion in that region. The reason why EQTN affects fusion of the OAM and PM in the more anterior zones is an area for more investigation.

Sperm equatorial segment protein 1 (SPESP1) is another ES-limited protein that is important for fertility in both expected and surprising ways. SPESP1-null and heterozygous males had delays in fertility and smaller litter sizes than wild-type males, and heterozygous and null sperm numbers in the oviduct were significantly reduced ( Fujihara et al. 2010). Interestingly, in vitro assays showed that the number of sperm, percent progressively motile sperm, and motility patterns were the same for SPESP1-null, heterozygous, and wild-type mice, but the percent of fertilized oocytes and the percent fusibility with zona-free oocytes were significantly less in SPESP1-null and heterozygotes than wild type. Furthermore, in SPESP1-null sperm following acrosomal exocytosis, the plasma and outer acrosomal membranes were found to undergo fusion throughout the entire equatorial segment; since these membranes are typically nonfusogenic, these results show that SPESP1 is important in maintaining the stability of the ES which has an impact on fusibility with the egg plasma membrane ( Fujihara et al. 2010).

2.8 Ten Questions

In light of recent developments in the field of fertilization using contemporary imaging techniques and molecular biology to create targeted mutations in genes considered to be important for fertilization, several issues remain that need to be addressed. These include, but are not limited to, the following ten questions: (1) Does acrosomal exocytosis need to occur in a specific region of the female reproductive tract or peri-oocyte environment for fertilization to take place? Current evidence suggests that most sperm have initiated acrosomal exocytosis prior to penetrating the zona pellucida of cumulus-enclosed oocytes. (2) Is binding of the zona pellucida by acrosomal matrix components exposed on the sperm surface required for fertilization or is the affinity of these proteins assistive but not essential for fertilization to take place? For example, ZP3R binds to the oocyte zona pellucida but is not required for fertilization. On the other hand, in the absence of ZAN, the species-specific filter that blocks heterospecific sperm from other phylo-genetic classes from binding to mouse oocytes is lost; it would be interesting to determine if ZAN operates to filter the binding of sperm related at the genus or strain level. (3) Can we develop better tools to assess the integrity of the membranes overlying the acrosomal contents during various stages of capacitation and acrosomal exocytosis? (4) Do the components of the acrosome influence the interactions of sperm with the oviductal epithelium, either independently on a single-cell level or perhaps more generally through influencing the reciprocal connectivity between neighboring sperm and the female reproductive tract? (5) Do "acrosome-reacted" sperm that are experimentally recovered from the perivitelline space and are used successfully to fertilize other eggs still contain functional acrosomal matrix material? These experiments raise questions concerning the functions of the acrosomal contents in fertilization (they may be assistive but not required). (6) Are the different terms used to describe the acrosomal matrix important or are they describing the same properties from varied perspectives? Some reports refer to the acrosomal matrix as a particulate compartment of the acrosomal contents ( Buffone et al. 2008). Others make reference to a crystalloid nature observed by transmission electron microscopy ( Fléchon 2015). Still others classify the acrosomal matrix as an amyloid ( Guyonnet et al. 2014). (7) What role does pH play in the intra-acrosomal dynamics and post-exocytotic dispersion of the contents? For example, the pH of the acrosome is acidic and gradually begins to alkalinize during the course of epididymal maturation; once exocytosis is initiated, the contents are rapidly exposed to the more alkaline pH of the milieu surrounding the sperm. (8) Does the change in pH occurring during these periods activate or inactivate acrosomal enzymes? This is true for some proteases such as the proacrosin zymogen activation, but there may be other enzymes exhibiting similar properties. (9) What are the substrates for acrosomal hydrolases? Initially, it was thought that acrosin acted upon the zona pellucida. However, it is clear that this serine protease is involved in the dispersion of the acrosomal matrix, but the actual substrates have yet to be elucidated. (10) Why are acrosomal proteins differentially compartmentalized? The component segregation could be important for differential binding and/or release during exocytosis. Alternatively, the different domains might simply influence the shape of the acrosome. Clearly, there is still much to learn concerning the involvement of acrosomal matrix components in directing the structure of the acrosome during spermatogenesis and epididymal maturation as well as the function of the acrosome in the fertilization process.

Acknowledgments

This work was supported in part by grants R21-ES024527 and P30-ES013508 from the National Institute of Environmental Health Sciences to GLG, and Chenery and Rashkind Research Grants from Randolph-Macon College to JAF.

Contributor Information

James A. Foster, Department of Biology, Randolph-Macon College, Ashland, VA 23005, USA.

George L. Gerton, Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6160, USA, ude.nnepu@notreg.

References

Bloom W, Fawcett DW, Maximow AA. A textbook of histology. 10th edn. Saunders; Philadelphia: 1975. [Google Scholar]

Holstein AF, Roosen-Runge EC. Atlas of human spermatogenesis. Grosse; Berlin: 1981. [Google Scholar]

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The acrosome is a single exocytotic vesicle overlying the nucleus of most animal sperm. While the sperm of some taxa (e.g., teleost fish) do not possess an acrosome, it is generally thought that mammalian sperm acrosomes may perform two principal exocytosis-dependent functions: (1) to serve as a site for the sequestration and release of proteins required for binding to and penetration of the zona pellucida and (2) to enable the sperm to fuse with the oolemma.

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Abstract

Fluorescence imaging techniques such as single molecule localization microscopy, high-content screening and light-sheet microscopy are producing ever-larger datasets, which poses increasing challenges in data handling and data sharing. Here, we introduce a real-time compression library that allows for very fast (beyond 1 GB/s) compression and de-compression of microscopy datasets during acquisition. In addition to an efficient lossless mode, our algorithm also includes a lossy option, which limits pixel deviations to the intrinsic noise level of the image and yields compression ratio of up to 100-fold. We present a detailed performance analysis of the different compression modes for various biological samples and imaging modalities.

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A real-time compression library for microscopy images

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Fluorescence imaging techniques such as single molecule localization microscopy, high-content screening and light-sheet microscopy are producing ever-larger datasets, which poses increasing challenges in data handling and data sharing. Here, we introduce a real-time compression library that allows for very fast (beyond 1 GB/s) compression and de-compression of microscopy datasets during acquisition.

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animation (mostly Bio moöl

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Resources, Animations, Interviews, + Tools :: DNA Learning Center

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Resources: DNALC 2-D and 3-D animation collections, video discussions of science news, and available products from the DNALC and CSHL Press.

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Quantitative Biology

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Krasnitz develops mathematical and statistical tools to characterize the cellular composition, genomic disruptions, evolutionary history, and invasive capacity of malignant tumors, often in collaboration with clinical oncologists.

Hammell studies the role of transposable element activation in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD).

Atwal is interested in diverse modeling and statistical inference problems having to do with cancer and immunology, often through consideration of single-cell sequencing data.

Genomic Technology Development

Levy, Krasnitz, and Iossifov work closely with the Wigler laboratory in the development of new DNA and RNA sequencing methods, single-cell genomic technologies, and cancer diagnostics.

Kinney is a pioneer in the development of massively parallel reporter assays for characterizing the relationship between regulatory sequences and gene expression, including both transcription and RNA splicing.

More detailed information about research at the SCQB is available from the faculty websites of the SCQB members and associate members.

In addition to its research activities, the SCQB serves as a hub for education, training and research in the quantitative life sciences.

For more information please contact SCQB@cshl.edu.

Events

SCQB Seminar Series

The SCQB Seminar Series is a weekly symposium featuring a rotating roster of graduate students, postdocs and invited guests. Seminars are held most Wednesdays at noon during the academic calendar year.

See a list of previously invited speakers (pdf).

QB Meetings and Conferences

Members and Associate Members of the SCQB faculty organize relevant QB Meetings and Conferences hosted at CSHL and around the NY area.

QB Scientific Tea

The SCQB community which includes faculty, postdocs, graduate students, staff and special guests are invited to attend weekly catered informal gatherings to discuss their research and other relevant topics.

Journal Clubs

Members of the SCQB host a bi-weekly Sequence/Function Journal club and a monthly Deep Learning journal club during the academic calendar year.

Opportunities for Postdoctoral Researchers

The Simons Center for Quantitative Biology Fellows Program

The CSHL Simons Center for Quantitative Biology Fellows Program supports research fellows, who function independently but with mentoring from the senior faculty. The program is designed for exceptional quantitative biologists who have recently received their Ph.D. or M.D. degree and who are sufficiently talented and experienced to forgo standard postdoctoral training.

Interdisciplinary Scholars in Experimental and Quantitative Biology Program (ISEQB)

The Interdisciplinary Scholars in Experimental and Quantitative Biology (ISEQB) is an innovative funding opportunity for postdoctoral research open to applications in all areas of research at CSHL, including genetics, cancer, plant biology and neuroscience. The ISEQB is designed to help recruit new postdocs or fund existing CSHL postdocs who are interested in both wet-lab and dry-lab research. This program aims to catalyze collaborative research as well as promote the growth of the QB community at CSHL. For details on how to apply, affiliates of CSHL can visit the ISEQB flyer.

Course Work

Watson School QB Bootcamp at CSHL

The Watson School QB Bootcamp is a 2.5-day rapid introduction to Python and the computer cluster at CSHL taught each Fall by the SCQB faculty to provide incoming Watson School students with working knowledge in programming in preparation for the full-semester Specialized Discipline Course in Quantitative Biology.

Specialized Discipline Course in Quantitative Biology at CSHL

The Specialized Discipline Course in Quantitative Biology is a 16-week course that aims to equip incoming Watson School students with basic training in computer programming, modern statistical methods and physical biology. Using a probabilistic and Bayesian approach, the course covers probabilities, statistical fluctuations, Bayesian inference, significance testing, fluctuations, diffusion, information theory, neural signal processing, dimensional reduction, Monte Carlo methods, population genetics and DNA sequence analyses.

Advanced Coursework in Quantitative Biology

The Simons Center for Quantitative Biology (SCQB) provides Advanced Coursework in Quantitative Biology to graduate students, postdocs and scientific staff. With support from the Watson School for Biological Sciences, we are currently offering Coursera's Machine Learning through a Massive Open Online Course with onsite teaching assistance provided by SCQB postdoctoral researchers

Alexander J, Kendall J, McIndoo J, Rodgers L, Aboukhalil R, Levy D, Stepansky A, Sun G, Chobardjiev L, Riggs M, Cox H, Hakker I, Nowak DG, Laze J, Llukani E, Srivastava A, Gruschow S, Yadav SS, Robinson B, Atwal G, Trotman L, Lepor H, Hicks J, Wigler M, Krasnitz A. Utility of single-cell genomics in diagnostic evaluation of prostate cancer. Cancer Res. 78(2):348-358, 2018.

Members of the SCQB have created a number of freely available software tools and web resources for the research community. Here is a list of all the available software tools.

Developed by the Kinney lab, SUFTware (Statistics Using Field Theory) provides fast and lightweight Python implementations of Bayesian Field Theory algorithms for low-dimensional statistical inference. SUFTware currently supports the one-dimensional density estimation algorithm DEFT (Density Estimation Using Field Theory)

Developed by the Hammell lab, TEToolkit is a package for including transposable elements in differential enrichment analysis of sequencing data sets.

Developed by the Hammell lab, Single-Cell RNA-seq Analysis and Klustering Evaluation (SAKE) is a robust platform for scRNA-seq analysis that provides quantitative statistical metrics at each step of the analysis pipeline.

Developed by the Gillis Lab, MetaNeighbor allows users to quantify cell type replicability across single-cell RNA data sets. For a broader array of software (including those packages outside of R) from the Gillis lab please visit: https://github.com/gillislab

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Quantitative Biology - Cold Spring Harbor Laboratory

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The Simons Center for Quantitative Biology is dedicated to the development of new mathematical tools and techniques for the analysis of biological data. Researchers at the Center work on a wide variety of applications, including autism, cancer, neuroscience, plant biology, gene regulation and evolution.