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Image from PrivateLearning, (n.d.) - Modified

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Image from London, (2008).

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Image from Hessler, (2006) - Figure 2.

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1. Where is miR-2954 expressed in the brain?

2. What are the consequences of its inhibition?

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The authors were the first in the field to observe consequences of miRNA manipulation on endogenous gene expression in zebra finch cells. They found that miR-2954, which is located on the Z sex chromosome, has greater than twofold expression level in males compared to females, which is larger than the difference reported in the study performed by Gunaratne et al. in 2011. This suggests that there must be other factors that contribute to the increased gene expression levels in males or suppression in females that need to be explored.

In addition, results reported that the inhibition of miR-2954 led to an increase in one of its targets, NR4A3, but not the other eight targets that have been predicted through TargetScan. Due to this, the authors suggest that they can only make a conclusion about the general profile of how cells respond and this response needs to be further analyzed to understand the role that miR-2954 plays.

Moreover, they found that the decrease in expression of genes involved in ribosomal and mitochondrial functions suggests the existence of a molecular pathway that links the finches' perceptual experience with energy metabolism. No such link has been proposed in a prior study so Lin et al.'s novel observation will go on to impact the field and inspire future studies that look into the relationship between miR manipulation and energetics.

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miR-2954 has the potential to mediate some of the neurogenic effects of song habituation

miR-2954's expression is not ubiquitous in the brain; it is localized to specific subsets

Inhibition of the microRNA was able to show a general profile of how cells respond and it caused large changes in ribosomal and mitochondrial gene expression

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Video file from Pycroft, 2016.

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Dahlia & Devangna - HMB360 Assignment.pptx

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The authors used statistical techniques to ensure that the results were significant and not due to chance alone

The Lin et al. (2014) study reported results that were consistent with previous studies in the field:

miR-2954 responds differently to song playback in the two sexes. It is down-regulated in females and upregulated in males after a short gap of time (Gunaratne et al., 2011)

The ubiquitous expression of the microRNA in bodily tissues was consistent with observations performed in male chickens and zebra finches (Zhao et al., 2010 and Luo et al., 2012)

After song exposure, there was a noticeable decline in mRNAs involved in ribosomal and mitochondrial functions (Dong et al., 2009)

When RT-qPCR did not give statistically significant results after miR-2954 inhibition, the authors conducted a genome-wide analysis using RNA sequencing to effectively study the repercussions of the miRNA inhibition. This technique has been used in previous studies to characterize zebra finch cell lines. (Balakrishnan et al., 2012)

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Balakrishnan, C. N., Lin, Y.-C., London, S. E., and Clayton, D. F. (2012). RNA-seq transcriptome analysis of male and female zebra finch cell lines. Genomics, 100, 363–369. doi: https://doi.org10.1016/j.ygeno.2012.08.002

Clayton, D. F. (2013). The genomics of memory and learning in songbirds. Annu. Rev. Genomics Hum. Genet., 14, 45–65. doi:
https://doi.org/10.1146/annurev-genom-090711-163809.

Clayton, N. S. (1988). Song discrimination learning in zebra finches. Anim. Behav., 36, 1016–1024. doi: https://doi.org/10.1016/S0003-3472(88)80061-7.

Dong, S., Replogle, K. L., Hasadsri, L., Imai, B. S., Yau, P. M., Rodriguez-Zas, S., et al. (2009). Discrete molecular states in the brain accompany changing responses to a vocal signal. Proc. Natl. Acad. Sci. U.S.A., 106, 11364–11369. doi: https://doi.org/10.1073/pnas.0812998106.

Gunaratne, P. H., Lin, Y.-C., Benham, A. L., Drnevich, J., Coarfa, C., Tennakoon, J. B., et al. (2011). Song exposure regulates known and novel microRNAs in the zebra finch auditory forebrain. BMC Genomics, 12:277. doi: https://doi.org/10.1186/1471-2164-12-277.

Hessler, N.A. (2006). The songbird brain [digital image]. Retrieved from http://www.brain.riken.jp/bsi-news/bsinews34/no34/speciale.html

Lewis, B. P., Burge, C. B., and Bartel, D. P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 120, 15–20. doi: https://doi.org/10.1016/j.cell.2004.12.035.

Lin, Y. C., Balakrishnan, C. N., & Clayton, D. F. (2014). Functional genomic analysis and neuroanatomical localization of miR-2954, a song-responsive sex-linked microRNA in the zebra finch. Front. Neurosci., 8, 1-20. doi: https://doi.org/10.3389/fnins.2014.00409.

London, S. (2008) Development of the Zebra Finch [digital image]. Retrieved from https://itg.beckman.illinois.edu/communications/iotw/2008-10-14/

Luo, G. Z., Hafner, M., Shi, Z. M., Brown, M., Feng, G. H., Tuschl, T., et al. (2012). Genome-wide annotation and analysis of zebra finch microRNA repertoire reveal sex-biased expression. BMC Genomics, 13:727. doi: https://doi.org/10.1186/1471-2164-13-727.

miRBase. (n.d.). miRNA gene family: mir-2954 (2 sequences). Retrieved from http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0001032.

PrivateLearning. (n.d.). Critical Analysis [digital image]. Retrieved from https://www.privatewriting.com/critical-analysis.

Pycroft, L. (2016, March 11). My Zebra Finch Brutus singing again [Video File]. Retrieved from https://www.youtube.com/watch?v=99Vk2JSTV5E.

Zhao, D., McBride, D., Nandi, S., McQueen, H. A., McGrew, M. J., Hocking, P. M., et al. (2010). Somatic sex identity is cell autonomous in the chicken. Nature, 464, 237–242. doi: https://doi.org/10.1038/nature08852.

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The authors in this study only focused on the miR-2954 gene involved in song perception and production in adult zebra finches in controlled isolation. However, the expression of this gene can differ in zebra finches in the wild.

A proposed experiment would be to do a comparison study which would compare zebra finches in the wild versus those in isolation. Ideally, it would be beneficial to follow the birds throughout their different stages of development but since that is difficult to carry out in the wild, the authors can let an egg grow to adulthood in isolation and capture different representatives for the development stages from the wild. Through analysis of the bird's brain tissue, the authors would be able to investigate whether or not gene expression varies in the neurons throughout development in the two environmental conditions.

We would expect miR-2954 levels to increase throughout the developmental process and see a higher expression level in the wild birds because of their socialization opportunities with others. The results of this experiment also have the potential to answer questions regarding the localization of the microRNA, which is one of the questions the authors wanted to answer.

If results are not as expected and both isolated and wild finches have similar levels of miR-2954 expression, then that would suggest that the environment does not play a role in this pathway and the process is purely due to genetic factors.

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Since the authors were only able to detect changes in 1/9 mRNAs that miR-2954 targets, there is a possibility that another microRNA may be compensating for its inhibition. In fact, there are 2 different sequences found on the Z chromosome that make up the family of miR02954. (miRBase) In the Lin et al. study, they do not specify which sequence of the miR they targeted, so it is possible that the one sequence that they inhibited was unable to affect all the targets because its counterpart was compensating for its loss.

A proposed experiment would be to provide an appropriate inhibitor for both sequences of miR-2954 to the zebra finch cell line. If compensation was taking place, then analysis with Illumina RNA sequencing would now reveal that most, if not all, targets predicted by TargetScan increase their expression levels in response to the two variants of miR-2954 being inhibited. This result would allow the authors to better analyze the results of inhibition and its associated downstream effects.

However, if the dual, spontaneous inhibition of both sequences of miR-2954 is unable to increase expression of the nine target mRNAs, then it is advisable to explore the inhibition effects of another song-regulated miRNA or the possibility of exogenous factors that may be meddling with the results.

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There were two major drawbacks to this paper:

The inhibition of miR-2954 failed to have an effect on all of its mRNA targets as predicted by TargetScan as well as the study conducted by Lewis et al. in 2005. Out of the 9 targets, miR inhibition only increased the level of NR4A3 while having no effect on the other eight. As a result, they changed the significance level to show a relevant change in three more targets. The authors should consider whether there is a possibility that another microRNA is compensating for the induced inhibition and thus not allowing the other targets to increase their expression.

The authors failed to differentiate between the two samples they retrieved tissues from. In the beginning of the paper, the authors state that they extracted 9 tissues from six wild birds. Then, they performed their isolation studies with 13 other birds. However, the rest of the paper focuses on the isolation studies with no reference to how the tissues of the six wild birds were used. This seems like a missed opportunity for the authors because they could have compared the miRNA expression between the two samples and analyzed whether there are environmental or somatic factors at play.

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Figure 3: Expression of miR-2954 at high magnification 

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Figure 3: Expression of miR-2954 at high magnification 

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This figure shows the overlapping of the 2 stains used with the gene.

The authors use DAPI staining to identify nuclei and NeuN staining to locate neurons

miR-2954 was highly expressed within cell nuclei in HVC, Area X and NCM, lower expression was seen in RA and it was absent from the LMAN core

In the male auditory forebrain, they observed a decrease in gene expression for genes involved in ribosomal and mitochondrial functions a day after repeated playbacks; which caused a decrease in miR-2954

Image from Lin et al., (2014)- Figure 3

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Figure 2: Expression of miR-2954 at low magnification 

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Figure 2: Expression of miR-2954 at low magnification 

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5 major areas of the brain involved in song perception and production were observed for miR-2954 expression: HVC, stratal nucleus Area X, lateral magnocellular nucleus of the anterior nidopallium (LMAN), robust nucleus of the arcopallium (RA) and caudomedial nidopallium (NCM)

These areas responsible for song production in males but reduced or absent in females.

Image from Lin et al., (2014) - Figure 2

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Figure 1: miR-2954 expression in the zebra finch tissues and cell line. 

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Figure 1: miR-2954 expression in the zebra finch tissues and cell line. 

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An ubiquitous expression pattern seen

Higher expression levels in males than females for the 9 major tissues and cell lines

Image from Lin et al., (2014) - Figure 1

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Figure 4: Effects of miR2954 on G266 Cells (A) and NR4A3 mRNA Levels (B)

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Figure 4: Effects of miR2954 on G266 Cells (A) and NR4A3 mRNA Levels (B)

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In the MAP Kinase pathway, there was an increase in the RNAs due to the inhibition of the miR-2954 gene

There was an increase in the transcription factor NR4A3, as seen in 4B below. This is a song responsive mRNA and one of the targets of miR-2954

Image from Lin et al., (2014) - Figure 4

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Subjects: Adult Zebra Finches

Birds were placed in isolation for 18 hours, after which they head a novel song and were then decapitated

9 bodily tissues were analyzed to study for miR-2954 expression

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Zebra finches are songbirds that communicate using learned vocalizations (Clayton N.S., 1988)

Their own singing and hearing vocalizations of others can cause complex changes in gene expression in the higher forebrain. (Clayton D.F., 2013)

Recent studies have found that some of the finches' miRNAs regulate themselves after song playbacks (Gunaratne et al., 2011)

miR-2954, one of the song-regulated miRNAs, is located on the avian Z chromosome and has sex-dependent response to song (Gunaratne et al., 2011)