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Dynamics of Meiotic Sex Chromosome Inactivation and Pachytene Activation in Mice Spermatogenesis

Ábel Vértesy, Javier Frias-Aldeguer, Zeliha Sahin, Nicolas Rivron, Alexander van Oudenaarden, Niels Geijsen

Preprint posted on June 10, 2019 https://www.biorxiv.org/content/10.1101/665372v1

Silencing and activating transcriptomic transitions during mouse spermatogenesis at single cell resolution

Selected by Sergio Menchero

Categories: genomics

Background & Summary:

Germ cell formation requires a regulated transition from mitosis to meiosis to generate haploid cells: oocytes and sperm. In males, there are specific features of meiosis given that the X and Y chromosomes only share the homologous region, and the non-homologous region stays unpaired. X and Y chromosomes undergo a process of silencing called meiotic sex chromosome inactivation (MSCI) and a subsequent reactivation of transcription of genes involved in spermiogenesis (final stage of spermatogenesis) (Turner, 2015).

In this work, Vertesy and colleagues use single-cell transcriptomics and single-molecule fluorescent in situ hybridisation (smFISH) to resolve the temporal dynamics that orchestrate this transition. The authors took advantage of a Dazl-GFP reporter mice which expresses the reporter gene in male germ cells spans from the spermatogonia up to the initiation of meiosis in spermatocytes (Chen et al., 2014). After validating a reconstructed temporal trajectory from their transcriptomic data, in-depth computational analysis allowed the authors to detect a functional order in the timing of silencing and reactivation of sex chromosome genes, rather than a positional order.

 

Key findings of the preprint:

  • Sharply timed gene activation during spermatogenesis.

Changes in gene expression programs driving the different steps of spermatogenesis take place in a sequential but sharp way temporally, rather than genes being progressively activated. Three different waves of gene activation can be clearly distinguished which correlate with three specific stages of differentiation: spermatogonia, (pre)leptotene and pachytene.

Figure 1 (Fig. 3C in the preprint) Z-score normalized rolling average of raw transcript counts of all genes with a single, sharp peak, ordered by their peaking times.

 

  • A transcriptional activation peak precedes meiotic sex chromosome inactivation.

MSCI is a silencing event and takes places rapidly. Many sex chromosomal genes are specifically activated right before they get silenced, dismissing the idea that a lack of transcription drives MSCI. Importantly, many of these genes are involved in gene regulation.

Figure 2 (Fig. 5B in the preprint). Both sex chromosomes show increasing relative expression before a rapid silencing that coincides with the induction of H2afx gene. Dashed lines indicate the start and maximum of H2afx transcription, also marking the start and end of MSCI.

 

  • Genes escaping MSCI are different from genes that escape X-chromosome inactivation in females.

A group of X chromosome genes are detected upon MSCI, and with an increased number of transcripts than before the general silencing. Interestingly, out of the 27 genes, only 1 is shared with known genes that escape the process of X-chromosome inactivation in female cells to balance dosage compensation. This suggests that the ability of certain genes to escape inactivation is not only based on their position in the chromosome.

  • The reactivation pattern of genes after MSCI follows a functional order.

Three behaviours of gene activation are detected: a cluster of genes that are gradually activated, another cluster of genes that are sharply activated (referred to as immediate genes) and a third one with an intermediate behaviour. The immediate genes were enriched in transcriptional activation and energy production functions but, interestingly, there was also a correlation with the order they will be used in the spermatozoa. Genes playing a role in flagella formation and cell movement were mostly found in the immediate group, while late activated genes are involved in fertilization.

Figure 3 (Fig. 6B,E,H in the preprint). Individual expression profiles of immediate genes show the step-like up regulation after MSCI (left). Gradual genes all showed a slowly increasing gene expression in pachytene (middle). Gradual-and immediate-class specific GO-terms map to different anatomical regions of the sperm. Gradual-class specific terms and corresponding anatomical structures are in red. Immediate-class specific terms and structures are in green. Shared terms and the related axoneme is indicated by the black arrow (right).

 

Why I chose this work:

Once the snapshots of different processes are known, the dynamics underlying these events are key to understand how they are regulated. The different steps that male germ cells undergo until the formation of haploid cells, including wide transcriptional silencing and reactivation, are a very good model to study dynamic transitions.

I chose this preprint because the authors nicely use state-of-the-art techniques, such as single-cell RNAseq and single-molecule FISH, to address those questions. They combine three reconstruction methods to generate a more reliable pseudotemporal trajectory which then, they validated by analysing expression of key genes by smFISH. Also, they have built an online tool that will help other interested researchers to explore the dynamic expression of specific genes: http://bit.ly/male_meiosis. After their first validation and general analyses, the authors go in depth to find detailed cues of how this process is temporally regulated, thus, harnessing the value of the data they have generated.

Questions to the authors:

  • The authors use single-molecule FISH to beautifully validate different patterns of gene expression according mainly to spatial changes. Does this technique allow to go deeper and quantify transcriptional differences within a spatially-separated population to try to detect a temporal scale? If this technique could detect finer differences, it could be used to see how synchronised are neighbouring cells in terms of the sequential peaks of gene activation or the transition to MSCI for instance.
  • Given that escapee genes after MSCI are different from escapee genes from other known X-chromosome silencing events (such as XCI in female cells), do the authors think those genes have specific functions in each case? Or could it be due to the lack of Xist expression in males that make the mechanism of silencing completely different? Since many escapee genes from the Xist locus are non-coding genes they could be involved in gene silencing directly, but if this is their function it could be the same than in other inactivation processes.
  • It is very curious that although genes involved in flagella formation and fertilization are transcribed in a specific order according to their function, they will not be used until a week later. Do the authors have an idea of why they follow this order to then be stored? Could this storage also be ordered to stay in a “prepared” mode and act more efficiently?

 

References:

Chen HH, Welling M, Bloch DB, Muñoz J, Mientjes E, Chen X, Tramp C, Wu J, Yabuuchi A, Chou YF, Buecker C, Krainer A, Willemsen R, Heck AJ, Geijsen N. 2014. DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells. Stem Cell Reports 3(5):892-904.

Turner JM. 2015. Meiotic silencing in mammals. Annu Rev Genet 49:395-412.

 

Posted on: 27th June 2019

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  • Author's response

    Ábel Vértesy shared

     

    • The authors use single-molecule FISH to beautifully validate different patterns of gene expression according mainly to spatial changes. Does this technique allow to go deeper and quantify transcriptional differences within a spatially-separated population to try to detect a temporal scale? 

    Although the spatial resolution of smFISH from an optics perspective is fantastic, sectioning of the testis only allows us to see cross-sections, because the seminiferous tubules criss-cross the tissue (Fig. 1). In any cross-section of the testis only a fraction of maturation stages are present, and these are not even subsequent stages (Fig. 2). If one could do a longitudinal scan along the tubules, fine ‘steps’ could be visualized.

    Fig.1: Seminiferous tubules criss-cross in the testis. Source: (wikipedia).

     

    Fig.2: Schematic for the cycle of the seminiferous epithelium and stages per cross section. The earliest A-spermatogonia is depicted at the bottom left, and undergo maturation towards the right – maturation is then continued in the second row, representing how germ cells are “stacked upon” A-spermatogonia as they are pushed towards the interior of the tubule. Adapted from: (Griswold 2016)

     

    • If this technique could detect finer differences, it could be used to see how synchronised are neighbouring cells in terms of the sequential peaks of gene activation or the transition to MSCI for instance.

    As described above, it would be challenging, but if synchronized, connected cells could be labeled, by large scale imaging (to get enough statistics), one may indeed see that synchronized sibling cells are more alike, than similarly staged, but non sibling cells.

    • Given that escapee genes after MSCI are different from escapee genes from other known X-chromosome silencing events (such as XCI in female cells), do the authors think those genes have specific functions in each case? 

    The functional role of escapee genes in female X-chromosome silencing (XCI) is adequately understood to say, that most genes escape silencing to provide sex-specific functions or, if the gene has copy on the Y chromosome (in the pseudoautosomal region), to reach similar expression in males and females (Balaton and Brown 2016).  Although the XCI is by far the most studied sex chromosome inactivation event, we are just at the beginning to understand why there is a strong variation between tissues or species (Tukiainen et al. 2017). In males, the functional role of escapee genes in post-meiotic silencing (PMSCI) is much less understood, and we are yet to discover the reason why certain genes escape meiotic silencing (MSCI).

    • Or could it be due to the lack of Xist expression in males that make the mechanism of silencing completely different? 

    The silencing mechanism in MSCI is clearly different from that in XCI. This mechanistic difference is most likely explained by different evolutionary origins – unlike XCI, MSCI originated from the meiotic silencing of (any) unsynapsed chromatin.

    • Since many escapee genes from the Xist locus are non-coding genes they could be involved in gene silencing directly, but if this is their function it could be the same than in other inactivation processes.

    That is an interesting possibility and may well explain a part of the escaping genes. I think that first we need to further confirm and further characterize the escape during MSCI, and once we have a consensus list of genes, we can start asking why this process is in place?

    • It is very curious that although genes involved in flagella formation and fertilization are transcribed in a specific order according to their function, they will not be used until a week later. Do the authors have an idea of why they follow this order to then be stored? Could this storage also be ordered to stay in a “prepared” mode and act more efficiently?

    At this point we can only speculate. mRNAs that share function and that are expressed at the same time might be stored, then released together. We see that the expression of mRNA binding proteins is very dynamic during spermatogenesis – early expressed mRNAs might be incorporated into different RNP’s as compared to late expressed mRNAs. Why these genes are transcribed so early, might be explained by a later bottleneck of the transcription, as the cell may need its energy for producing other mRNAs and proteins. Why and how this happens is indeed one of the most interesting questions raised by our observation.

     

    Bibliography

    Balaton, B.P. and Brown, C.J. 2016. Escape artists of the X chromosome. Trends in Genetics 32(6), pp. 348–359.

    Griswold, M.D. 2016. Spermatogenesis: the commitment to meiosis. Physiological Reviews 96(1), pp. 1–17.

    Tukiainen, T., Villani, A.-C., Yen, A., et al. 2017. Landscape of X chromosome inactivation across human tissues. Nature 550(7675), pp. 244–248.

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