The Spatio-Temporal Control of Zygotic Genome Activation
Preprint posted on December 06, 2018 https://www.biorxiv.org/content/biorxiv/early/2018/12/06/488056.full.pdf
It has been thought that activation of the zygotic genome (ZGA) in amphibians depends on a specific Nuclear-to-Cytoplasm ratio. Gentsch et al challenge the old thinking and discover links between morphogen signalling and ZGA.Meng Zhu
The maternal to zygotic transition (MZT) is an event that each single embryo has to experience to start its own gene expression program. The timing of MZT is species-specific, but it is always represented by the clear-out of maternal transcripts and proteins and the activation of the zygotic genome. The later (ZGA) is fundamental to unlock the embryonic development and yet the molecular mechanisms are still not fully understood.
In frog embryos, the early development starts by 12 rounds of rapid and synchronised cleavage divisions, in which the cells do not grow in volume and the cell cycle is a simple turnover between S and M phases. This is followed by the critical “mid-blastula transition” (MBT) which is characterised by a series of cellular transitions, including the lengthening of cell cycle, the switch of primary metabolites, the change of cell motility properties and the appearance of zygotic transcripts (ZGA). ZGA has been postulated to be a result of the elongation of G2 phase, as in somatic cells G2 is usually the time when transcription activity peaks, and the lack of G2 before MBT may simply block transcription. This hypothesis is also supported by the observation that the earliest zygotic transcripts are short – either lack or possess short-length introns – and that the delay of the cell cycle transition by over-expressing DNA replication factors disrupts ZGA. However, although these experiments show that a coordinated cell cycle is required for ZGA, they fall short in demonstrating the sufficiency. To gain a deeper mechanistic insight into ZGA regulation would arguably require a more detailed characterisation of its dynamics.
In this preprint, the authors characterised RNAPII chromatin engagement at 6 developmental stages spanning pre- to post-MBT of X. tropcalis embryos. Combining the information obtained from this method with the data from RNA-seq they can differentiate the zygotic transcripts from those that are maternal, and thus gain a more precise view of ZGA with high temporal resolution. The results presented in this preprint provide novel insights into ZGA regulation in frog embryos.
ZGA does not depend on N/C ratio of MBT
Earlier studies by Newport and his colleagues suggest that the timing of MBT depends on a crucial N/C ratio, as the injection of exogenous plasmid DNA or the reduction of cell volume seems to be able to accelerate MBT including ZGA. One would expect that the initiation of ZGA would occur at a specific developmental time right after reaching such N/C ratio. However, in this study by applying the new pipeline to examine the number of zygotic genes transcribed, the authors observed that zygotic transcription already took place a few cell cycles earlier than MBT (~32-cell stage) and exponentially increased thereafter, with the highest activity reached around MBT. These results suggest that reaching MBT N/C ratio is not a necessary condition for ZGA, against what was proposed earlier. This MBT N/C ratio independent behaviour is also in great contrast to the regulation of other MBT events, such as the slow down of DNA replication as reported by the same group. Interestingly, the authors also found that many of the early ZGA genes are with certain functions, such as nucleosome assembly and mRNA metabolism, indicating that the early ZGA genes may help to amplify the zygotic transcription activity.
Cell-cycle length cannot account for the constraints of transcript length
The short cell-cycle length has been thought to hinder the transcription of long genes. Although the authors indeed confirmed that the earliest ZGA genes are generally short, they observed that the lengthening of transcripts also happens before MBT. Thus, in contrast to what has been conceived, the cell cycle per see may not account for the transcripts lengthening.
Morphogen signalling “patterns” ZGA
By performing stage-dependent transcriptome analysis, the authors found that many early ZGA genes (at 32-cell stage) are associated with Nodal signalling. The authors further interrogate whether other morphogen signalling pathways can play a role in regulating ZGA. They examined Wnt Bmp and Nodal, and found that in general regional morphogen activities correlate with local zygotic transcription and they account for a great proportion of ZGA. For instance, Wnt and Bmp, the two morphogens patterning the dorsal and ventral sides of the embryo, together can account for the zygotic transcription activity alongside the D-V axis. These observations that the zygotic genome at the early stages are highly configured by morphogen signalling may explain the fact that in the frog embryo the immediate early post-MBT event is to establish the body plan.
Besides morphogen signalling, the zygotic genome is also regulated by the maternal transcription factors (TFs) such as Pou53/Sox3 and VegT, consistent with another recent preprint by the same group. Interestingly, the activation of the entire zinc finger protein cluster is not regulated by any of those factors, for which the authors proposed KLF4 as the putative upstream activator. Overall, these results give genomic level mechanistic insight into earlier identified crucial developmental factors in regulating post-MBT events.
Questions to the authors
1. the observation that Wnt and Bmp act synergistically to regulate D-V axis patterning genes is quite surprising, as from the gain or loss-of function experiments (the Dorsalisation or Ventralisation phenotypes obtained by dysregulating these two pathways) normally people would expect an antagonistic relationship between them. When turned-down one pathway, have the authors noticed any upregulation of targets of the other?
2. Besides foxi4.2 did the authors find any potential novel factors controlling “animal specific fate” that may be like VegT in the vegetal pole?
3. Is the morphogen gradient read by altering the gene expression level or the identities of genes activated?
4. It is easy to conceive the reasons for the exponential increase of zygotic gene numbers over-time, but what leads to the increase of transcripts’ length?
5. What determines the timing of specific morphogen signals to activate downstream targets?For example, Nodal downstream targets are activated at the 32-cell stage. On the contrary, despite b-catenin is already translocated to the nucleus at a similar stage, the transcriptional outcome only become evident at a much later stage (~1K cell).
6. Although it could be slightly beyond the scope of this paper, but is there any master regulators for “ubiquitous ZGA”?
Gentsch, G.E., Spruce, T., Owens, N.D.L., and Smith, J.C. (2018b). The role of maternal pioneer factors in predefining first zygotic responses to inductive signals. bioRxiv 306803.
Collart, C., Allen, G.E., Bradshaw, C.R., Smith, J.C., and Zegerman, P. (2013). Titration of four replication factors is essential for the Xenopus laevis midblastula transition. Science 341, 893–896.
Newport, J., and Kirschner, M.W. (1982a). A major developmental transition in early xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell 30, 675–686.
Newport, J., and Kirschner, M.W. (1982b). A major developmental transition in early xenopus embryos: II. control of the onset of transcription. Cell 30, 687–696.
Heyn, P., Kircher, M., Dahl, A., Kelso, J., Tomancak, P., Kalinka, A.T., and Neugebauer, K.M. (2014). The earliest transcribed zygotic genes are short, newly evolved, and different across species. Cell Rep. 6, 285–292.
Posted on: 20th February 2019 , updated on: 22nd February 2019Read preprint
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