Marshall, K. E. | Search bioRxiv

bioRxiv2018

Transcriptional dynamics following freezing stress reveal selection for mechanisms of freeze tolerance at the poleward range margin in the cold water intertidal barnacle Semibalanus balanoides

The ability to survive freezing has repeatedly evolved across multiple phyla. This suggests that the mechanisms of freeze tolerance must be readily evolvable from basal physiological traits. While several biochemical correlates to freeze tolerance have been described, the mechanism that confers freeze tolerance is still not well understood. To understand both the basic biochemical mechanisms of freeze tolerance as well as their role in local adaptation at the poleward range edge, we conducted a transcriptomic study on two populations (one from the poleward range margin in the White Sea, Russia, and one from the central coast of British Columbia, Canada) of the cold water acorn barnacle Semibalanus balanoides on a time series following a freezing event. We found that the British Columbia population (at the equatorward range margin) was significantly less freeze tolerant than the White Sea population (at the poleward range margin). After assembling and annotating a de novo transcriptome for S. balanoides, we found that the patterns of differential transcript expression following freezing were almost entirely non-overlapping between the two populations, with the White Sea population expressing a series of heat shock proteins in response to freezing stress as well as several aquaporins, while the British Columbia population expressed a series of proteases instead, indicating severe protein damage. We found strong evidence of purifying selection on the significantly upregulated transcripts in the White Sea population, suggesting local adaptation to freezing threat. Taken together, this shows the importance of freeze tolerance to population survival at the poleward range margin, and highlights the central roles of aquaporins and heat shock proteins to the trait of freeze tolerance across taxa.

physiology↗

bioRxiv2017

A conserved maternal-specific repressive domain in Zelda revealed by Cas9-mediated mutagenesis in Drosophila melanogaster

In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome becomes transcriptionally active hours after fertilization. Transcriptional activation during this maternal-to-zygotic transition (MZT) is tightly coordinated with the degradation of maternally provided mRNAs. In Drosophila melanogaster, the transcription factor Zelda plays an essential role in widespread activation of the zygotic genome. While Zelda expression is required both maternally and zygotically, the mechanisms by which it functions to remodel the embryonic genome and prepare the embryo for development remain unclear. Using Cas9-mediated genome editing to generate targeted mutations in the endogenous zelda locus, we determined the functional relevance of protein domains conserved amongst Zelda orthologs. We showed that neither a conserved N-terminal zinc finger nor an acidic patch were required for activity. Similarly, a previously identified splice isoform of zelda is dispensable for viability. By contrast, we identified a highly conserved zinc-finger domain that is essential for the maternal, but not zygotic functions of Zelda. Animals homozygous for mutations in this domain survived to adulthood, but embryos inheriting these loss-off-function alleles from their mothers died late in embryogenesis. These mutations did not interfere with the capacity of Zelda to activate transcription. Unexpectedly, these mutations generated a hyperactive form of the protein and enhanced Zelda-dependent gene expression. These data have defined a protein domain critical for controlling Zelda activity during the MZT, but dispensable for its roles later in development, for the first time separating the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are exclusively required for establishing the developmental program during the MZT. We propose that tightly regulated gene expression is essential to navigate the MZT and that failure to precisely execute this developmental program leads to embryonic lethality.\n\nAUTHOR SUMMARYFollowing fertilization, the one-celled zygote must be rapidly reprogrammed to enable the development of new, unique organism. During these initial stages of development there is little or no transcription of the zygotic genome, and maternally deposited products control this process. Among the essential maternal products are mRNAs that encode transcription factors required for preparing the zygotic genome for transcriptional activation. This ensures that there is a precisely coordinated hand-off from maternal to zygotic control. In Drosophila melanogaster, the transcription factor Zelda is essential for activating the zygotic genome and coupling this activation to the degradation of the maternally deposited products. Nonetheless, the mechanism by which Zelda functions remains unclear. Here we used Cas9-mediated genome engineering to determine the functional requirements for highly conserved domains within Zelda. We identified a domain required specifically for Zeldas role in reprogramming the early embryonic genome, but not essential for its functions later in development. Surprisingly, this domain restricts the ability to Zelda to activate transcription. These data demonstrate that Zelda activity is tightly regulated, and we propose that precise regulation of both the timing and levels of genome activation is required for the embryo to successfully transition from maternal to zygotic control.

developmental biology↗