Carter M. Takacs

Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition

After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here we show that Nanog, Pou5f1 (also called Oct4) and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest before gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

Dual positive and negative regulation of wingless signaling by adenomatous polyposis coli.

The evolutionarily conserved Wnt/Wingless signal transduction pathway directs cell proliferation, cell fate, and cell death during development in metazoans and is inappropriately activated in several types of cancer. The majority of colorectal carcinomas contain truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor, a negative regulator of Wnt/Wingless signaling. Here, we demonstrate that Drosophila Apc homologs also have an activating role in both physiological and ectopic Wingless signaling. The Apc amino terminus is important for its activating function, whereas the β-catenin binding sites are dispensable. Apc likely promotes Wingless transduction through down-regulation of Axin, a negative regulator of Wingless signaling. Given the evolutionary conservation of APC in Wnt signal transduction, an activating role may also be present in vertebrates with relevance to development and cancer.

Testing putative hemichordate homologues of the chordate dorsal nervous system and endostyle: expression of NK2.1 (TTF-1) in the acorn worm Ptychodera flava (Hemichordata, Ptychoderidae)

SUMMARY Recent phylogenetic investigations have confirmed that hemichordates and echinoderms are sister taxa. However, hemichordates share several cardinal characteristics with chordates and are thus an important taxon for testing hypotheses of homology between key chordate characters and their putative hemichordate antecedents. The chordate dorsal nervous system (DNS) and endostyle are intriguing characters because both hemichordate larval and adult structures have been hypothesized as homologues. This study attempts to test these purported homologies through examination of the expression pattern of a Ptychodera flava NK2 gene, PfNK2.1, because this gene is expressed both in the DNS and endostyle/thyroid in a wide range of chordate taxa. We found that PfNK2.1 is expressed in both neuronal and pharyngeal structures, but its expression pattern is broken up into distinct embryonic and juvenile phases. During embryogenesis, PfNK2.1 is expressed in the apical ectoderm, with transcripts later detected in presumable neuronal structures, including the apical organ and ciliated feeding band. In the developing juvenile we detected PfNK2.1 signal throughout the pharynx, including the stomochord, and later in the hindgut. We conclude that the similar utilization of NK2.1 in apical organ development and chordate DNS is probably due to a more general role for NK2.1 in neurogenesis and that hemichordates do not possess a homologue of the chordate DNS. In addition, we conclude that P. flava most likely does not possess a true endostyle; rather during the evolution of the endostyle NK2.1 was recruited from its more general role in pharynx development.

MicroRNAs as genetic sculptors: Fishing for clues

microRNAs (miRNAs) encode small RNA molecules of approximately 22nts in length that regulate the deadenylation, translation, and decay of their target mRNAs. The identification of miRNAs in plants and animals has uncovered a new layer of gene regulation with important implications for development, cellular homeostasis and disease. Because each miRNA is predicted to regulate several hundred genes, a major challenge in the field remains to elucidate the precise roles for each miRNA and to understand the physiological relevance of individual miRNA-target interactions in vivo. Despite the wide variety of biological contexts where miRNAs function, a common theme emerges, whereby miRNAs shape gene expression within both spatial and temporal dimensions by removing messages from previous cellular states as well as modulating the levels of actively transcribed genes. This review will focus on the role that the teleost Danio rerio (zebrafish) has played in shaping our understanding of miRNA function in vertebrates.

Adenomatous polyposis coli is present near the minimal level required for accurate graded responses to the Wingless morphogen.

The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, we reduced Apc activity to different degrees, and analyzed the effects on three concentration-dependent responses to Arm signaling that specify distinct retinal photoreceptor fates. We find that both Apc1 and Apc2 negatively regulate Arm activity in photoreceptors, but that the relative contribution of Apc1 is much greater than that of Apc2. Unexpectedly, a less than twofold reduction in total Apc activity, achieved by loss of Apc2, decreases the effective threshold at which Wg elicits a cellular response, thereby resulting in ectopic responses that are spatially restricted to regions with low Wg concentration. We conclude that Apc activity is not present in vast excess, but instead is near the minimal level required for accurate graded responses to the Wg morphogen.

miR-430 regulates oriented cell division during neural tube development in zebrafish.

MicroRNAs have emerged as critical regulators of gene expression. Originally shown to regulate developmental timing, microRNAs have since been implicated in a wide range of cellular functions including cell identity, migration and signaling. miRNA-430, the earliest expressed microRNA during zebrafish embryogenesis, is required to undergo morphogenesis and has previously been shown to regulate maternal mRNA clearance, Nodal signaling, and germ cell migration. The functions of miR-430 in brain morphogenesis, however, remain unclear. Herein we find that miR-430 instructs oriented cell divisions in the neural rod required for neural midline formation. Loss of miR-430 function results in mitotic spindle misorientation in the neural rod, failed neuroepithelial integration after cell division, and ectopic cell accumulation in the dorsal neural tube. We propose that miR-430, independently of canonical apicobasal and planar cell polarity (PCP) pathways, coordinates the stereotypical cell divisions that instruct neural tube morphogenesis.

RESA identifies mRNA-regulatory sequences at high resolution

Gene expression is extensively regulated at the levels of mRNA stability, localization and translation. However, decoding functional RNA-regulatory features remains a limitation to understanding post-transcriptional regulation in vivo. Here, we developed RNA-element selection assay (RESA), a method that selects RNA elements on the basis of their activity in vivo and uses high-throughput sequencing to provide a quantitative measurement of their regulatory functions at near-nucleotide resolution. We implemented RESA to identify sequence elements modulating mRNA stability during zebrafish embryogenesis. RESA provides a sensitive and quantitative measure of microRNA activity in vivo and also identifies novel regulatory sequences. To uncover specific sequence requirements within regulatory elements, we developed a bisulfite-mediated nucleotide-conversion strategy for large-scale mutational analysis (RESA–bisulfite). Finally, we used the versatile RESA platform to map candidate protein–RNA interactions in vivo (RESA–CLIP).

Analyses of mRNA structure dynamics identify embryonic gene regulatory programs

RNA folding plays a crucial role in RNA function. However, knowledge of the global structure of the transcriptome is limited to cellular systems at steady state, thus hindering the understanding of RNA structure dynamics during biological transitions and how it influences gene function. Here, we characterized mRNA structure dynamics during zebrafish development. We observed that on a global level, translation guides structure rather than structure guiding translation. We detected a decrease in structure in translated regions and identified the ribosome as a major remodeler of RNA structure in vivo. In contrast, we found that 3′ untranslated regions (UTRs) form highly folded structures in vivo, which can affect gene expression by modulating microRNA activity. Furthermore, dynamic 3′-UTR structures contain RNA-decay elements, such as the regulatory elements in nanog and ccna1, two genes encoding key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.Characterization of mRNA structure during the zebrafish maternal-to-zygotic transition identifies the ribosome as a major RNA structure remodeler in vivo and reveals that structural dynamics can affect gene expression, partly by modulating miRNA activity.

mRNA structure dynamics identifies the embryonic RNA regulome

RNA folding plays a crucial role in RNA function. However, our knowledge of the global structure of the transcriptome is limited to steady-state conditions, hindering our understanding of how RNA structure dynamics influences gene function. Here, we have characterized mRNA structure dynamics during zebrafish development. We observe that on a global level, translation guides structure rather than structure guiding translation. We detect a decrease in structure in translated regions, and we identify the ribosome as a major remodeler of RNA structure in vivo. In contrast, we find that 3’-UTRs form highly folded structures in vivo, which can affect gene expression by modulating miRNA activity. Furthermore, we find that dynamic 3’-UTR structures encode RNA decay elements, including regulatory elements in nanog and cyclin A1, key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.

Genetic compensation is triggered by mutant mRNA degradation

Genetic compensation by transcriptional modulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems 1–3; however, whether and how such a response can be activated in the absence of protein feedback loops is unknown. Here, we develop and analyze several models of transcriptional adaptation in zebrafish and mouse that we show are not caused by loss of protein function. We find that the increase in transcript levels is due to enhanced transcription, and observe a correlation between the levels of mutant mRNA decay and transcriptional upregulation of related genes. To assess the role of mutant mRNA degradation in triggering transcriptional adaptation, we use genetic and pharmacological approaches and find that mRNA degradation is indeed required for this process. Notably, uncapped RNAs, themselves subjected to rapid degradation, can also induce transcriptional adaptation. Next, we generate alleles that fail to transcribe the mutated gene and find that they do not show transcriptional adaptation, and exhibit more severe phenotypes than those observed in alleles displaying mutant mRNA decay. Transcriptome analysis of these different alleles reveals the upregulation of hundreds of genes with enrichment for those showing sequence similarity with the mutated gene’s mRNA, suggesting a model whereby mRNA degradation products induce the response via sequence similarity. These results expand the role of the mRNA surveillance machinery in buffering against mutations by triggering the transcriptional upregulation of related genes. Besides implications for our understanding of disease-causing mutations, our findings will help design mutant alleles with minimal transcriptional adaptation-derived compensation.