The neural crest is a vertebrate stem cell population with broad developmental potential. While a gene regulatory network describing establishment of these cells has been generated, much remains to be learned about the dynamics of this process. Here, we use fluorescent in situ hybridization chain reaction to quantify the spatiotemporal dynamics of neural crest formation in Xenopus. We find that the initial onset of neural crest genes is broad and partially overlapping, with distinct anterior-posterior and medio-lateral biases. A shared neural crest domain emerges, but some genes retain relative expression differences that persist into migratory stages, producing stream-specific gene expression patterns. These differences correlate with dynamic expression of the neural plate border factors pax3 and zic1. Correlating relative intensities of pax3 and zic1 with the presence or absence of nascent neural crest transcripts predicts that these factors can differentially regulate snai2 and sox8, which we confirm experimentally. Strikingly, later stages display an inverse correlation between neural crest and neural plate border factors, suggesting that pax3 and zic1 initially promote neural crest gene activation but are downregulated as neural crest identity emerges.

Wdr5, a multifunctional scaffolding protein, with established roles in chromatin regulation and pluripotency, but its functions in early development remain poorly understood. Here, we show that Xenopus wdr5 is expressed in blastula stem cells and enriched in neural crest cells. Depletion of wdr5 abolished neural crest gene expression in embryos and in reprogrammed explants while expanding neural plate border and neural plate domains. Gain-of-function experiments revealed striking dose-dependent effects: low Wdr5 enhanced neural crest formation, whereas high levels suppressed it, suggesting a requirement for precise stoichiometry with interacting partners. We identify Myc as an essential co-factor for Wdr5 in neural crest - Wdr5 and Myc physically interact and co-expression at defined ratios rescues neural crest formation. We further show that the Wdr5 WBM site is required for Myc-dependent activation of neural crest genes, whereas the WIN site regulates myc expression itself; both domains are necessary to rescue wdr5 depletion. These findings reveal that Wdr5 orchestrates neural crest development through multiple, domain-specific mechanisms, integrating stoichiometric control with partner-specific transcriptional regulation, and underscores the importance of precise co-factor ratios in cell fate decisions.

FoxQ2 is a highly conserved Forkhead-box transcription factor expressed anteriorly in cnidarians and bilaterians, yet its evolution is marked by rapid divergence and lineage-specific duplications or losses. Moreover, its presence and localization in vertebrate groups remains unclear. To reconcile these conflicting reports of conservation and divergence, we combine phylogenetic and synteny analyses of FoxQ2 sequences from 21 animal phyla. We uncover three ancient FoxQ2 paralogs in bilaterians-FoxQ2I, FoxQ2II, and FoxQ2III. All three were present in the chordate ancestor, and two are retained in vertebrates, indicating a richer FoxQ2 repertoire in vertebrates than previously recognized. To assess FoxQ2 expression, we analyzed mollusk, acoel, amphioxus, and zebrafish single-cell transcriptomic datasets, and conducted fluorescent in situ hybridization in amphioxus, lamprey, skate, zebrafish, and chicken. FoxQ2I and FoxQ2II show conserved anterior expression, while FoxQ2III is expressed in the gut endoderm in chordates, including amphioxus, lamprey, and skate. We also predict conserved transcription factor binding sites across amphioxus genera, revealing stage- and cell-type-specific regulatory interactions for FoxQ2I in deuterostomes. Overall, this work clarifies FoxQ2's evolutionary history, identifies the endodermally expressed paralog FoxQ2III, and proposes that early duplication of FoxQ2I/II enabled subfunctionalization, driving the fast evolutionary rate of FoxQ2 sequences observed in bilaterians.

The evolution of complex vertebrate body plans was driven by the acquisition of the neural crest, a stem cell population that retains broad, multi-germ layer potential after most embryonic cells have become lineage restricted. We have previously shown that neural crest cells share significant gene regulatory architecture with pluripotent blastula stem cells. Here, we examine the roles that two Krüppel-like Family (Klf) transcription factors, Klf2 and Klf17, play in these cell populations. We found that inhibition of either klf2 or klf17 expanded expression of pluripotency, neural plate border and neural crest factors in neurula stage Xenopus embryos, suggesting that Klf factors regulate the exit from pluripotency and proper establishment of the boundary of the neural crest domain. To gain further insights into the role of Klf factors in the evolution of the neural crest, we examined their expression in sea lamprey, a jawless vertebrate, and show that ectopic expression of lamprey klf17 in Xenopus embryos phenocopies Xenopus klf17. These data suggest that klf17 may have been the ancestral Klf factor that functioned in these gene regulatory networks in stem vertebrates.

FoxQ2 is a highly conserved class of Forkhead-box transcription factors expressed on the anterior side of the body in cnidarians and bilaterians. Despite this conserved expression pattern, recent phylogenetic analyses have revealed a complex and rapid evolution of this class, with several taxon-specific duplications and losses. Until recently, FoxQ2 was thought to be lost in most vertebrate lineages, and its presence and localization in different vertebrate groups remains unclear. To reconcile these conflicting reports of conservation and divergence, here we present a comprehensive analysis of the phylogenetic relationships and expression patterns of FoxQ2 genes across metazoans. By combining phylogenetics and synteny analyses of FoxQ2 sequences from 21 animal phyla, we uncover the presence of three ancient FoxQ2 paralogs in bilaterians, which we name FoxQ2a, FoxQ2b and FoxQ2c . All three FoxQ2 paralogs are present in the chordate lineage and two are conserved in vertebrates, indicating a richer repertoire of vertebrate Fox genes than previously estimated. To investigate the expression of FoxQ2 genes across bilaterians, we mined expression data from existing single cell transcriptomic datasets of mollusk, acoel, amphioxus and zebrafish development, and expanded it using fluorescent in situ hybridization in amphioxus, lamprey, skate, zebrafish and chicken. Our analysis demonstrates the conserved anterior expression of FoxQ2a and FoxQ2b paralogs while also revealing a novel domain of FoxQ2c expression within the chordate endoderm, including in amphioxus, lamprey and skate. Finally, we devise a method to predict conserved transcription factor binding sites across the three extant amphioxus genera with specificity to developmental stage and cell-type identity. This suggests conserved regulatory interactions for the expression of FoxQ2a across deuterostomes. Overall, this work clarifies the complex evolutionary history of FoxQ2 genes and identifies a newly discovered endodermally-expressed Fox gene, FoxQ2c . We further propose that the early duplication of FoxQ2a and FoxQ2b , along with their redundant functions, provided the ideal background for subfunctionalization, contributing to the fast evolutionary rate of FoxQ2 sequences observed in bilaterians. ### Competing Interest Statement EBG has been an employee of Genentech since September 2022. All other authors declare no competing interests. Cancer Research UK Cambridge Institute, https://ror.org/05ybzg123, C9545/A29580 National Institutes of Health, , R01GM116538 National Science Foundation, , 1764421 Simons Foundation, , SFARI 597491-RWC Academia Sinica, , AS-GC-111-L01 National Science and Technology Council, , 113-2621-B-001-004-MY3 Royal Society, , UF130182, URF\R\191007, RG140377

The evolutionary transition from simple chordate body plans to complex vertebrate body plans was driven by the acquisition of the neural crest, a stem cell population that retains broad, multi-germ layer developmental potential long after most embryonic cells have become lineage restricted. We have previously shown that neural crest cells share significant gene regulatory architecture with pluripotent blastula stem cells. Here we examine the roles that Krüppel-like Family (Klf) transcription factors play in these stem cell populations. Although Klf4 has established roles in regulating pluripotency in mammalian stem cells cultures, we find that in Xenopus it is klf2 that is highly expressed in pluripotent blastula stem cells. klf2 expression is down-regulated as cells transition to a neural crest state while a related klf factor, klf17, is significantly up regulated in response to neural crest induction. We used gain and loss of function studies to compare the activities of these closely related factors and found that they have both shared and distinct activities. Inhibition of either klf2 or klf17 activity led to significantly expanded expression of pluripotency, neural plate border and neural crest factors in neurula stage embryos, leading us to hypothesize that klf factors regulate the exit from pluripotency and proper establishment of the boundary of the neural crest domain. To gain further insights into the role of klf factors in the evolution of the neural crest, we examined their expression in the jawless vertebrate, Petromyzon marinus ( sea lamprey). We find that lamprey have a klf2/4 and a klf17 gene, but that only klf17 is expressed in blastula and neural crest stem cells. Moreover, ectopic expression of lamprey klf17 in Xenopus embryos phenocopies Xenopus klf17 activity. These data suggest that klf17, rather than klf4, may have been the ancestral klf factor that functioned in these GRNs in stem vertebrates.

Neural crest cells are a stem cell population unique to vertebrates that give rise to a diverse array of derivatives, including much of the peripheral nervous system, pigment cells, cartilage, mesenchyme, and bone. Acquisition of these cells drove the evolution of vertebrates and defects in their development underlies a broad set of neurocristopathies. Moreover, studies of neural crest can inform differentiation protocols for pluripotent stem cells and regenerative medicine applications. Xenopus embryos are an important system for studies of the neural crest and have provided numerous insights into the signals and transcription factors that control the formation and later lineage diversification of these stem cells. Pluripotent animal pole explants are a particularly powerful tool in this system as they can be cultured in simple salt solution and instructed to give rise to any cell type including the neural crest. Here we report a protocol for small molecule-mediated induction of the neural crest state from blastula stem cells and validate it using transcriptome analysis and grafting experiments. This is an powerful new tool for generating this important cell type that will facilitate future studies of neural crest development and mutations and variants linked to neurocristopathies.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

The neural crest is vertebrate-specific stem cell population that helped drive the origin and evolution of the vertebrate clade. A distinguishing feature of these stem cells is their multi-germ layer potential, which has drawn developmental and evolutionary parallels to another stem cell population-pluripotent embryonic stem cells (animal pole cells or ES cells) of the vertebrate blastula. Here, we investigate the evolutionary origins of neural crest potential by comparing neural crest and pluripotency gene regulatory networks (GRNs) in both jawed ( Xenopus ) and jawless (lamprey) vertebrates. Through comparative gene expression analysis and transcriptomics, we reveal an ancient evolutionary origin of shared regulatory factors between neural crest and pluripotency GRNs that dates back to the last common ancestor of extant vertebrates. Focusing on the key pluripotency factor pou5 (formerly oct4), we show that the lamprey genome encodes a pou5 ortholog that is expressed in animal pole cells, as in jawed vertebrates, but is absent from the neural crest. However, gain-of-function experiments show that both lamprey and Xenopus pou5 enhance neural crest formation, suggesting that pou5 was lost from the neural crest of jawless vertebrates. Finally, we show that pou5 is required for neural crest specification in jawed vertebrates and that it acquired novel neural crest-enhancing activity after evolving from an ancestral pou3 -like clade that lacks this functionality. We propose that a pluripotency-neural crest GRN was assembled in stem vertebrates and that the multi-germ layer potential of the neural crest evolved by deploying this regulatory program.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank'of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in predicted enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization. Editor's evaluation This paper reports fundamental findings that substantially advance our understanding of a major research question – how hybridization events influence gene regulatory programs and how evolutionary pressures have shaped these programs in response to such events. This convincing work uses appropriate and validated methodology in line with the current state-of-the-art. https://doi.org/10.7554/eLife.83952.sa0 Decision letter Reviews on Sciety eLife's review process Introduction In animals, zygotic genome activation (ZGA) is triggered after an initial period of transcriptional quiescence following fertilization of the egg, during the maternal-to-zygotic transition (MZT; Lee et al., 2014; Vastenhouw et al., 2019). In mammals, this occurs during the slow first cleavages (Svoboda, 2018), a few days removed from the subsequent induction of pluripotent stem cells in the blastocyst by a core network of factors including NANOG, OCT4, and SOX2 (Li and Belmonte, 2017; Takahashi and Yamanaka, 2016). In contrast, faster-dividing taxa including zebrafish, Xenopus, and Drosophila activate their genomes in the blastula hours after fertilization (Foe and Alberts, 1983; Kane and Kimmel, 1993; Newport and Kirschner, 1982a; Vastenhouw et al., 2019), which leads immediately to pluripotency. In zebrafish, maternally provided homologs of NANOG, OCT4, and SOX2 are required for a large share of genome activation (Lee et al., 2013; Leichsenring et al., 2013; Miao et al., 2022); thus, vertebrate embryos deploy conserved pluripotency induction mechanisms at different times during early development. Beyond vertebrates, unrelated maternal factors direct genome activation and the induction of stem cells, for example Zelda (Liang et al., 2008), CLAMP (Colonnetta et al., 2021; Duan et al., 2021), and GAF (Gaskill et al., 2021) in Drosophila, although they seem to share many functional aspects with vertebrate pluripotency factors, including pioneering roles in opening repressed embryonic chromatin and establishing activating histone modifications (Blythe and Wieschaus, 2016; Gaskill et al., 2021; Hug et al., 2017). This diversity of strategies implies that the gene network regulating pluripotency has been extensively modified over evolutionary time (Endo et al., 2020; Fernandez-Tresguerres et al., 2010), though it is unknown when and under what circumstances major modifications arose. We sought to understand how recent genome upheaval has affected the pluripotency regulatory network in the allotetraploid Xenopus laevis, by deciphering how embryonic genome activation is coordinated between its two subgenomes. X. laevis's L (long) and S (short) subgenomes are inherited from each of two distinct species separated by ~34 million years that hybridized ~18 million years ago (Session et al., 2016; Figure 1A). A subsequent whole-genome duplication restored meiotic pairing. Despite extensive rearrangements and deletions, most genes are still encoded as two copies (homeologs) on parallel, non-inter-recombining chromosomes (Session et al., 2016). Previously, homeologs had been challenging to distinguish due to high functional and sequence similarity; however, the recent high-quality X. laevis genome assembly has made it feasible to resolve differential expression and regulation genome-wide between the two subgenomes (Elurbe et al., 2017; Session et al., 2016). Figure 1 with 1 supplement see all Download asset Open asset Identifying the first wave of genome activation across the two subgenomes. (A) The allotetraploid X. laevis genome contains two distinct subgenomes "L" and "S" due to interspecific hybridization of ancestral diploids. (B) Triptolide inhibits genome activation, as measured in the late blastula, while cycloheximide inhibits only secondary activation, distinguishing genes directly activated by maternal factors. NF = Nieuwkoop and Faber. (C) Heatmap of RNA-seq coverage over exons (left) and introns (right) of activated genes. Allopolyploidy often provokes acute effects on gene expression (Hu and Wendel, 2019; Moran et al., 2021), leading to regulatory shifts over time to reconcile dosage imbalances and incompatibilities between gene copies (Grover et al., 2012; Song et al., 2020; Swamy et al., 2021). This phenomenon has been explored primarily in plants (Adams and Wendel, 2005; Husband et al., 2013; Mable, 2004), but the extent to which this has occurred in the few characterized allopolyploid vertebrates is unclear (Chen et al., 2019a; Li et al., 2021; Luo et al., 2020). For X. laevis, there is a broad trend toward balanced homeolog expression across development and adult tissues, with a subtle bias in favor of the L homeolog that emerges after genome activation (Session et al., 2016), and an overall ontogenetic and transcriptomic trajectory similar to 48 million-years diverged diploid X. tropicalis (Harland and Grainger, 2011; Yanai et al., 2011). Initial observations in X. laevis have demonstrated differential homeologous enhancer activity in the eye (Ochi et al., 2017) and a divergent cis-regulatory landscape of histone modifications and recruitment of transcriptional machinery in the early gastrula (Elurbe et al., 2017), suggesting that embryonic genome activation is likely also asymmetric between the two subgenomes. Although Xenopus embryos have long been a model for understanding the MZT, for example (Amodeo et al., 2015; Charney et al., 2017; Chen et al., 2019b; Gentsch et al., 2019; Gibeaux et al., 2018; Gurdon et al., 1958; Kimelman et al., 1987; Newport and Kirschner, 1982a; Newport and Kirschner, 1982b; Paraiso et al., 2019; Skirkanich et al., 2011; Veenstra et al., 1999; Yanai et al., 2011), ZGA regulators have not previously been identified in X. laevis. Here, we identify maternal Pou5f3 and Sox3 as top-level regulators of X. laevis pluripotency and ZGA and elucidate the predicted enhancer architecture that differentially recruits them to homeologous gene copies between the two subgenomes. Despite differential subgenome activation, combined transcriptional output converges to proportionally resemble the diploid state, maintaining gene dosage for the embryonic pluripotency program. Results Identifying divergently activated homeologous genes At genome activation, the X. laevis pluripotency network consists of maternal regulators acting directly on the first embryonic genes (Figure 1B). To identify these genes, we performed a total RNA-seq early embryonic time course using our X. laevis-specific ribosomal RNA depletion protocol (Phelps et al., 2021; Figure 1A and B, Supplementary file 1). Subtle gene activation is observed in the blastula at Nieuwkoop and Faber (N.F.) stage 8, culminating in 4772 genes with significant activation by the middle of stage 9 (8 hours post fertilization [h.p.f.] at 23 °C) (Figure 1C, Supplementary file 2). Gene activation was detected through a combination of exon- and intron-overlapping sequencing reads deriving from nascent pre-mRNA (Lee et al., 2013) – indeed, two-thirds of these genes had substantial maternal contributions that masked their activation when quantifying exon-overlapping reads alone (Figure 1C). These genes fail to be activated in embryos treated at 1 cell (stage 1) with the transcription inhibitor triptolide (Gibeaux et al., 2018) when compared to DMSO vehicle control embryos (Figure 1B and C, Figure 1—figure supplement 1). To distinguish direct targets of maternal factors (primary activation) (Figure 1B), we then performed RNA-seq on stage 9 embryos treated with cycloheximide at stage 8, to inhibit translation of newly synthesized embryonic transcription factors that could regulate secondary activation (Harvey et al., 2013; Lee et al., 2013). A total of 2662 genes (56% of all activated genes) were still significantly activated in cycloheximide-treated embryos compared to triptolide-treated embryos, representing the first wave of genome activation in the embryo (Figure 1C, Supplementary file 1A). We analyzed subgenome of origin for activated genes and found that they are preferentially encoded as two homeologous copies in the genome (p=2.3 × 10–225, χ-squared test, 10 d.o.f.; Figure 2A). However, a majority of these genes have asymmetric expression between the two homeologs, often with transcription deriving from only the L or S copy alone (Figure 2B–C). This asymmetry is more pronounced at stage 8, but balances somewhat as genome activation progresses, suggesting timing differences for homeolog activation that could result from subtle regulatory divergence (Figure 2A, Figure 2—figure supplement 1A–C); and slightly less pronounced for strictly zygotic genes compared to maternal-zygotic genes (maternal contribution ≥1 TPM; Figure 2D, Figure 2—figure supplement 1D), which are often reactivated with different homeolog expression patterns compared to their maternal contribution (Figure 2E). Figure 2 with 2 supplements see all Download asset Open asset Homeologous genes are differentially activated in the early embryo. (A) Proportion of genes encoded as homeologs on both subgenomes versus only one subgenome (singleton) (left), as compared to expression patterns in the early embryo. p Values are from χ-squared tests, 10 d.o.f., comparing genomic to expressed proportions, 16 d.o.f., comparing proportions between activated genes and the maternal contribution, 12 d.o.f., comparing proportions at subsequent stages of activation. (B) Browser tracks showing log2 reads-per-million RNA-seq coverage of equivalently activated homeologs (top) and differentially activated homeologs (L-specific, middle; S-specific, bottom). Trip = triptolide, CHX = cycloheximide. (C) Biplot comparing log2 fold primary activation over triptolide treated embryos for the S homeolog (x axis) versus the L homeolog. (D) Left, proportion of genes activated symmetrically or asymmetrically from the L or S subgenomes, stratified into whether there is a maternal contribution for either homeolog (MZ) or not (Z) (p=0.02, χ-squared test, 4 d.o.f.); and whether a gene is activated only in the stage 9 blastula or is additionally increased in only one or more than one differentiated lineage from stages 10–13 (p=1.3 × 10–22, χ-squared test, 8 d.o.f.). Right, homeolog proportions of later gene activation in epidermal (Epi), neural progenitor (Neur), ventral mesodermal (Meso), and endodermal (Endo) lineages from stages 10–13 (p=3.3 × 10–64, χ-squared test comparing stage 9 and the four lineages, 16 d.o.f.). Lineage-specific gene expression data are from Johnson et al., 2022. (E) Homeolog-specific stage 9 activation proportions, versus maternal contribution homeolog expression patterns, for maternal-zygotic genes. (F) Concordance of homeolog activation patterns across the differentiated lineages at stages 10–13, for genes initially activated at stage 9 and also increased in at least two differentiated lineages. (G) Browser track showing strand-separated reads-per-million RNA-seq coverage over the mir-427 encoding locus on the distal end of Chr1L (v10.1). After genome activation, a heightened imbalance in favor of the L homeolog emerges, as measured by activation patterns in four differentiated cell lineages (Johnson et al., 2022; Figure 2D, Figure 2—figure supplement 1E–I), that appears to indicate a shift toward more divergent homeolog regulation as development proceeds, as was observed previously (Session et al., 2016). However, it is likely that some of the shared homeolog activation as measured in the whole blastula is actually composed of homeolog-specific regional activation (Chen and Good, 2022). Indeed, for one-third of genes activated in more than one lineage, different homeologs are activated in different lineages (Figure 2F, Figure 2—figure supplement 1F and G), and for those genes that are already activated at stage 9, this seems to result in a higher proportion of both-homeolog activation, as compared to genes with single-lineage or blastula-specific activation (p=1.3 × 10–22, χ-squared test, 8 d.o.f.). Overall, this indicates a high degree of divergent cis-regulatory architecture between gene homeologs throughout early development. Genes activated from both subgenomes are enriched in transcriptional regulators (p<0.01, Fisher's exact test, two-sided) (Figure 2—figure supplement 1J), suggesting that gene function may have influenced homeolog expression patterns. However, there is no evidence for stronger functional divergence between homeologs expressed asymmetrically between the subgenomes, as estimated by non-synonymous versus synonymous mutation rate in coding regions (dN/dS ratio; Figure 2—figure supplement 1K and L). The microRNA mir-427 is encoded on only one subgenome Among the first-wave genes is the microRNA mir-427, which plays a major role in clearance of maternally contributed mRNA (Lund et al., 2009). Similar to X. tropicalis mir-427 (Owens et al., 2016) and the related zebrafish mir-430 (Lee et al., 2013), mir-427 is one of the most strongly activated genes in the X. laevis embryonic genome (Figure 2D, Figure 2—figure supplements 1C and 2A). In version 9.2 of the X. laevis genome assembly, the miR-427 precursor hairpin sequence is found in only five copies overlapping a Xenbase-annotated long non-coding RNA on chr1L (Figure 2—figure supplement 2B). To better capture the genomic configuration of the mir-427 primary transcript, we aligned the miRBase-annotated precursor sequence (Kozomara et al., 2019) to the version 10.1 X. laevis genome assembly. This revealed an expanded mir-427 locus at the distal end of Chr1L composed of 33 precursor copies, encoded in both strand orientations over 55 kilobases (Figure 2D, Figure 2—figure supplement 2A and B). The corresponding region on Chr1S is unalignable (Figure 2—figure supplement 2C), suggesting that mir-427 is encoded on only the L subgenome. We additionally found two mir-427 hairpin sequence matches to the distal end of Chr3S, but these loci were not supported by substantial RNA-seq coverage (Figure 2—figure supplement 2D). This is reminiscent of the X. tropicalis mir-427 genomic configuration (Owens et al., 2016), although smaller in scale and on a non-homologous chromosome. In X. tropicalis, 171 tandemly arrayed mir-427 precursors are found on the distal end of Chr03, which is thought to accelerate mature miR-427 accumulation during the MZT to facilitate rapid maternal clearance (Owens et al., 2016). Zebrafish similarly encodes a large array of more than 2000 mir-430 precursors, which begin to target maternal mRNA for clearance shortly after ZGA (Bazzini et al., 2012; Giraldez et al., 2006; Hadzhiev et al., 2023; Lee et al., 2013). These results strongly suggest that the mir-427 locus has undergone genomic remodeling, resulting in absence from the S subgenome, but possibly also translocation between chromosomes in the tropicalis or laevis lineages. Subgenomes differ in their regulatory architecture To discover the maternal regulators of differential homeolog activation, we first profiled embryonic chromatin using Cleavage Under Target & Release Using Nuclease (CUT&RUN) (Hainer and Fazzio, 2019; Skene and Henikoff, 2017), which we adapted for blastulae. We found that cell dissociation was necessary for efficient nuclear isolation to carry out the on-bead CUT&RUN chemistry (Figure 3A, Figure 3—figure supplement 1A–D). At stages 8 and 9, the active marks H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 27 acetylation (H3K27ac) were enriched in the transcription start site (TSS) regions of activated genes, and differential homeolog activation measured by RNA-seq significantly correlates with differential histone modification profiles, with a slight overall bias toward stronger L homeolog chromatin activity (Figure 3B and C, Figure 3—figure supplement 1E–G, Supplementary file 3). Differential promoter engagement by transcriptional machinery likely underlies the differential histone modification levels; however, we found no promoter sequence differences between homeologs that would implicate differential recruitment of specific transcription factors (Supplementary file 4). Figure 3 with 2 supplements see all Download asset Open asset Differential homeolog activation is regulated by subgenome-specific enhancers. (A) CUT&RUN coverage over all annotated transcription-start site (TSS) regions, sorted by descending stage 8 H3K27ac signal. (B) Bee-swarm plots showing the log2 ratio of L versus S homeolog coverage among genes where only one homeolog is activated (L only, S only), or both homeologs are activated. TSS region is 1 kb centered on the TSS; upstream region is 500 bp to 3 kb upstream of the TSS. Horizontal bars show medians. Individual category p values are from two-sided paired t-tests of log2 L homeolog coverage vs log2 S homeolog coverage, p values across the three categories are from a one-way ANOVA on the log2 ratios. (C) Stage 9 H3K4me3 CUT&RUN coverage over paired homeologous gene regions around the TSS (left) and maps comparing high-confidence predicted enhancer density near homeologous TSSs (middle). Differential predicted enhancers are active in only one subgenome, conserved predicted enhancers are active in both. Average densities are plotted to the right of each paired map. Gene pairs are sorted according to L versus S subgenome RNA-seq activation ratio (right). (D) Schematics showing aligned predicted enhancers and their homeologous regions (gray) mapped onto L (red, top lines) and S (blue, bottom lines) chromosomes. Comparable schematics show Xenbase annotated homeologous gene pairs (lavender). (E) Heatmap of stage 9 ATAC-seq and stage 8 H3K27ac CUT&RUN over L & S homeologous regions for equivalently active high-confidence predicted enhancers (top) and subgenome-specific predicted enhancers. (F) Top enriched transcription factor motif families in L-specific and S-specific active high-confidence predicted enhancers compared to inactive homeologous regions. FDR-corrected p-values from Homer are shown. RPM = reads per million. Instead, we searched for differences in gene-distal regulatory elements – that is enhancers – between the two subgenomes. To identify regions of open chromatin characteristic of enhancers, we performed Assays for Transposase-Accessible Chromatin with sequencing (ATAC-seq) on dissected animal cap explants; the high concentration of yolk in vegetal cells inhibits the Tn5 transposase (Esmaeili et al., 2020). Accessible chromatin is already evident at stage 8 in putative enhancer regions, though the overall signal is weak, and by stage 9, these regions exhibit robust accessibility (Figure 3—figure supplement 2A). We called peaks of elevated sub-nucleosome sized fragment coverage at stage 9, then intersected the open regions with our H3K27ac CUT&RUN. This yielded 15,654 putative open and acetylated gene-distal regulatory regions at genome activation, of which we classified 5047 as high confidence predicted enhancers that had ≥2 fold signal enrichment in each of at least three H3K27ac replicates and three ATAC-seq replicates individually (Figure 3—figure supplement 2A, Supplementary file 5). To identify homeologous L and S enhancer regions, we constructed a subgenome chromosome-chromosome alignment using LASTZ (Harris, 2007). This yielded a syntenic structure consistent with genetic maps (Figure 3D; Session et al., 2016), recapitulating the large inversions between chr3L/chr3S and chr8L/chr8S. Seventy-nine percent of predicted enhancer regions successfully lifted over to homeologous chromosomes, and of these, >92% of these are flanked by the same homeologous genes (Figure 3—figure supplement 2B), confirming local synteny. Among the paired regions involving high-confidence predicted enhancers, only 21% had conserved activity in both homeologs, with the remaining pairs exhibiting differential H3K27ac and chromatin accessibility (Figure 3E, Figure 3—figure supplement 2C). Differential predicted enhancer density around genes significantly correlated with differential activation (p=1.3 × 10–16, Pearson's correlation test; Figure 3C, middle, Figure 3—figure supplement 2D), with greater L enhancer density around differentially activated L genes, and similarly for S enhancers and S genes. In contrast, conserved enhancers had equivalent density near both homeologs regardless of activation status (p=0.67, Pearson's correlation test; Figure 3C, right). Thus, differences in enhancer activity likely underlie divergent gene homeolog transcription at genome activation. Maternal pluripotency factors differentially engage the subgenomes Given that these paired enhancer regions are differentially active despite having similar base sequences, we searched for transcription factor binding motifs that distinguished active enhancers from their inactive homeolog. Two motifs were strongly enriched in both active L enhancers and active S enhancers, corresponding to the binding sequences of the pluripotency factors OCT4 and SOX2/3 (SOXB1 family; Figure 3F, Supplementary file 4). Since mammalian OCT4 and SOX2 are master regulators of pluripotent stem cell induction (Li and Belmonte, 2017; Takahashi and Yamanaka, 2016), and zebrafish homologs of these factors are maternally provided and required for embryonic genome activation (Lee et al., 2013; Leichsenring et al., 2013; Miao et al., 2022), we hypothesized that differential enhancer binding by maternal X. laevis OCT4 and SOXB1 homologs underlies asymmetric activation of the L and S subgenomes. RNA-seq confirms high maternal levels of sox3 and pou5f3.3 (OCT4 homolog) mRNA, as well as lower levels of paralog pou5f3.2, each deriving from both subgenomes (Figure 3—figure supplement 2E). To assess their roles in genome activation, we inhibited their translation using previously validated antisense morpholinos (Morrison and Brickman, 2006; Takebayashi-Suzuki et al., 2007; Zhang et al., 2003) injected into stage 1 embryos. Combinations of pou5f3.3+sox3 morpholinos and pou5f3.2+pou5 f3.3 morpholinos led to mild and severe gastrulation defects, respectively, while combining all three morpholinos led to developmental arrest with a complete failure to close the blastopore (Figure 4—figure supplement 1A), consistent with what has been reported in X. tropicalis (Gentsch et al., 2019). RNA-seq of morpholino-treated embryos at stage 9 revealed extensive misregulation of genome activation, though only 15% of genes exhibited deficient activation, while 43% of genes actually exhibited slightly higher levels in the morphants (Figure 4A, Figure 4—figure supplement 1B and G–K), which could be due to direct or indirect transcriptional repression mediated by Pou5f3 and Sox3. Increases were predominantly detected from intron signal (Figure 4—figure supplement 1J), which would largely rule out post-transcriptional effects. A larger proportion of strictly zygotic genes were down-regulated in the morphants compared to maternal-to-zygotic genes (p=6.6 × 10-18, χ-squared test, 3 d.o.f.), perhaps reflecting a more complex regulatory network that regulates maternal gene reactivation (Figure 4—figure supplement 1L). Figure 4 with 2 supplements see all Download asset Open asset Pou5f3.3 and Sox3 binding drives genome activation. (A) Heatmap showing log2 fold activation differences for exonic and intronic regions of primary-activated genes for combinations of pou5f3.2, pou5f3.3, and sox3 morpholino-treatments, or Triptolide treatment, compared to controls. Right panel is in the presence of cycloheximide (CHX). (B) Biplot comparing exonic expression levels in cycloheximide-treated control embryos versus embryos also injected with pou5f3.2, pou5f3.3, and sox3 morpholinos. Primary-activated genes with maternal contribution <1 TPM (strictly zygotic) are purple circles, maternal-zygotic genes detected by exonic increases are orange triangles. TPM = transcripts per million. (C) Barplot summarizing the proportion of genes affected by morpholino treatment with cycloheximide on primary-activated genes (left bar), without cycloheximide (middle bar), and all stage 9 activated genes without cycloheximide (right bar). Down = significantly decreased in one of the morpholino treatments, up = significantly increased. (D, F) Biplots showing genes with >2 fold L or S biased activation (upper red and lower blue points, respectively) in control embryos (left panel) versus their activation in pou5f3.2, pou5f3.3, and sox3 morpholino-treated embryos (right panel, maintaining the same color per gene). (E, G) Quantification of the biplots in (D, F) in before-and-after plots. Y-axis is the absolute value of the log2 L vs S activation difference. p Values are from Wilcoxon signed-rank tests (paired). Overlaid boxplots show median, upper and lower quartiles, and 1.5 x interquartile range. (H) Regulatory networks consistent with direct regulation of embryonic gene activation by Pou5f3 and Sox3 (1) versus additional regulation by zygotic factors (2), which likely accounts for genes up-regulated in MO treatments. (I) Stage 8 Pou5f3.3 (left) and Sox3 (right) CUT&RUN coverage near TSSs for genes down-regulated in morpholino-treated embryos with or without cycloheximide (top), genes up-regulated (middle), and genes not significantly affected in any morpholino treatment (bottom). Top enriched motifs for each factor are shown below with p-values from Homer de novo discovery. (J) Aggregate plots of the binding signal in (I), with down-regulated genes further separated into genes down-regulated with morpholino treatment and cycloheximide (1°) or only down-regulated without cycloheximide (2°). p Values are from Kruskal-Wallis tests on summed signal per TSS. (K) Cumulative distributions of distance from a Pou5f3/Sox3-bound regulatory element for genes strongly (≥8 fold) and less strongly (<8 fold) down-regulated in morpholino-treated embryos with or without cycloheximide, compared to up-regulated, unaffected and unactivated genes. p Value is from a Kruskal-Wallis test. (L) Maps showing density of Pou5f3/Sox3-bound regulatory elements around paired homeologous TSSs, divided into elements with differential homeologous L & S binding (left panels) versus both bound (right panels). TSSs are grouped according to L versus S homeolog sensitivity to morpholino treatment. (M) Browser tracks showing CUT&RUN enrichment and ATAC-seq coverage near active homeolog hes3.L and inactive homeolog hes3.S. Seven L-specific high-confidence regulatory regions are highlighted with their homeologous S regions (bold 'L'), as well as two lower-confidence enhancers, one of which also has weak activity in S, but minimal Pou5f3 or Sox3 binding (labeled 'LS'). To further clarify the regulatory network, we also performed morpholino treatments followed by cycloheximide treatment at stage 8, collecting at stage 9 for RNA-seq, to focus the loss of function on primary activation. In these embryos, nearly 70% of first-wave genes were down regulated, including the mir-427 transcript (Figure 4A–C, Figure 4—figure supplement 1B–F, I), suggesting that maternal Pou5f3 and Sox3 directly activate a large proportion of first-wave genome activation, but newly synthesized zygotic factors rapidly mobilize to refine target gene expression levels (Figure 4H). In the absence of wild-type Pou5f3 and Sox3 activity, divergent homeolog activation is reduced for a subset of genes, indicating that these factors at least partially underlie differential subgenome activation (Figure 4D–G). Among primary-activated genes, there does not seem to be a strong bias toward greater regulation of either homeolog; however, a significantly larger proportion of strictly zygotic genes encoded on both subgenomes is activated by Pou5f3 and Sox3 compared to singleton genes (p=0.0020, χ-squared test, 5 d.o.f.; Figure 4—figure supplement 1M, N), which may reflect a reliance on these factors to mediate homeolog-specific expression when two copies exist. To interrogate Pou5f3 and Sox3 chromatin binding across the subgenomes, we performed CUT&RUN on stage 8 embryos injected with mRNA encoding V5 epitope-tagged pou5f3.3.L and sox3.S. Peak calling revealed thousands of binding sites for each factor

Natriuretic peptide signaling has been implicated in a broad range of physiological processes, regulating blood volume and pressure, ventricular hypertrophy, fat metabolism, and long bone growth. Here, we describe a completely novel role for natriuretic peptide signaling in the control of neural crest (NC) and cranial placode (CP) progenitors formation. Among the components of this signaling pathway, we show that natriuretic peptide receptor 3 (Npr3) plays a pivotal role by differentially regulating two developmental programs through its dual function as clearance and signaling receptor. Using a combination of MO-based knockdowns, pharmacological inhibitors and rescue assays we demonstrate that Npr3 cooperate with guanylate cyclase natriuretic peptide receptor 1 (Npr1) and natriuretic peptides (Nppa/Nppc) to regulate NC and CP formation, pointing at a broad requirement of this signaling pathway in early embryogenesis. We propose that Npr3 acts as a clearance receptor to regulate local concentrations of natriuretic peptides for optimal cGMP production through Npr1 activation, and as a signaling receptor to control cAMP levels through inhibition of adenylyl cyclase. The intracellular modulation of these second messengers therefore participates in the segregation of NC and CP cell populations.

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

Neural crest cells are central to vertebrate development and evolution, endowing vertebrates with a “new head” that resulted in morphological, physiological, and behavioral features that allowed vertebrates to become active predators. One remarkable feature of neural crest cells is their multi-germ layer potential that allows for the formation of both ectodermal (pigmentation, peripheral glia, sensory neurons) and mesenchymal (connective tissue, cartilage/bone, dermis) cell types. Understanding the cellular and evolutionary origins of this broad cellular potential in the neural crest has been a long-standing focus for developmental biologists. Here, we review recent work that has demonstrated that neural crest cells share key features with pluripotent blastula stem cells, including expression of the Yamanaka stem cell factors (Oct3/4, Klf4, Sox2, c-Myc). These shared features suggest that pluripotency is either retained in the neural crest from blastula stages or subsequently reactivated as the neural crest forms. We highlight the cellular and molecular parallels between blastula stem cells and neural crest cells and discuss the work that has led to current models for the cellular origins of broad potential in the crest. Finally, we explore how these themes can provide new insights into how and when neural crest cells and pluripotency evolved in vertebrates and the evolutionary relationship between these populations.