Pluripotency of a founding field: rebranding developmental biology.

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.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

The atypical RNA-binding protein Taf15 regulates dorsoanterior neural development through diverse mechanisms in Xenopus tropicalis.

The FET family of atypical RNA-binding proteins includes Fused in sarcoma (FUS), Ewing's sarcoma (EWS) and the TATA-binding protein-associate factor 15 (TAF15). FET proteins are highly conserved, suggesting specialized requirements for each protein. Fus regulates splicing of transcripts required for mesoderm differentiation and cell adhesion in Xenopus, but the roles of Ews and Taf15 remain unknown. Here, we analyze the roles of maternally deposited and zygotically transcribed Taf15, which is essential for the correct development of dorsoanterior neural tissues. By measuring changes in exon usage and transcript abundance from Taf15-depleted embryos, we found that Taf15 may regulate dorsoanterior neural development through fgfr4 and ventx2.1. Taf15 uses distinct mechanisms to downregulate Fgfr4 expression, namely retention of a single intron within fgfr4 when maternal and zygotic Taf15 is depleted, and reduction in the total fgfr4 transcript when zygotic Taf15 alone is depleted. The two mechanisms of gene regulation (post-transcriptional versus transcriptional) suggest that Taf15-mediated gene regulation is target and co-factor dependent, contingent on the milieu of factors that are present at different stages of development.

The neurodevelopmental disorder risk gene DYRK1A is required for ciliogenesis and control of brain size in Xenopus embryos.

DYRK1A [dual specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 A] is a high-confidence autism risk gene that encodes a conserved kinase. In addition to autism, individuals with putative loss-of-function variants in DYRK1A exhibit microcephaly, intellectual disability, developmental delay and/or congenital anomalies of the kidney and urinary tract. DYRK1A is also located within the critical region for Down syndrome; therefore, understanding the role of DYRK1A in brain development is crucial for understanding the pathobiology of multiple developmental disorders. To characterize the function of this gene, we used the diploid frog Xenopus tropicalis We discover that Dyrk1a is expressed in ciliated tissues, localizes to ciliary axonemes and basal bodies, and is required for ciliogenesis. We also demonstrate that Dyrk1a localizes to mitotic spindles and that its inhibition leads to decreased forebrain size, abnormal cell cycle progression and cell death during brain development. These findings provide hypotheses about potential mechanisms of pathobiology and underscore the utility of X. tropicalis as a model system for understanding neurodevelopmental disorders.

A protein scaffold plays matchmaker for chordin.

In this issue, Inomata et al. (2008) report that the scaffold protein Olfactomedin 1 (ONT1) recruits the Tolloid proteases to their substrate Chordin, an antagonist of bone morphogenetic proteins (BMPs), during development of the frog embryo. Consequently, ONT1 expression in the organizer of the late gastrula stabilizes the gradient of BMP signaling that is essential for dorsoventral patterning.

Integration of Wnt and FGF signaling in the Xenopus gastrula at TCF and Ets binding sites shows the importance of short-range repression by TCF in patterning the marginal zone.

During Xenopus gastrulation, Wnt and FGF signaling pathways cooperate to induce posterior structures. Wnt target expression around the blastopore falls into two main categories: a horseshoe shape with a dorsal gap, as in Wnt8 expression; or a ring, as in FGF8 expression. Using ChIP-seq, we show, surprisingly, that the FGF signaling mediator Ets2 binds near all Wnt target genes. However, ß-catenin preferentially binds at the promoters of genes with horseshoe patterns, but further from the promoters of genes with ring patterns. Manipulation of FGF or Wnt signaling demonstrated that 'ring' genes are responsive to FGF signaling at the dorsal midline, whereas 'horseshoe' genes are predominantly regulated by Wnt signaling. We suggest that, in the absence of active ß-catenin at the dorsal midline, the DNA-binding protein TCF binds and actively represses gene activity only when close to the promoter. In contrast, genes without functional TCF sites at the promoter may be predominantly regulated by Ets at the dorsal midline and are expressed in a ring. These results suggest recruitment of only short-range repressors to potential Wnt targets in the Xenopus gastrula.

New insights into Xenopus sex chromosome genomics from the Marsabit clawed frog X. borealis.

In many groups, sex chromosomes change frequently but the drivers of their rapid evolution are varied and often poorly characterized. With an aim of further understanding sex chromosome turnover, we investigated the polymorphic sex chromosomes of the Marsabit clawed frog, Xenopus borealis, using genomic data and a new chromosome-scale genome assembly. We confirmed previous findings that 54.1 Mb of chromosome 8L is sex-linked in animals from east Kenya and a laboratory strain, but most (or all) of this region is not sex-linked in natural populations from west Kenya. Previous work suggests possible degeneration of the Z chromosomes in the east population because many sex-linked transcripts of this female heterogametic population have female-biased expression, and we therefore expected this chromosome to not be present in the west population. In contrast, our simulations support a model where most or all of the sex-linked portion of the Z chromosome from the east acquired autosomal segregation in the west, and where much genetic variation specific to the large sex-linked portion of the W chromosome from the east is not present in the west. These recent changes are consistent with the hot-potato model, wherein sex chromosome turnover is favoured by natural selection if it purges a (minimally) degenerate sex-specific sex chromosome, but counterintuitively suggest natural selection failed to purge a Z chromosome that has signs of more advanced and possibly more ancient regulatory degeneration. These findings highlight complex evolutionary dynamics of young, rapidly evolving Xenopus sex chromosomes and set the stage for mechanistic work aimed at pinpointing additional sex-determining genes in this group.

A chromosome-scale genome assembly and dense genetic map for Xenopus tropicalis.

The Western clawed frog Xenopus tropicalis is a diploid model system for both frog genetics and developmental biology, complementary to the paleotetraploid X. laevis. Here we report a chromosome-scale assembly of the X. tropicalis genome, improving the previously published draft genome assembly through the use of new assembly algorithms, additional sequence data, and the addition of a dense genetic map. The improved genome enables the mapping of specific traits (e.g., the sex locus or Mendelian mutants) and the characterization of chromosome-scale synteny with other tetrapods. We also report an improved annotation of the genome that integrates deep transcriptome sequence from diverse tissues and stages. The exon-intron structures of these genes are highly conserved relative to both X. laevis and human, as are chromosomal linkages ("synteny") and local gene order. A network analysis of developmental gene expression will aid future studies.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

miR-34/449 miRNAs are required for motile ciliogenesis by repressing cp110.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

Alternative polyadenylation coordinates embryonic development, sexual dimorphism and longitudinal growth in Xenopus tropicalis.

RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.

Deletion of the sclerotome-enriched lncRNA PEAT augments ribosomal protein expression.

To define a complete catalog of the genes that are activated during mouse sclerotome formation, we sequenced RNA from embryonic mouse tissue directed to form sclerotome in culture. In addition to well-known early markers of sclerotome, such as Pax1, Pax9, and the Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 enhancer antisense transcript) was induced in sclerotome-directed samples. Strikingly, PEAT is located just upstream of the Pax1 gene. Using CRISPR/Cas9, we generated a mouse line bearing a complete deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant embryos showed that loss of PEAT modestly increases bone morphogenetic protein target gene expression and also elevates the expression of a large subset of ribosomal protein mRNAs.

fus/TLS orchestrates splicing of developmental regulators during gastrulation.

Here we investigated the function of the atypical RNA-binding protein fus/TLS (fused in sarcoma/translocated in sarcoma) during early frog development. We found that fus is necessary for proper mRNA splicing of a set of developmental regulatory genes during early frog development and gastrulation. Upon fus knockdown, embryos fail to gastrulate and show mesodermal differentiation defects that we connect to intron retention in fgf8 (fibroblast growth factor 8) and fgfr2 (fgf receptor 2) transcripts. During gastrulation, the animal and marginal regions dissociate, and we show that this is caused, at least in part, by intron retention in cdh1 transcripts. We confirm the specificity of splicing defects at a genomic level using analysis of RNA sequencing (RNA-seq) and show that 3%-5% of all transcripts display intron retention throughout the pre-mRNA. By analyzing gene ontology slim annotations, we show that the affected genes are enriched for developmental regulators and therefore represent a biologically coherent set of targets for fus regulation in embryogenesis. This shows that fus is central to embryogenesis and may provide information on its function in neurodegenerative disease.

Katanin-like protein Katnal2 is required for ciliogenesis and brain development in Xenopus embryos.

Microtubule remodeling is critical for cellular and developmental processes underlying morphogenetic changes and for the formation of many subcellular structures. Katanins are conserved microtubule severing enzymes that are essential for spindle assembly, ciliogenesis, cell division, and cellular motility. We have recently shown that a related protein, Katanin-like 2 (KATNAL2), is similarly required for cytokinesis, cell cycle progression, and ciliogenesis in cultured mouse cells. However, its developmental expression pattern, localization, and in vivo role during organogenesis have yet to be characterized. Here, we used Xenopus embryos to reveal that Katnal2 (1) is expressed broadly in ciliated and neurogenic tissues throughout embryonic development; (2) is localized to basal bodies, ciliary axonemes, centrioles, and mitotic spindles; and (3) is required for ciliogenesis and brain development. Since human KATNAL2 is a risk gene for autism spectrum disorders, our functional data suggest that Xenopus may be a relevant system for understanding the relationship of mutations in this gene to autism and the underlying molecular mechanisms of pathogenesis.

Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis.

The Wnt signaling pathway is crucial for tissue morphogenesis, participating in cellular behavior changes, notably during the process of convergent-extension. Interactions between Wnt-secreting and receiving cells during convergent-extension remain elusive. We investigated the role and genetic interactions of Wnt ligands and their trafficking factors Wls, Gpc4 and Frzb in the context of palate morphogenesis in zebrafish. We describe that the chaperon Wls and its ligands Wnt9a and Wnt5b are expressed in the ectoderm, whereas juxtaposed chondrocytes express Frzb and Gpc4. Using wls, gpc4, frzb, wnt9a and wnt5b mutants, we genetically dissected the Wnt signals operating between secreting ectoderm and receiving chondrocytes. Our analysis delineates that non-canonical Wnt signaling is required for cell intercalation, and that wnt5b and wnt9a are required for palate extension in the anteroposterior and transverse axes, respectively.

Genome-wide identification of Wnt/ß-catenin transcriptional targets during Xenopus gastrulation.

The canonical Wnt/ß-catenin signaling pathway plays multiple roles during Xenopus gastrulation, including posteriorization of the neural plate, patterning of the mesoderm, and induction of the neural crest. Wnt signaling stabilizes ß-catenin, which then activates target genes. However, few targets of this signaling pathway that mediate early developmental processes are known. Here we sought to identify transcriptional targets of the Wnt/ß-catenin signaling pathway using a genome-wide approach. We selected putative targets using the criteria of reduced expression upon zygotic Wnt knockdown, ß-catenin binding within 50kb of the gene, and expression in tissues that receive Wnt signaling. Using these criteria, we found 21 novel direct transcriptional targets of Wnt/ß-catenin signaling during gastrulation and in addition have identified putative regulatory elements for further characterization in future studies.

sall1 and sall4 repress pou5f3 family expression to allow neural patterning, differentiation, and morphogenesis in Xenopus laevis.

The embryonic precursor of the vertebrate central nervous system, the neural plate, is patterned along the anterior-posterior axis and shaped by morphogenetic movements early in development. We previously identified the genes sall1 and sall4, known regulators of pluripotency in other contexts, as transcriptional targets of developmental signaling pathways that regulate neural development. Here, we demonstrate that these two genes are required for induction of posterior neural fates, the cell shape changes that contribute to neural tube closure, and later neurogenesis. Upon sall1 or sall4 knockdown, defects are associated with the failure of the neural plate to differentiate. Consistent with this, sall-deficient neural tissue exhibits an aberrant upregulation of pou5f3 family genes, the Xenopus homologs of the mammalian stem cell maintenance factor Pou5f1 (Oct4). Furthermore, overexpression of pou5f3 genes in Xenopus causes defects in neural patterning, morphogenesis, and differentiation that phenocopy those observed in sall1 and sall4 morphants. In all, this work shows that both sall1 and sall4 act to repress pou5f3 family gene expression in the neural plate, thereby allowing vertebrate neural development to proceed.

Noggin is required for first pharyngeal arch differentiation in the frog Xenopus tropicalis.

The dorsal ventral axis of vertebrates requires high BMP activity for ventral development and inhibition of BMP activity for dorsal development. Presumptive dorsal regions of the embryo are protected from the ventralizing activity of BMPs by the secretion of BMP antagonists from the mesoderm. Noggin, one such antagonist, binds BMP ligands and prevents them from binding their receptors, however, a unique role for Noggin in amphibian development has remained unclear. Previously, we used zinc-finger nucleases to mutagenize the noggin locus in Xenopus tropicalis. Here, we report on the phenotype of noggin mutant frogs as a result of breeding null mutations to homozygosity. Early homozygous noggin mutant embryos are indistinguishable from wildtype siblings, with normal neural induction and neural tube closure. However, in late tadpole stages mutants present severe ventral craniofacial defects, notably a fusion of Meckel's cartilage to the palatoquadrate cartilage. Consistent with a noggin loss-of-function, mutants show expansions of BMP target gene expression and the mutant phenotype can be rescued with transient BMP inhibition. These results demonstrate that in amphibians, Noggin is dispensable for early embryonic patterning but is critical for cranial skeletogenesis.

Precise spatial restriction of BMP signaling is essential for articular cartilage differentiation.

The articular cartilage, which lines the joints of the limb skeleton, is distinct from the adjoining transient cartilage, and yet, it differentiates as a unique population within a contiguous cartilage element. Current literature suggests that articular cartilage and transient cartilage originate from different cell populations. Using a combination of lineage tracing and pulse-chase of actively proliferating chondrocytes, we here demonstrate that, similar to transient cartilage, embryonic articular cartilage cells also originate from the proliferating chondrocytes situated near the distal ends of skeletal anlagen. We show that nascent cartilage cells are capable of differentiating as articular or transient cartilage, depending on exposure to Wnt or BMP signaling, respectively. The spatial organization of the articular cartilage results from a band of Nog-expressing cells, which insulates these proliferating chondrocytes from BMP signaling and allows them to differentiate as articular cartilage under the influence of Wnt signaling emanating from the interzone. Through experiments conducted in both chick and mouse embryos we have developed a model explaining simultaneous growth and differentiation of transient and articular cartilage in juxtaposed domains.