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.

Xenopus leads the way: Frogs as a pioneering model to understand the human brain.

From its long history in the field of embryology to its recent advances in genetics, Xenopus has been an indispensable model for understanding the human brain. Foundational studies that gave us our first insights into major embryonic patterning events serve as a crucial backdrop for newer avenues of investigation into organogenesis and organ function. The vast array of tools available in Xenopus laevis and Xenopus tropicalis allows interrogation of developmental phenomena at all levels, from the molecular to the behavioral, and the application of CRISPR technology has enabled the investigation of human disorder risk genes in a higher-throughput manner. As the only major tetrapod model in which all developmental stages are easily manipulated and observed, frogs provide the unique opportunity to study organ development from the earliest stages. All of these features make Xenopus a premier model for studying the development of the brain, a notoriously complex process that demands an understanding of all stages from fertilization to organogenesis and beyond. Importantly, core processes of brain development are conserved between Xenopus and human, underlining the advantages of this model. This review begins by summarizing discoveries made in amphibians that form the cornerstones of vertebrate neurodevelopmental biology and goes on to discuss recent advances that have catapulted our understanding of brain development in Xenopus and in relation to human development and disease. As we engage in a new era of patient-driven gene discovery, Xenopus offers exceptional potential to uncover conserved biology underlying human brain disorders and move towards rational drug design.

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.

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.

Autism gene variants disrupt enteric neuron migration and cause gastrointestinal dysmotility.

The comorbidity of autism spectrum disorders and severe gastrointestinal symptoms is well-established, yet the molecular underpinnings remain unknown. The identification of high-confidence large-effect autism risk genes offers the opportunity to identify convergent, underlying biology by studying these genes in the context of the gastrointestinal system. Here we show that the expression of these genes is enriched in human prenatal gut neurons as well as their migratory progenitors, suggesting that the development and/or function of these neurons may be disrupted by autism-associated pathogenic variants, leading to gastrointestinal dysfunction. Here we document the prevalence of gastrointestinal issues in patients with large-effect variants in sixteen of these genes, highlighting dysmotility, consistent with potential enteric neuron dysfunction. Using the high-throughput diploid frog Xenopus tropicalis , we individually target five of these genes ( SYNGAP1, CHD8, SCN2A, CHD2 , and DYRK1A ) and observe disrupted enteric neuronal progenitor migration for each. More extensive analysis of DYRK1A reveals that perturbation causes gut dysmotility in vivo , which can be ameliorated by treatment with a selective serotonin reuptake inhibitor (escitalopram) or a serotonin receptor 6 agonist, identified by in vivo drug screening. This work suggests that atypical development of enteric neurons contributes to the gastrointestinal distress commonly seen in individuals with autism and that increasing serotonin signaling may be a productive therapeutic avenue.

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience.

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.