BackgroundThe mechanisms by which the conserved genetic “toolkit” for development generates phenotypic disparity across metazoans is poorly understood. Echinoderm larvae provide a great resource for understanding how developmental novelty arises. The sea urchin pluteus larva is dramatically different from basal echinoderm larval types, which include the auricularia-type larva of its sister taxon, the sea cucumbers, and the sea star bipinnaria larva. In particular, the pluteus has a mesodermally-derived larval skeleton that is not present in sea star larvae or any outgroup taxa. To understand the evolutionary origin of this structure, we examined the molecular development of mesoderm in the sea cucumber, Parastichopus parvimensis.ResultsBy comparing gene expression in sea urchins, sea cucumbers and sea stars, we partially reconstructed the mesodermal regulatory state of the echinoderm ancestor. Surprisingly, we also identified expression of the transcription factor alx1 in a cryptic skeletogenic mesenchyme lineage in P. parvimensis. Orthologs of alx1 are expressed exclusively within the sea urchin skeletogenic mesenchyme, but are not expressed in the mesenchyme of the sea star, which suggests that alx1+ mesenchyme is a synapomorphy of at least sea urchins and sea cucumbers. Perturbation of Alx1 demonstrates that this protein is necessary for the formation of the sea cucumber spicule. Overexpression of the sea star alx1 ortholog in sea urchins is sufficient to induce additional skeleton, indicating that the Alx1 protein has not evolved a new function during the evolution of the larval skeleton.ConclusionsThe proposed echinoderm ancestral mesoderm state is highly conserved between the morphologically similar, but evolutionarily distant, auricularia and bipinnaria larvae. However, the auricularia, but not bipinnaria, also develops a simple skelotogenic cell lineage. Our data indicate that the first step in acquiring these novel cell fates was to re-specify the ancestral mesoderm into molecularly distinct territories. These new territories likely consisted of only a few cells with few regulatory differences from the ancestral state, thereby leaving the remaining mesoderm to retain its original function. The new territories were then free to take on a new fate. Partitioning of existing gene networks was a necessary pre-requisite to establish novelty in this system.

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 was 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 function.

No abstract

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.

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, patients 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.