Generation of high throughput transduction tools and evaluation of the role od BMP2/4 in late morphogenesis in sea urchin embryos

Abstract

The sea urchin embryo has recently emerged as a powerful model organism for systems-level studies aimed at understanding developmental complexity; however, the tools available for high throughput transgenic approaches are limited. Toward that end, the goal of part one of this study was to engineer pantropic retroviruses (PRVs) for genetic perturbations of urchin embryos at a scale suitable for systems-level measures. To insert sea urchin enhancers and simultaneously disrupt the endogenous viral enhancer, we used a viral self-inactivation (SIN) strategy with the Otx cis-regulatory module 3 (CRM3) enhancer, which drives ubiquitous and maternal-plus-zygotic expression in sea urchin embryos. We found that such PRVs are genomically integrated into sea urchin zygotes at a copy number of one, and that Otx CRM3-driven transgene expression initiates in the 2-cell stage, and is global and persistent. To transduce bicistronic messages, we validated the cricket paralysis virus (CrPV) internal ribosomal entry site (IRES) in sea urchins, and successfully developed SIN PRVs that express CrPV IRES-dependent bicistronic messages. Finally, we utilized Otx CRM3 to successfully drive functional antisense (AS) sequences for bone morphogenetic protein 2/4 (BMP2/4) or Delta; embryos transduced with these AS-expressing viruses exhibit the expected phenotypes, validating this approach. Together these results establish a foundation for SIN PRVs as a tool for studying sea urchin development. Part two of this study focuses on the role of BMP2/4 signaling in sea urchin development. BMP2/4 is required for dorsal specification, and our early study showed that Chordin, a dedicated BMP antagonist, inhibits BMP2/4 at the dorsal-ventral (DV) boundary, promoting "peripheral" neurogenesis in that territory. Our more recent efforts utilized blastomere injections to generate embryos in which BMP2/4 was blocked in half of the embryo, which results in expansion of the ventral territory, but overall retention of DV patterning. The results demonstrate that BMP2/4 signals via SMAD1/5/8 to induce the morphogenesis of the oral hood. This structure develops from the apical plate, and BMP2/4 is not required for apical specification, indicating that BMP2/4 is required downstream of that early step. The apical plate also gives rise to the "central" serotonergic neurons, and our results indicate that BMP2/4 is directly required for serotonergic neural development.