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Description
Throughout chordate evolution, patterns of peripheral nervous system (PNS) development have been largely conserved, however there have been many examples of regulatory sequence rearrangement and divergence. To better understand the evolution of gene regulation during development, sequencing the genomes and transcriptomes of closely related taxa is performed and sequence conservation analyzed. Ascidians, having no whole genome duplication events and small, rapidly evolving genomes, can be excellent natural laboratories for studying the evolution of gene regulation. Here, we have assembled nascent transcriptomes and reference genomes for two ascidian species, Ascidia zara and Ascidia species. The genomes were assembled with three different programs, Meraculous, SOAP de novo, and Discovar; ultimately utilizing the assembly from Discovar as it had the fewest scaffolds and best coverage according to BUSCO analysis. VISTA analysis of the Pou4 loci, a gene required for PNS development, revealed areas of deep conservation as well as interesting differences between the two Ascidia species and across genera with two Ciona species. Additionally, we adapted transgenic technologies to these animals, demonstrating that future functional analysis was possible. The transcriptome, assembled using Trinity, was a key factor in identifying genes expressed in PNS development for in situ hybridization. Several genes could be cloned and we found that Pou4 exhibits similar staining patterns in A. species as in C. robusta. Interestingly, the expression pattern of ETR, a pan-neural marker, was not similar to A. zara or C. robusta, indicating that there may be important differences during PNS development between these species. While conducting immunohistochemistry for acetylated tubulin, we found that A. species and A. zara both have ciliated ESNs, and surprisingly, A. species has an additional, novel cell type that exhibits a curious circular acetylated tubulin staining pattern while DAPI staining reveals condensed, kidney-beanshaped nuclei. Because of the overall lack of pattern and condensed nuclei, we hypothesize that these cells may be neural and migratory in nature. The immunohistochemistry results also led us to search the annotated transcriptomes where, fittingly, we identified a number of migratory genes expressed during the development of A. species that are not expressed in A. zara or C. robusta.
