Functional analysis of Pitx during asexual regeneration in a basal chordate.

Embryogenesis in ascidians is the classic example of mosaic development, yet within this phyla a number of colonial species exist which as adults can reproduce entire bodies asexually. The colonial ascidian Botryllus schlosseri is an excellent model to study this process: on a weekly basis it regenerates all somatic and germline tissues, and while these processes have been characterized morphologically at high-resolution over the last 70 years, almost nothing is known regarding the genetic basis of asexual development and its relationship to embryogenesis. In this study, we functionally characterized the role of the paired-related homeobox transcription factor, Pitx, during this regenerative process. During ascidian embryogenesis Pitx seems to be multifunctional and involved in the formation of multiple tissues, including the stomodeum, pituitary gland, and determination of left-right asymmetry, similar to other deuterostomes. Previous spatial-temporal expression studies during asexual regeneration in Botryllus adults suggest the same roles in this developmental program. Here, we analyzed Pitx function using RNA interference at distinct stages of asexual development. Pitx phenotypes were described focusing on each developmental stage both in vivo, and via histological analysis, and were found to correspond to expression patterns; with the exception that normal asymmetries in the gut were not affected by knockdown. As mRNA destruction is not instantaneous, we found that by tailoring our short interfering double-stranded RNA delivery different developmental processes could be studied independently. This allows a reverse genetic approach to dissect asexual developmental pathways, even in cases involving multifunctional, ubiquitously expressed genes like Pitx.