Pou4 Function in an Invertebrate Chordate

San Diego State University Foundation, San Diego CA

Investigators

Abstract

Transcription factors (TF) are proteins that control the expression of genes. This project uses a simple animal that is closely related to vertebrates like ourselves, the ascidian, to study a TF called Pou4 that is important for the development of sensory neurons, which include the hair cells of the inner ear. It was previously found that expressing ascidian Pou4 in ascidian cell types where it is not usually found converts those cells into sensory neurons. Likewise, forced expression of any of the three mouse Pou4 genes in the wrong ascidian cell types also converts those cells into sensory neurons. However, in mice, expressing Pou4 in the wrong cell type does not convert those cells into sensory neurons. The present project examines the mechanistic basis for the differences between the ascidian and mouse Pou4 genes in order to better understand evolutionary changes in development that give rise to new cell types like vertebrate sensory neurons. To answer these questions, the researchers are adapting a new gene editing system (CRISPR/Cas9) to the ascidian to decipher the mechanisms behind Pou4 function. Broader Impacts: San Diego State University ranks among the top US universities for economic and campus ethnic diversity. The proposed study will provide laboratory research opportunities for undergraduate and graduate students. To broaden the overall understanding of the function and experimental use of transgenic organisms to SDSU's larger undergraduate population, the researchers are working with the coordinator of their introductory cell and molecular biology courses to develop a relatively simple, "hands on" exercise where students can generate and assay transgenic ascidian embryos. In ascidians, the single Pou4 (CiPou4) transcription factor (TF) is a key regular of the PNS, and ectopic expression of this gene produces ectopic sensory neurons. In mammals, there are three Pou4s; all predominately expressed in sensory neurons. The investigators used RNA-SEQ to identify CiPou4 transcriptional targets and many are known to be expressed in vertebrate hair cells, suggesting a deep evolutionary linkage between these cells types. In mammals, ectopic expression of the hair cell Pou4F3 gene does not produce new hair cells; but when expressed in ascidians ectopic sensory neurons are generated. This observation suggests that while protein function is conserved, Pou4 transcriptional targets have been lost and/or are differentially regulated in vertebrates. Here we will use RNA-SEQ to determine if the mouse Pou4 TFs activate similar transcriptional targets compared to CiPou4. We will test if a CRISPR/Cas9-mediated CiPou4 knock-out can be rescued by the mouse Pou4 genes. This project is transformative because the ascidian larva provides a unique system to examine the evolution of TF function and the role this TF has played during the evolution of a novel vertebrate cell type, the sensory hair cell.