Description
The formation of epidermal sensory neurons (ESNs) in the ascidian peripheral nervous system is dependent on coordinated interactions of transcription factors (TFs) encoded in a proneural gene regulatory network. The first ESN-specific TF in the network is Pou4. Pou4 genes are involved in sensory neuron differentiation throughout chordate evolution, including sensory hair cells in the vertebrate inner ear. In ascidians, Pou4 overexpression can convert the entire epidermis into ESNs. To understand the regulation and evolution of Pou4, we need to understand its cis-regulatory modules (CRMs). CRMs are composed of clustered, combinatorial binding sites for TFs to direct spatiotemporal expression of a gene. In the ascidians studied here, Ciona robusta, Ciona savignyi, Ascidia species and Ascidia zara, sequence alignments revealed deep conservation within each genus and highlighted clusters of conserved sequence in the non-coding region ~1.8kb 5’ of the start codon through the first intron (5’CRM-Intron) and in the ~3.5kb region 3’ of the stop codon (3’CRM). Electroporation allows for the expression of CRMs linked to a fluorescent reporter to produce thousands of transgenic embryos and optimizing transgenic technology to be used in all four species allowed for a comparative study of Pou4 CRM expression. Across genera, sequence conservation outside of the coding region decreased significantly. However, transgenes containing the CRMs from each species produced spatiotemporal expression indistinguishable from endogenous gene expression when electroporated in the opposite species, indicating that the changes in sequence had little impact on expression. Individual electroporation of the 5’CRM-Intron and 3’CRM from each species suggested a shift in the location of TF binding sites required for ESN specificity over evolutionary time. Expression of the 5’ CRM-Intron of A. species alone was sufficient to recapitulate ESN-specificity of Pou4, similar to a 5’ CRM region of Pou4f3 in mouse sensory hair cells. Contrastingly, expression of the 3’CRM of the other three species was ESN-specific while the 5’CRM-Intron was not. Our findings suggest that a comparative study of the regulatory mechanisms of proneural genes like Pou4 between Ascidia and Ciona provides us with a unique opportunity to further understand the evolution and development of sensory neurons in chordates.
