Onecut is a direct neural-specific transcriptional activator of Rx in Ciona intestinalis

Abstract

Retinal homeobox (Rx) genes play a crucial and conserved role in the development of the anterior neural plate of metazoans. During chordate evolution, they have also acquired a novel function in the control of eye formation and neurogenesis. To characterize the Rx genetic cascade and shed light on the mechanisms that led to the acquisition of this new role in eye development, we studied Rx transcriptional regulation using the ascidian, Ciona intestinalis. Through deletion analysis of the Ci-Rx promoter, we have identified two distinct enhancer elements able to induce Ci-Rx specific expression in the anterior part of the CNS and in the photosensory organ at tailbud and larva stages. Bioinformatic analysis highlighted the presence of two Onecut binding sites contained in these enhancers, so we explored the role of this transcription factor in the regulation of Ci-Rx. By in situ hybridization, we first confirmed that these genes are co-expressed in the same cells. Through a series of in vivo and in vitro experiments, we then demonstrated that the two Onecut sites are responsible for enhancer activation in Ci-Rx endogenous territories. We also demonstrated in vivo that Onecut misexpression is able to induce ectopic activation of the Rx promoter. Finally, we demonstrated that Ci-Onecut is able to promote Ci-Rx expression in the sensory vesicle. Together, these results support the conclusion that in Ciona embryogenesis, Ci-Rx expression is under the control of the Onecut transcription factor and that this factor is necessary and sufficient to specifically activate Ci-Rx through two enhancer elements.

The central nervous system of ascidian larvae

“…The cell lineage of the photoreceptors in Ciona thus requires clarification. Specification of the ocellus‐associated photoreceptor lineages involves a Neurogenin ‐to‐ Onecut ‐to‐ Rx genetic cascade with Rx then activating Arrestin and Opsin1 gene expression . Nodal expression on the left side of the larvae represses Rx gene expression (and thus Arrestin and Opsin1 ), restricting photoreceptor cell formation to only the right side (Figure (b)).…”

A cis-regulatory change underlying the motor neuron-specific loss of terminal selector gene expression in immotile tunicate larvae

“…To survey CNS development in M. occulta embryos, we performed whole-mount mRNA in situ hybridizations for genes that have been well characterized during CNS development in other tunicates with swimming larvae, notably Ciona spp., Halocynthia roretzi, and Molgula occidentalis. The first candidates we analyzed were Celf3/4/5 (also known as ETR-1) Onecut, and Neurogenin, all relatively broad markers of neuronal fate in swimming tunicate larvae (Figure 2A-C) (D'Aniello et al, 2011;Imai et al, 2009;Lowe and Stolfi, 2018;Satou et al, 2001;Yagi and Makabe, 2001). Their mRNA in situ patterns clearly revealed the developing embryonic neural tube around the tailbud stage (~7.5-9.5 hours post-fertilization, hpf).…”

Regulatory elements retained during chordate evolution: Coming across tunicates

“…C. intestinalis transgenic embryos were obtained via electroporation experiments as previously described (D'Aniello et al ., ). LacZ staining of transgenic embryos was observed by histochemical detection of β‐galactosidase activity.…”