Zebrafish Rfx4 controls dorsal and ventral midline formation in the neural tube

Abstract

Background: Rfx winged‐helix transcription factors, best known as key regulators of core ciliogenesis, also play ciliogenesis‐independent roles during neural development. Mammalian Rfx4 controls neural tube morphogenesis via both mechanisms. Results: We set out to identify conserved aspects of rfx 4 gene function during vertebrate development and to establish a new genetic model in which to analyze these mechanisms further. To this end, we have generated frame‐shift alleles in the zebrafish rfx 4 locus using CRISPR/Cas9 mutagenesis. Using RNAseq‐based transcriptome analysis, in situ hybridization and immunostaining we identified a requirement for zebrafish rfx 4 in the forming midlines of the caudal neural tube. These functions are mediated, least in part, through transcriptional regulation of several zic genes in the dorsal hindbrain and of foxa 2 in the ventral hindbrain and spinal cord (floor plate). Conclusions: The midline patterning functions of rfx 4 are conserved, because rfx 4 regulates transcription of foxa 2 and zic 2 in zebrafish and in mouse. In contrast, zebrafish rfx 4 function is dispensable for forebrain morphogenesis, while mouse rfx 4 is required for normal formation of forebrain ventricles in a ciliogenesis‐dependent manner. Collectively, this report identifies conserved aspects of rfx 4 function and establishes a robust new genetic model for in‐depth dissection of these mechanisms. Developmental Dynamics 247:650–659, 2018. © 2017 Wiley Periodicals, Inc.