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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 rfx4 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 rfx4 locus using CRISPR/Cas9 mutagenesis. Using RNAseq-based transcriptome analysis, in situ hybridization and immunostaining we identified a requirement for zebrafish rfx4 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 foxa2 in the ventral hindbrain and spinal cord (floor plate)., Conclusions: The midline patterning functions of rfx4 are conserved, because rfx4 regulates transcription of foxa2 and zic2 in zebrafish and in mouse. In contrast, zebrafish rfx4 function is dispensable for forebrain morphogenesis, while mouse rfx4 is required for normal formation of forebrain ventricles in a ciliogenesis-dependent manner. Collectively, this report identifies conserved aspects of rfx4 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., (© 2017 Wiley Periodicals, Inc.)

The vertebrate retina develops in close proximity to the forebrain and neural crest-derived cartilages of the face and jaw. Coloboma, a congenital eye malformation, is associated with aberrant forebrain development (holoprosencephaly) and with craniofacial defects (frontonasal dysplasia) in humans, suggesting a critical role for cross-lineage interactions during retinal morphogenesis. ZIC2, a zinc-finger transcription factor, is linked to human holoprosencephaly. We have previously used morpholino assays to show zebrafish zic2 functions in the developing forebrain, retina and craniofacial cartilage. We now report that zebrafish with genetic lesions in zebrafish zic2 orthologs, zic2a and zic2b, develop with retinal coloboma and craniofacial anomalies. We demonstrate a requirement for zic2 in restricting pax2a expression and show evidence that zic2 function limits Hh signaling. RNA-seq transcriptome analysis identified an early requirement for zic2 in periocular neural crest as an activator of alx1, a transcription factor with essential roles in craniofacial and ocular morphogenesis in human and zebrafish. Collectively, these data establish zic2 mutant zebrafish as a powerful new genetic model for in-depth dissection of cell interactions and genetic controls during craniofacial complex development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cilia are cell surface organelles with key roles in a range of cellular processes, including generation of fluid flow by motile cilia. The axonemes of motile cilia and immotile kinocilia contain 9 peripheral microtubule doublets, a central microtubule pair, and 9 connecting radial spokes. Aberrant radial spoke components RSPH1, 3, 4a and 9 have been linked with primary ciliary dyskinesia (PCD), a disorder characterized by ciliary dysmotility; yet, radial spoke functions remain unclear. Here we show that zebrafish Rsph9 is expressed in cells bearing motile cilia and kinocilia, and localizes to both 9 + 2 and 9 + 0 ciliary axonemes. Using CRISPR mutagenesis, we show that rsph9 is required for motility of presumptive 9 + 2 olfactory cilia and, unexpectedly, 9 + 0 neural cilia. rsph9 is also required for the structural integrity of 9 + 2 and 9 + 0 ciliary axonemes. rsph9 mutant larvae exhibit reduced initiation of the acoustic startle response consistent with hearing impairment, suggesting a novel role for Rsph9 in the kinocilia of the inner ear and/or lateral line neuromasts. These data identify novel roles for Rsph9 in 9 + 0 motile cilia and in sensory kinocilia, and establish a useful zebrafish PCD model.