The initial tube-like shape of the vertebrate brain primordium is generated when the flat neural plate folds to form the neural tube. An essential step in this process, termed neurulation, is formation of dorsolateral hinge points (DLHPs). While higher vertebrate DLHP formation is well established, we were the first to show that DLHPs are also formed in teleosts. We showed that the zebrafish zic2a expression pattern predicts the location of future DLHPs (Nyholm et al., 2007) and that zic2a function is required for DLHP formation through regulation of apical actomyosin contraction and of apical junction integrity (Nyholm et al., 2009). Our studies revealed a level of deep conservation of neurulation mechanisms operating in higher and lower vertebrates, since actomyosin contraction is required for DLHP formation in higher vertebrates.
Brain ventricle formation, another key aspect of neural tube morphogenesis, depends on ciliary motility. To interrogate the underlying genetic mechanisms, which are poorly understood, we have targeted two genes with CRISPR-mediated mutagenesis. Our first gene of interest, rsph9, encodes a structural component of ciliary radial spokes found in motile cilia and is causally linked to occurrence of primary ciliary dyskinesia (PCD) in humans. Using CRISPR-generated mutant alleles of rsph9, we demonstrated that Rsph9 protein is required for structural integrity and function of motile cilia in the developing zebrafish brain primordium. Remarkably, structural deficits in zebrafish rsph9 mutants closely resemble those in respiratory epithelia of human PCD patients, suggesting utility of zebrafish for PCD modeling (Sedykh et al., 2016).
Our second gene of interest, rfx4, is a member of a conserved transcription factor gene family with critical roles in ciliogenesis. Analysis of CRISPR-induced rfx4 mutant zebrafish has identified requirements for Rfx4 function in both the ventral (floor plate) and dorsal (roof plate) midlines of the brain primordium. Rfx4 function is required to activate transcription of zic2a and zic2b, among others. Notably, our analysis suggests that Rfx4 does not play a significant role in primary ciliogenesis (Sedykh et al., submitted).
Zic2, a transcription factor gene essential for normal development of the human forebrain, has two homologs in zebrafish, zic2a and zic2b. We have shown that zic2a and zic2b are required for forebrain development in zebrafish (Sanek and Grinblat, 2008; Sedykh et al., 2017). Using zebrafish as a model organism, we have gained important novel insights into the mechanism of zic2 function the developing forebrain.
We identified a novel negative feedback loop that modulates activity of an essential signaling pathway (Hh) and showed that zic2a function is required for this loop to operate properly (Sanek et al. 2009). We also uncovered a novel role for the Zic2/Hh interaction during retinal morphogenesis (Sanek et al., 2009; Sedykh et al., 2017) and identified roles for zic2a and zic2b in craniofacial skeleton development (TeSlaa et al., 2013; Sedykh et al., 2017). RNAseq-based transcriptome analysis of Zic2 mutant zebrafish has generated a set of candidate transcriptional targets of Zic2; functional analysis of these targets is our current focus.
Ultimately, this work will aid in understanding the complex etiology of birth defects that affect human craniofacial complex development: holoprosencephaly, coloboma and frontonasal dysplasia.