Abstract
We have previously observed the predominant expression of nucleoporin 62-like (Nup62l) mRNA in the pharyngeal region of zebrafish, which raises the question whether Nup62l has important implications in governing the morphogenesis of pharyngeal arches (PA) in zebrafish. Herein, we explored the functions of Nup62l in PA development. The disruption of Nup62l with a CRISPR/Cas9-dependent gene knockout approach led to defective PA, which was characterized by a thinned and shortened pharyngeal region and a significant loss of pharyngeal cartilages. During pharyngeal cartilage formation, prechondrogenic condensation and chondrogenic differentiation were disrupted in homozygous nup62l-mutants, while the specification and migration of cranial neural crest cells (CNCCs) were unaffected. Mechanistically, the impaired PA region of nup62l-mutants underwent extensive apoptosis, which was mainly dependent on activation of p53-dependent apoptotic pathway. Moreover, aberrant activation of a series of apoptotic pathways in nup62l-mutants is closely associated with the inactivation of Wnt/β-catenin signaling. Thus, these findings suggest that the regulation of Wnt/β-catenin activity by Nup62l is crucial for PA formation in zebrafish.
Highlights
Craniofacial abnormalities are common congenital defects and usually result from the aberrant development of pharyngeal arches (PA) [1]
We have previously shown that nup62l was expressed ubiquitously at early stages and the strongest signals were enriched in some specific organs/tissues at posterior stages, in PA region [37]
DNA sequencing results indicated that homozygous nup62l-mutants harbored an 11-bp insertion within genomic DNA at the target site (Figure 1B; Supplementary Figure S1A), leading to a frame-shift mutation of open reading frame and a premature stop codon that can abolish all functions of nucleoporin 62-like (Nup62l) (Figure S1B)
Summary
Craniofacial abnormalities are common congenital defects and usually result from the aberrant development of pharyngeal arches (PA) [1]. The advantages of high fecundity, transparent embryos, and small size make zebrafish a powerful model for specialized mutagenesis screens for the identification of genes whose counterparts can regulate craniofacial development in humans [10]. Both the ventral arch defects in zebrafish mutations suc/et and cartilage fusions in vgo mutants, as well as the phenotypes of et1-/mice, resemble the phenotypes observed in humans suffering from DiGeorge and velocardiofacial syndromes [11,12]. Conditional inactivation of β-catenin with Wnt1-Cre leads to defective formation of craniofacial structures derived from NCCs [24]. We demonstrated that the aberrant activation of these apoptotic pathways was closely associated with the suppression of Wnt/β-catenin signaling in nup62l-mutants
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