Zebrafishwnt9bSynteny and Expression During First and Second Arch, Heart, and Pectoral Fin Bud Morphogenesis

Abstract

Roles for Wnt9b in craniofacial development are indicated by the cleft lip mutant phenotype observed in the A=WySn mouse strain, 1 caused by a retrotransposon insertion mutation at the Wnt9b locus. Analyses of the zebrafish Wnt9b ortholog, wnt9b, were pursued to provide insight into early vertebrate craniofacial patterning events mediated by Wnt9b signaling. Zebrafish wnt9b cDNA clones were isolated and found to encode an open reading frame of 358 amino acids, with 68% amino acid identity to mouse Wnt9b and 70% amino acid identity to human WNT9B. Syntenic analyses demonstrated that wnt9b and wnt3 exist as a contiguous pair in amniote vertebrate species, and that these genes are separate in the zebrafish and Takifugu genomes. During the pharyngula period, a time of extensive growth and morphogenesis, zebrafish wnt9b exhibits discrete expression in dorsal and ventral first and second branchial arch tissues, the heart, and pectoral fin buds. These analyses suggest that in zebrafish, as in humans, wnt9b plays distinct roles in directing morphogenetic movements of developing branchial arch elements, and identify the zebrafish as a useful developmental model for the study of human craniofacial cleft lip and palate.

Zebrafish Wnt9b Patterns the First Pharyngeal Arch into D-I-V Domains and Promotes Anterior-Medial Outgrowth

“…Our prior report of Wnt9b expression demonstrates Wnt9b is expressed maternally, early zygotically and even later during zebrafish craniofacial development [41]. Of particular note Wnt9b is expressed within the first pharyngeal arch (PA1) ectodermal epithelia during the time when CNCC are patterned by the edn1 pathway from Dorsal-Ventral subdomains into Dorsal-Intermediate-Ventral domains.…”

“…In zebrafish, we and others showed that Wnt9b is expressed maternally and during early zygotic phases [41]- [43]. Later, from about one day post fertilization (dpf) onwards, Wnt9b is expressed within the first pharyngeal arch (PA) ectodermal epithelia that overlies the arch's cranial neural crest cells (CNCCs), which have mostly finished their migration into the ventral half of the embryo by 24 hours post fertilization (hpf) [41].…”

“…Later, from about one day post fertilization (dpf) onwards, Wnt9b is expressed within the first pharyngeal arch (PA) ectodermal epithelia that overlies the arch's cranial neural crest cells (CNCCs), which have mostly finished their migration into the ventral half of the embryo by 24 hours post fertilization (hpf) [41]. Robust expression of Wnt9b in the first arch ectoderm persists past 40hpf, surrounding the primary mouth as it initially opens at 38 -40hpf [44].…”

“…Synthetic mRNA was generated from linearized Wnt9b plasmid used in our previous publication [41] and the mMessage mMachine T7 ULTRA Transcription Kit (Ambion). The concentration of purified Wnt9b mRNA was determined using a spectrophotometer and the mRNA was diluted in nuclease-free water with 12.5% phenol red dye to deliver either 140 pg or 280 pg in a 3 nl injection pulse.…”

Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development

“…The gene expression of zebrafish wnt9b has been examined but limited signal was detected in the first and second pharyngeal arches (Jezewski et al, 2008). Study of vertebrate gene synteny identified conservation of wnt9a across human, mouse, Xenopus and zebrafish genomes (Garriock et al, 2007).…”