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Human palatal clefting is debilitating and difficult to rectify surgically. Animal models enhance our understanding of palatogenesis and are essential in strategies designed to ameliorate palatal malformations in humans. Recent studies have shown that the zebrafish palate, or anterior neurocranium, is under similar genetic control to the amniote palatal skeleton. We extensively analyzed palatogenesis in zebrafish to determine the similarity of gene expression and function across vertebrates. By 36 hours postfertilization (hpf) palatogenic cranial neural crest cells reside in homologous regions of the developing face compared with amniote species. Transcription factors and signaling molecules regulating mouse palatogenesis are expressed in similar domains during palatogenesis in zebrafish. Functional investigation of a subset of these genes, fgf10a, tgfb2, pax9, and smad5 revealed their necessity in zebrafish palatogenesis. Collectively, these results suggest that the gene regulatory networks regulating palatogenesis may be conserved across vertebrate species, demonstrating the utility of zebrafish as a model for palatogenesis. Developmental Dynamics 240:2204–2220, 2011. © 2011 Wiley-Liss, Inc.
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
In humans disruption of palatal development can result in the congenital disorder cleft palate. Cleft palate has a prevalence of approximately 1 in 700 live births and leads to problems with feeding, speaking, hearing, and social integration (Dixon et al., 2011). Our understanding of human palatogenesis and cleft palate relies on studies of palate development across diverse vertebrate species.
The morphology of the palatal skeleton varies greatly in extinct and extant vertebrate species (Ferguson, 1988; Kimmel et al., 2009). Morphogenesis of the palate has been studied extensively in alligators, chicken, mice, and human. Recently, the zebrafish anterior neurocranium, the functional equivalent of the mammalian palate (discussed in more detail below), has been used as a model for palatogenesis. Although there are inter-species differences in palatogenesis, the earliest events are highly similar. Segmented streams of cranial neural crest cells (CNCC) migrate from the dorsal neural tube to populate the pharyngeal arches in all vertebrate species. In this manuscript, we focus on mouse and zebrafish for simplicity.
In both mouse and zebrafish the first stream of CNCC splits and a portion migrate over the eye to reside just below the eye and are referred to as frontonasal CNCC (Osumi-Yamashita et al., 1994; Wada et al., 2005; Eberhart et al., 2006; Kimmel and Eberhart, 2008) (Fig. 1A,B). The remainder of the first stream of migrating CNCC move into the first pharyngeal arch and populate two domains (Osumi-Yamashita et al., 1994; Trainor et al., 2003; Wada et al., 2005; Eberhart et al., 2006). The maxillary domain occupies the space ventral to the eye and dorsal to the oral ectoderm while the mandibular domain occupies the space ventral to the oral ectoderm to the limit of the first arch (Fig. 1A,B). Within the pharyngeal arches of both species CNCC condense on the oral ectoderm where reciprocal signals between the neural crest and the ectoderm drive fusion events necessary for palatogenesis.
Figure 1. Comparison of zebrafish and mouse palatogenesis with a detailed look at zebrafish palate formation. A,B: Outlines of embryos (lateral view), after CNCC condensation in the frontonasal (yellow), maxillary (turquoise), and mandibular (green) domains of zebrafish (A) and mouse (B). C,D: Drawings of cross-sections through heads of zebrafish (C) and mouse (D) after formation of the palate. Colors correspond to similar structures across both species. E,F,H: In situ hybridization of sox9a expression marking the palatal CNCC in zebrafish, ventral view with anterior to the left is shown, and mandibular crest have been removed to allow full view of the developing palate. E: At 36 hours postfertilization (hpf), sox9a expression marks the CNCC that line the maxillary (arrow) and the frontonasal (arrowhead) domains before their migration to the midline. F: Migration of frontonasal cells to the midline to begin the formation of the ethmoid plate (arrowhead) at 44 hpf and maxillary cells (arrow) begin rearrangements to form the trabeculae rods of the palate. G: By 48 hpf, most, if not all, frontonasal CNCC have migrated to the midline (arrowhead) and trabeculae rods are well formed in the palate (arrow). fn, frontonasal CNCC; mx, maxillary CNCC; md, mandibular CNCC; e, eye; ea, ear; b, brain; n, nasal opening; p, palate; o, oral opening; t, tongue; M, Meckel's.
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The mammalian palatal skeleton forms by the fusion of a series of CNCC-derived bones in the primary and secondary palate. In mice, the palatal shelves initiate from the maxillary processes on embryonic day (E) 11 and grow vertically, lateral to the tongue between E12 and E13. At E14.5, the palatal shelves rapidly re-orientate to a horizontal position above the tongue and contact one another at the midline. The medial edge epithelia of the apposed palatal shelves adhere to form a midline epithelial seam, which subsequently degenerates to allow mesenchymal continuity across the palate by E15. The palatal skeleton is formed by fusion of the secondary palate with the primary palate and the nasal septum, creating a separation between the oral and nasal cavities (Dixon et al., 2011) (Fig. 1D).
In fish, a series of bones forms in the roof of the mouth (Kesteven, 1922; Shah et al., 1995; Cubbage and Mabee, 1996), separating the oral cavity from the brain (Fig. 1C). In fish species where palatogenesis has been studied, the fusion events appear to happen directly, without a midline epithelial seam, although the presence of rudimentary palatal shelves has been suggested in salmon (Shah et al., 1990). In the early larval zebrafish, a simple palatal skeleton, which consists of the cartilaginous paired trabeculae and ethmoid plate as well as the dermal parasphenoid bone, forms by 4 days postfertilization (dpf), the latter two being midline structure (Fig. 2A,C) (Schilling and Kimmel, 1997). These elements are either maintained or contribute to the juvenile and adult palatal skeleton (Cubbage and Mabee, 1996; Kimmel et al., 1998). The oral cavity of zebrafish contains teeth that are situated in the back of the oral cavity on the fifth ceratobrachial (Huysseune et al., 1998). Recently, investigation of the oral cavity in zebrafish has revealed a muscular, pseudo-stratified epithelia covered tongue initially present by 1 dpf and maintained into adulthood (Abbate et al., 2006; Hu et al., 2010). The nasal canals, used in olfactory reception, are functionally similar to other vertebrates and are situated dorsal to the palate and anterior to the eyes (Weth et al., 1996), but do not appear to connect to the pharyngeal cavity as they do in amniotes. Some authors have argued for homology of individual elements in the palatal skeletons of fish and amniotes (Kesteven, 1922; Shah et al., 1990, 1995). While we make no such claims for homology of palatal skeletal elements, there is growing evidence that the amniote and fish palatal skeletons are under similar genetic control, warranting a deeper exploration of the palatogenic gene regulatory network in zebrafish.
Figure 2. Wild-type craniofacial cartilage and bone. A–D: Alcian blue and Alizarin red-stained cartilages and bones, respectively, of 5 days postfertilization (dpf) zebrafish, anterior is to the left. A: Whole-mount dorsal view showing the neurocranium. B: Ventral view showing the pharyngeal elements. C: Dorsal flat mount view of the neurocranium. D: Lateral view of first and second arch pharyngeal elements, dorsal is up. A,C: Palate is anterior to the dashed line and comprised of the ethmoid plate and trabeculae. e, eye; ep, ethmoid plate; t, trabeculae; p, palate; M, Meckel's cartilage; pq, palatoquadrate; c, ceratohyl; h, hyosymplectic; pt, pharyngeal teeth; pp, pterogoid process (upperjaw); ps, paresphenoid.
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Several recent genetic studies have illustrated the usefulness of zebrafish in the study of palate development. For example, disruption of SHH or SATB2 causes defects in the palatal skeleton in human, mouse, and zebrafish (Belloni et al., 1996; Chiang et al., 1996; Roessler et al., 1996; FitzPatrick et al., 2003; Wada et al., 2005; Britanova et al., 2006; Eberhart et al., 2006; Sheehan-Rooney, unpublished data). Ghassibe-Sabbagh and colleagues recently revealed the importance of FAF1, for proper palate formation in humans and its importance was verified in zebrafish by morpholino analysis, highlighting the utility of quick verification of human studies in zebrafish (Ghassibe-Sabbagh et al., 2011). Importantly, work in zebrafish has recently been shown to be predictive for genes contributing to cleft palate in humans. Mutation of Pdgfra leads to cleft palate in both zebrafish and mouse (Xu et al., 2005; Eberhart et al., 2008). Research by Eberhart and colleagues (2008) showed that the microRNA mir140 negatively regulates pdgfra, thereby providing the first evidence of a specific microRNA involved in palatogenesis.
Subsequently, sequence variants in this same microRNA were found to be associated with cleft palate in humans (Li et al., 2010), demonstrating the importance of zebrafish as a model of palatogenesis.
In amniotes, such as mouse, a highly regulated gene network drives palatogenesis (Cobourne and Sharpe, 2003; Hilliard et al., 2005). Mouse mutants have revealed many genes involved in palatogenesis such as the transcription factors Msx1, Pax9, Lhx8, Tbx22, and Osr2, and signaling molecules including Tgfb3, Bmp4, Shh, Fgf10, and Bmp2 (Satokata and Maas, 1994; Kaartinen et al., 1995; Proetzel et al., 1995; Peters et al., 1998; Zhao et al., 1999; Zhang et al., 2002b; Lan et al., 2004; Rice et al., 2004; Liu et al., 2005; Pauws et al., 2009). Notably, rescue of the cleft palate in Msx1 knockout mice by means of Bmp4 expression revealed a gene regulatory network involving several factors. In the anterior palate, interplay between Bmp4 and Msx1 in the mesenchyme maintains Shh in the ectoderm. In turn, Shh then induces Bmp2 in the mesenchyme to promote proliferation and palatal shelf growth (Zhang et al., 2002b). Subsequent research demonstrated that Fgf10 regulates Shh, adding to this network of genes (Rice et al., 2004). Despite these observations, a comprehensive understanding of the gene regulatory networks regulating palatogenesis has not been achieved.
Zebrafish has proven to be an extremely useful model system in which to achieve a deeper understanding of craniofacial development, particularly, with regard to anterior/posterior and dorsal/ventral patterning (David et al., 2002; Hunter and Prince, 2002; Crump et al., 2004a, b, 2006; Miller et al., 2004, 2007; Walker et al., 2006, 2007; Talbot et al., 2010). In the current study, we sought to determine the usefulness of zebrafish for studies of palatal development by examining homologs of members of the amniote palatogenic gene regulatory network in zebrafish. Our results show that the vast majority of the signaling molecules and transcription factors known to be expressed in the developing palate of mouse are similarly expressed in zebrafish. The genes osr1 and osr2 show partitioning of gene expression in the maxillary domain. Both the maxillary and mandibular domains express the zebrafish homolog of Msx1, msxe (Postlethwait, 2006). Other transcription factors including the paralogs lhx6 and lhx8 as well as tbx22 are expressed in a similar pattern to mouse with expression in the maxillary and mandibular domains. We also show that many of the signaling molecules, such as Bmps and Tgfbs that have been shown to be important in mouse palatogenesis have similar expression patterns in zebrafish.
Not only are there similarities in gene expression but these same genes drive development of the zebrafish palate. Knock-down of the signaling molecules fgf10a and tgfb2 causes disruptions to the palate. Pharyngeal pouch endoderm and CNCC of the maxillary and mandibular domains express pax9 in zebrafish. Morpholino (MO) knock-down of pax9 in zebrafish causes highly penetrant defects in the palate, hyoid arch and teeth. Furthermore, our analysis of our smad5 mutant zebrafish clearly shows that CNCC require the reception of bone morphogenetic protein (Bmp) signaling for proper palatogenesis. Taken together, our results suggest conservation of gene expression and function in the palatogenic gene regulatory network across vertebrates.