Requirement of endoderm for craniofacial cartilage formation

A previous study showed that pharyngeal endoderm is important in cartilage development and patterning of the CNC-derived pharyngeal skeleton. The pharyngeal pouches are segmental series of epithelial structures derived from pharyngeal endoderm.

In vertebrate, the formation of endoderm and mesoderm is specified by Nodal signaling (Schier and Shen, 2000). sox32/Casanova (cas), which is a transcription factor

downstream of Nodal signaling is essential for endoderm development (Alexander et al., 1999). The zebrafish cas mutants lacked all pharyngeal arch cartilages and absence of trabeculae by Alcian Blue staining at 4-5 days. Formation of three streams of dlx2a-labeled CNCs was initially identified in cas mutants at 23s stage. However, three

streams of dlx2a-positive neural crest cells progressively lost from stream III to stream I in cas mutants during 24 to 48 hpf. Transplantation results further demonstrated that the

sox-related transcription factor, cas, is required non-autonomously to maintain the identify and survival of pharyngeal arch cartilages (David et al., 2002).

Characterization of zebrafish mutants further demonstrates the involvement of several genes in the pharyngeal pouch development. Tbx1 is a member of T-box transcription factor family. tbx1 is expressed in the primordia of the pharyngeal arches (pa1 to pa7) at 20s stage and is localized to the arch epithelium and mesodermal core of pharyngeal arches by 27 hpf (Piotrowski et al., 2003). At 30 hpf, tbx1 is detected in the endodermal pouches and arch muscles, between 24 hpf and 72 hpf tbx1 was expressed in the cardiac region, pharyngeal arch and otic vesicle in zebrafish. Retinoid acid (RA) signaling is essential for patterning the endoderm of the posterior pharyngeal arches.

The defects produced by a loss of Tbx1 highly resemble those induced by hyper- and hypo- RA. Treat different dose RA could produce an altered tbx1 expression pattern.

Repression of tbx1 expression was most evident at 36 hpf, 24 hours after RA treatment at 12.5 hpf for 1.5 hours. In addition, RA could repress tbx1 expression in a dose-dependant manner (Zhang et al., 2006). Embryos of tbx1/van gogh (vgo) mutants revealed defects in the ear, thymus, and pharyngeal arches. Alcian Blue staining revealed drastically reduced mandibular and hyoid arches, and the absence of five branchial arches in vgo/tbx1 mutants. Tbx1 is shown to be required non-autonomously in neural crest derived pharyngeal structures. Results of in situ hybridization and rescue

experiments demonstrated that vgo/tbx1 regulates edn1 expression and Edn1 further control expression of hand2, a bHLH transcription factor expressed in the neural crest cells (Piotrowski et al., 2003). Function of Tbx1 in pharyngeal pouch morphogenesis was later characterized in vgo/tbx1 mutant, revealing failure in the initiation of pouch outpocketing (Choe and Crump, 2014). vgo/tbx1 mutant also retained expression of the immunoglobulin-domain protein Alcama, a marker of maturing pouches, despite the absence of morphological pouches. Transgenic rescue experiments by stably integration of nkx2.5: Tbx1 versus nkx2.3: Tbx1 transgenes indicate that mesodermal Tbx1 is adequate for pouch morphogenesis but not later cartilage formation. A two-step model describing Tbx1 function in pouch morphogenesis was then proposed. Tbx1 promotes expression of wnt11r and fgf8a in different domains of the mesoderm where Wnt11r initiates pouch morphogenesis via epithelial destabilization and Fgf8a directs following pouch outgrowth. Loss of wnt11r and fgf8a phenocopies vgo/tbx1 mutant phenotype (Choe and Crump, 2014).

sucker (suc)/endothelin 1 (edn1) encodes a 21-amino-acid secreted ligand and is

expressed in central mesenchymal cores of arch paraxial mesoderm, ventral epithelia of surface ectoderm and pharyngeal endodermal pouches (Miller et al., 2000). Within the pharyngeal pouches, expression is restricted to posterior, ventral epithelia. In suc/edn1 mutant embryos, ventral cartilages of mandibular and hyoid arches such as Meckel’s

cartilage and ceratohyal are reduced and fused to the dorsal cartilages of the same arches. Before chondrogenesis, suc/edn1 mutant embryos have severe defects in expression of hand2, dlx2a, msx1a, msx1b, gsc, dlx3b and epha3 in the ventral arch neural crest cells. The role of Edn1 in the formation of ventral cartilages and joints in the anterior pharyngeal arches is further analyzed (Miller et al., 2003). Ventral

pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Two Edn1 downstream target genes, bapx1/nkx3.2 and hand2, specify joints and ventral pharyngeal fates. Additionally, Edn1 can bind to Ednrb receptor expressed in pharyngeal pouches to stabilize Alcama protein. Alcama then binds to Nadl1.1 expressed in neural crest cells to regulate expression of hand2, dlx3b, dlx5a, dlx6a which are essential for neural crest differentiation (Choudhry et al., 2011). Edn1 binds to one or both of the two known mammalian G protein-coupled endothelin receptor, Ednra (endothelin type A receptor) and Ednrb. There are two zebrafish ednra genes, ednraa (ednra2) and ednrab (ednra1). ednrab is expressed in the migrating and

postmigratory neural crest cells of the pharyngeal arches and ectodermal epithelium while ednraa is identified in postmigratory neural crest cells within arches at 24 hpf.

Combined loss of Ednraa and Ednrab eliminates the lower jaw similar to suc/edn1 mutants (Nair et al., 2007). ednrb1 expression in the endoderm is compatible with our hypothesis that Edn1 signals to the endoderm to regulate Alcama levels (Choudhry et

al., 2011).

Embryos treated with SU5402 inhibitor to prevent Fgf signaling failed to develop any viscerocranial cartilage and form severely reduced neurocranium at 96 hpf (David et al., 2002). fgf3 expression is detected in the mid-hindbrain boundary, rhombomere 4 and in pharyngeal endoderm region beneath rhomboneres 1-3 at 12s stage. Endodermal expression of fgf3 is restricted to three formed endoderm pouches at 25s stage. At 24 hpf, fgf3 expression in the anterior pharyngeal pouches decreases but is maintained in two posterior pharyngeal pouches. fgf3 morphants displayed hyoid arch with inverted AP polarity and loss of branchial arches except the 7th arch. Down regulation of dlx2a expression in stream III cranial neural crest cells was identified in fgf3 morphant at 24 hpf, indicating that in fgf3 is required for posterior cranial neural crest cells to maintain dlx2a expression (David et al., 2002; Walshe and Mason, 2003a). fgf8 started to be

expressed in the first pouch endoderm and endoderm associated with posterior arches at 16 hpf. Both fgf8 and fgf3 are expressed by endoderm associated with the developing jaw such as first endodermal pouch at 30 hpf. fgf8 mutant phenotype in pharyngeal pouches and cartilages is relatively mild, suggesting other Fgfs such as Fgf3 functions redundantly in patterning pharyngeal arches. Although fgf8 mutnats and fgf3 morphants had largely normal pouches, fgf8 mutant; fgf3 morphant embryos did not develop

pouches and had severe reductions in size of mandibular cartilages and absence of hyoid

cartilages and posterior branchial cartilages. Therefore, Fgf signaling in the mesoderm and segmented hindbrain controls the segmentation of the pharyngeal endoderm into pouches; this is essential for the subsequent patterning of pharyngeal cartilages (Crump et al., 2004a; Walshe and Mason, 2003b).

integrinα5 (itga5) is expressed in pharyngeal endoderm with a pattern that spatially

and temporally corresponding to regions of pouch formation and cranial neural crest cells. Expression of itga5 is found in cranial neural crest, otic placode and pharyngeal endoderm at 5s stage. Strong itga5 expression throughout pharyngeal endoderm, including the first pouch is identified at 18 hpf. By 26 hpf, expression of itga5 is found in the fourth pouch whereas itga5 is no longer expressed in the first pouch. At 38 hpf, strong itga5 expression is identified in the sixth pouch while itga5 is not expressed in the fourth pouch. Expression of itga5 is also detected in patchy zones of pharyngeal crests. Specific defects in the formation of the first pouch and loss of the anterior

Hyomandibula (ahm)and Symplectic (sy) cartilages of hyoid cartilages was identified in itga5 mutant larvae; this phenotype is attributed to lack of pouch-derived signals from

the first pouch to promote the growth and survival of neural crest cells in the adjacent ahm and sy of hyoid cartilage (Crump et al., 2004b). Itga5 was also implicated in posterior pharyngeal arch development. Prdm1a was shown to be required for posterior ceratobrachial cartilage development. Overlapping expression of prdm1a and itga5 was

identified in the posterior arches and decreased itga5 expression was detected in prdm1a mutants, indicating prdm1a acts upstream of itga5. Loss of dlx2a expression in the ceratobranchial cartilage 2-5, and cell proliferation in prdm1a mutants can be rescued with itga5 mRNA injection. Therefore, Prdm1a and Itga5 are both required for posterior pharyngeal arch development (LaMonica et al., 2015).