Neural crest morphogenesis

All pharyngeal skeletons are derived from cranial neural crest cells. Neural crest cells characterized as a stem/progenitor cell population are unique to vertebrate embryos and are able to migrate extensively and differentiate into a variety of derivatives

including sensory and autonomic ganglia, cartilage and bone of the face and pigment cells of the skin (Simoes-Costa and Bronner, 2015).

During gastrulation stage, neural crest cells are induced from ectodermal germ layer. When the neural plate closes to form the neural tube, the dorsal part of the neural fold expressing neural crest genes (FoxD3 and Sox10), that reflect neural crest

specification (Labosky and Kaestner, 1998; Southard-Smith et al., 1998). Neural crest cells initially reside in the neural plate border territory, where the lateral edges of the central nervous system. The neural plate border induction is regulated by the interaction of proneural gene activator FGFs signaling and inhibitors of WNT and BMP and Notch signaling (Streit et al., 2000). In response to these signaling module, several neural plate border specifier genes (Tfap2a, Msx1, Zic1, Gbx2, Pax3/7, Dlx5/6, Gata2/3, foxi1/2 and Hairy2) are expressed (Khudyakov and Fraser, 2009; Meulemans and

Bronner-Fraser, 2004; Nichane et al., 2008). These transcription factors further conduct mutual cross-regulator interaction to stabilize their expression in the neural plate border and

perform inhibitory interactions with neural transcription factor. As a result, a sharp boundary between the neural plate and the neural plate border is generated.

The neural plate border becomes segregated into medial premigratory neural crest and lateral preplacodal domains. The expression of key placodal regulators (Six1, Eya1/2 and Irx1), which is induced by lateral neural plate border specifiers (Foxi1/3,

Gata2/3 and Dlx5/6), is established lateral to the neural crest domain (Grocott et al.,

2012; Sato et al., 2010).

The first neural crest specifier genes expressed in the neural plate border are FoxD3, Ets1 and Snai1/2 in chick (Khudyakov and Bronner-Fraser, 2009). The neural

plate border specifiers Pax3/7 and Msx1 regulate FoxD3 expression (Simões-Costa et al., 2012). Pax3/7, Msx1 and Zic1 are conserved and crucial genes for the acquisition of neural crest identity directly activate a number of neural crest specifier genes in multiple species. A full suite of neural crest specifier genes (Myb, Myc, prdm1a, FoxD3, Snai2, Ets1, Sox8/9/10 and Pax3/7) are then expressed to maintain neural crest identity.

Next, these neural crest specifiers activate the epithelial-to-mesenchymal transition (EMT) program, which allows delamination of the neural crest from the ectoderm and become a migratory cell type. The delamination of neural crests involves cell surface changes resulting in the dissociation of adherens junctons. A study in chick showed that a switch between type 1 cadherins (Ecad and Ncad) and type 2 cadherins (Cad7 and

Cad11) is required for neural crests delamination. FoxD3 also represses expression of

Ncad and activates Cad7 expression (Cheung et al., 2005; Dottori et al., 2001).

Similarly Snail1/2 cooperates with Lmo4 to downregulate Ncad expression (Ferronha et al., 2013). Furthermore, Snail1/2 interacts with phosphorylated Sox9 to facilitate trunk neural crest delamination (Liu et al., 2013a). In addition, Snail2 represses another type 2 cadherin (Cad6b) which needs to be downregulated before neural crest delamination (Taneyhill et al., 2007).

Delamination and cell dispersion of epithelial-to-mesenchymal transition (EMT) are partially uncoupled in neural crest development. A study in X. laevis showed the dissociation of neural crest cells is mediated partly by Twist via repression of Ecad in the delaminating cells (Barriga et al., 2013). Another transcriptional repressor (Zeb2) identified in Chick plays similar role as Twist. Knockdown of Zeb2 maintains Ecad expression in migratory neural crest cells and results in aggregates of adherent neural crest cells in the vicinity of the neural tube (Rogers et al., 2013). Therefore, neural crest EMT is a two-step process. Neural crest cells first delaminate from the neural tube, a process involved in repression of Cad6b by Snail2 which allows neural crest cells to leave the neural tube, followed by cell dispersion which allows separation of neural crest into individual migratory cells, a process involved repression of Ecad by Twist and Zeb2.

After delamination, migrating neural crest cells express various transcription factors for cell migration and for initiating differentiation. Neural crest specifier genes (such as FoxD3, Est1 and Sox8/9/10), retained expression in some or all migratory cells, however, their expression in the migratory neural crest cells are regulated by different enhancer (Simões-Costa et al., 2012). In addition, they also positively self-regulated in order to continuously express in the migratory neural crest cells (Wahlbuhl et al., 2012).

In zebrafish, at 12 hpf(hours post fertilization), migratory cranial neural crest cells arise from dorsal and lateral region of neural ectoderm. CNC migrates in three streams at 15 hpf. The first (anterior-most) stream emanated from rhombomere (r) 1 and r2 enters the first (mandibular) pharyngeal arch (pa1), which gives rise to the jaw. The second (middle) stream emanated from r4 populates the second (hyoid) pharyngeal arch (pa2), which gives rise to the face. The third (posterior) stream emanated from r6, which progressively splits into two, three, four and eventually five cell groups, each separated by endodermal pouches of pharynx. The third stream populates the third and more caudal pharyngeal arches (pa3-7), which give rise to the components of the neck and the gills. These streams are separated by two cranial NC-free zones, which are located lateral to r3 and r5 (Yelick and Schilling, 2002). By larval stages, each pharyngeal arch contains specific cartilage derived from CNC and muscle derived from mesoderm core.

The CNC skeletal derivatives are patterned through a combination of neural crest cell

differences and adjacent tissue signals, including endoderm and ectoderm.

1.3 Pharyngeal anterior-posterior and dorsoventral patterning

Study in mouse indicates that nested expression of homeodomain factors, Hox genes, provides a combinatorial “Hox-code” to specify regional properties within anterior-posterior (AP) axis (Parker et al., 2016). Neural crest cells (NCCs) adjacent to rhombomere 2 (r2), which colonize PA1, do not express any Hox genes.

Migrating NCCs from r4 into PA2 continues to express Hoxa2 and Hoxb3 but cease Hoxb1 expression (Hunt et al., 1991; Hunter and Prince, 2002). By later stages (mouse

E10.0) a pharyngeal NCC Hox code reveals nested gene expression. Hoxa2 and Hoxb2 expressed in PA2–4, Hoxa3 in PA3–4 and Hoxb3 and Hoxd3 weakly in PA3 but strongly in PA4, and Hoxd4 strongly expressed in PA4.

Hoxa2-null mice fail to correctly form PA2 skeletal derivatives, showing

duplicated PA1-like structures (Gendron-Maguire et al., 1993; Minoux and Rijli, 2010).

Conversely, ectopic expression of Hoxa2 in PA1 suppresses the formation of PA1-derived jaw structures, and results in duplication of PA2 derivatives (Kitazawa et al., 2015). The evolutionarily conserved function of Hoxa2 gene in craniofacial

morphogenesis was identified across jawed vertebrates, such as in chick, Xenopus and zebrafish (Grammatopoulos et al., 2000; Pasqualetti et al., 2000).

Studies in zebrafish indicate that skeletal precursors of dorsoventral (DV) axis are

determined by complex interactions between multiple signaling pathways, in particular Endothelin-1 (Edn1), Bone Morphogenetic Protein (BMP), and Jagged–Notch

signaling. These signals appear to act through several families of transcription factors, including Dlx, Msx, and Hand, to establish dynamic zones of skeletal differentiation.

The diversification of the vertebrate facial skeleton, including the evolution of the jaw, was driven largely by modifications downstream of a conversed pharyngeal DV patterning program (Medeiros and Crump, 2012).

1.4 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).

1.5 Pax1

Vertebrate Paired-box containing transcription factor Pax genes were identified based on their homology in DNA-binding motif with the Drosophila melanogaster paired gene. Pax transcription factor family members contain two DNA-binding

domains (the paired domain and the homeodomain), a co-factor-interacting domain (the octapeptide) and C-terminal activation and repression domains. In mammals, nine Pax members are grouped into four subfamilies such as Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6, according to their structural similarity and the sequence homology (Underhill, 2000). All Pax proteins contain paired domain and transactivation domain. Pax1/9 members contain no homeodomain but contain octapeptide which is highly conserved eight-amino acid [(H/Y)S(I/V)(N/S)G(I/L)LG] domain as protein-protein interaction motif. Pax2/5/8 members contain partial homeodomain and octapeptide. Homeodomain is helix-turn-helix DNA-binding motif. Pax3/7 members contain full homeodomain and

octapeptide; Pax4/6 members contain full homeodomain but no octapeptide. Pax gene family are important regulatory genes and are essential for cell lineage specification and the development of a variety of organs and tissues. Mutation in human Pax3 gene results in Waardenburg syndrome while human Pax6 mutation causes Aniridia.

Mutation in human Pax2 or Pax8 gene leads to Renal coloboma or congenital Hypothyroidism while human Pax9 mutation causes Oligodontia (Underhill, 2000).

Several Pax genes are required for the differentiation of various neural crest derivatives.

For examples, Pax3 and Pax7 regulate proliferation of craniofacial neural crest cells, Pax9 controls tooth and craniofacial skeleton development while Pax1 and Pax9 are

essential for thymus, thyroid and parathyroid development. (Monsoro-Burq, 2015).

Human PAX1 is essential for the development of vertebral column. Patients with Klippel-Feil syndrome was identified by shortness of the neck and cervical vertebrae fusion due to alteration ofPAX1 sequence such as missense, silent, intronic changes or a silent change within the paired box (McGaughran et al., 2003). While PAX1

homozygous variant G166V within paired box is identified in the autosomal recessively inherited OFCS (otofaciocervical syndrome) disorder characterized by facial

dysmorphism, external ear anomalies, branchial cysts, anomalies of vertebrae and the shoulder girdle, and mild intellectual disability (Pohl et al., 2013).

In mouse embryos, Pax1 is expressed in the developing sclerotomes and later in

the anlagen of intervertebral discs along whole vertebral column. Mice undulated (un) homozygous mice containing a Gly-Ser replacement in the paired box of Pax1 exhibit vertebral abnormalities along the entire anterior-posterior axis (Balling et al., 1988). A Pax1 null mutant (Un^s) was created by gene targeting and homozygous Pax1 null mice

showed similar phenotype as natural undulated mutant mice. Analysis of heterozygous Pax1 knockout (Un^s) mice further demonstrated that Pax1 is haploinsufficient in the

development of some skeletal elements of vertebral column and sternum (Wilm et al., 1998). Pax1 is also transiently expressed in the developing limb buds that are important for the development of pectoral girdle demonstrated by different alleles of undulated mutants (Timmons et al., 1994). Pax1 was also shown to be required for the

chondrogenic differentiation of sclerotomal cells by transactivating Bapx1/Nkx3.2 gene expression in the sclerotome. Pax1;Pax9 double mutant mice lack Bapx1 expression in the sclerotome, indicating Bapx1 is a direct target of Pax1 and Pax9 (Rodrigo et al., 2003). A study using Pax1;Pax9 double mutant mice further demonstrated functional redundancy between Pax1 and Pax9 during vertebral column development and their roles in the control of cell proliferation during early sclerotome development (Peters et al., 1999). Pax1 is also expressed in the embryonic thymus and expression of Pax1 protein can be identified in the epithelium of the third pharyngeal pouch which produce part of the thymus epithelium. Pax1 expression continues during thymus development

and Pax1 expression in thymus epithelium is essential to form the thymus

microenvironment which is required for normal T cell maturation (Wallin et al., 1996).

Chick Pax1 is detected in the sclerotome, limbs and shoulder girdle, (Takimoto et al., 2013). Although mouse Pax1 was implicated in promoting chornodrogenic

differentiation of sclerotomal cells through induction of Bapx1/Nkx3.2 expressed, chick Pax1 was identified to inhibit chondrocyte maturation, independently of Bapx1/Nkx3.2.

Forced expression of Pax1 in chick forelimb produced shortened skeletal elements with reduction of proteoglycan accumulation in cartilage. Overexpression of Pax1 in cultured chondrocytes lacked accumulation of cartilaginous proteoglycan which is accompanied by downregulation of Sox9, Col2a1, Chm1 and Agc1. These results indicate that chick Pax1 acts as a negative regulator of chondrocyte maturation via antagoning Sox9 expression (Takimoto et al., 2013). Similar expression of pax1 in the sclerotome and in the pharyngeal pouch was identified in Xenopus embryos (Sanchez and Sanchez, 2013).

In medaka fish, similar expression of pax1 and pax9 was observed in the

In medaka fish, similar expression of pax1 and pax9 was observed in the