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Nelms BL, Labosky PA. Transcriptional Control of Neural Crest Development. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Transcriptional Control of Neural Crest Development.

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Chapter 6Homeobox Genes

6.1. Alx

The aristaless-related homeobox (Alx) family consists of four members in mammals: Alx1, Alx3, Alx4, and Arx. The Alx paired-type homeodomain binds DNA at specific sites either as a homodimer or as a heterodimer with other paired-type homeodomain factors. Both Alx1 and Alx4 are expressed in the NC-derived first pharyngeal arch and craniofacial mesenchyme and their derivatives and in limb bud mesenchyme. In vitro, Alx1 and Alx4 can form heterodimers and can each activate transcription of reporter genes in a similar manner, suggesting functional redundancy. Alx1-null; Alx4-null compound mutants demonstrate a genetic interaction indicating a role for both genes in nasal cartilage fusion, mandible patterning, and other aspects of craniofacial development (Qu et al., 1999). Similar redundancy is seen with Alx3 and Alx4. Alx3-null mice are seemingly normal, but compound Alx3-null; Alx4-null mutants have severe craniofacial defects not observed in Alx4-null single mutants (Beverdam et al., 2001). These defects include cleft nasal regions and malformation or loss of facial bones and other NC-derived skull elements. In these Alx3-null; Alx4-null compound mutants, an increase in apoptosis in the outgrowing frontonasal process is the likely cause of the craniofacial defects (Beverdam et al., 2001).

6.2. Barx1

The homeobox transcription factor Barx1 (with some homology to Drosophila Bar, expressed in the eye and other sensory organs) was identified in the mouse as a protein that binds to a regulatory element of the Ncam1 promoter. Barx1 is strongly expressed in parts of the head and neck mesenchyme, especially in parts of the first and second pharyngeal arches where it is generally restricted to NC-derived tissues, including mesenchyme associated with the olfactory epithelium, the primary and secondary palate, the stroma of the submandibular gland, and molar papillae. By E16.5, Barx1 expression in the head is only detected in the developing molars and may be involved in delineating molar identity from developing incisor identity (Tissier-Seta et al., 1995). Human Barx1 is expressed in testis, heart, iris, craniofacial mesenchyme of NC origin, and developing teeth (Gould and Walter, 2000). During development of the pharyngeal arch derivatives, Barx1 is downstream of members of the Dlx gene family. In Dlx1-null; Dlx2-null compound mutant embryos, Barx1 expression is lost in favor of the chondrogenic marker Sox9 (Thomas et al., 1997). Loss of Dlx2a in zebrafish causes ectopic Barx1 expression in the dorsal ceratohyal arch (Sperber et al., 2008). In zebrafish, Barx1 is expressed in the rhombencephalic NC and in NC-derived pharyngeal arch mesenchyme, and this expression can be induced by BMP4 and is maintained through FGF signaling. Barx1 expression is necessary for proliferation of osteochondrogenic progenitors. Knockdown of Barx1 results in reduced and malformed cartilage elements due to reductions in chondrocyte differentiation and condensation in conjunction with loss of osteochondrogenic markers Col2a1, Runx2a and Chondromodulin, and the odontogenic marker Dlx2 (Sperber and Dawid, 2008). Like many genes expressed in the NC-derived pharyngeal arch mesenchyme, Barx1 is also regulated by Endothelin signaling and its expression is significantly reduced in Edn1-null embryos (Clouthier et al., 2000).

6.3. Dlx1, Dlx2, Dlx3, Dlx5, Dlx6

The distalless homeobox (Dlx) gene family has six homologs in mammals. The six mouse Dlx genes are primarily expressed in the developing forebrain and cranial NC derivatives, particularly during NC migration, patterning of the orofacial skeleton, and tooth initiation and development (Berdal et al., 2000; Davideau et al., 1999; McGuinness et al., 1996; Merlo et al., 2000; Weiss et al., 1998). The mammalian Dlx genes are expressed at various times during tooth development in a manner related to their genomic organization, much like an “odontogenic Hox” code (Thomas et al., 1997; Weiss et al., 1998). Null alleles of Dlx1, Dlx2, Dlx3, and Dlx5 in the mouse have revealed functions in craniofacial patterning, sensory organ morphogenesis, osteogenesis, and placental formation (Merlo et al., 2000). However, there is a substantial degree of functional redundancy shared between members of the Dlx family, making functional analysis of these genes more difficult without using compound-null mutants (Kraus and Lufkin, 2006). In zebrafish, loss of Dlx genes correlates with loss or abnormal morphology of craniofacial cartilage elements except for those that originate from NCCs of the midbrain region and do not express Dlx genes. Similar to Hox genes, the expression of zebrafish Dlx genes in NCCs migrating from the hindbrain and in visceral arch primordia has also been shown to be sensitive to treatment with RA (Ellies et al., 1997).

6.4. Dlx1 AND Dlx2

Dlx2 is required for differentiation of subsets of cranial NC and forebrain cells (McGuinness et al., 1996) and is particularly important in the NC-derived pharyngeal arch mesenchyme. Dlx1 and Dlx2 pattern the teeth by specifying a subpopulation of cranial NC as odontogenic, allowing for molar development. In mice carrying null mutations for both Dlx1 and Dlx2, the NC-derived ectomesenchyme underlying the maxillary molar epithelium loses its odontogenic potential, changing fate from odontogenic to chondrogenic. These mice do not develop maxillary molars, but the incisors and mandibular molars are normal (Thomas et al., 1997). Dlx2 is also expressed in the mandible and maxilla, and is induced by signals from the mandibular and maxillary arch epithelia (Ferguson et al., 2000). Dlx1 expression extends to the third pharyngeal arch, which contributes to the thymus. Dlx1-null mice do not have any obvious thymus developmental defects, but there may be functional redundancy shared by other Dlx family members, particularly Dlx2. Transcripts for multiple Dlx family members were detected in a screen of an E13.5 thymus cDNA library, and Dlx1 and Dlx2 were detected in adult murine thymus and Thy1-positive thymocytes (Woodside et al., 2004).

6.5. Dlx3

In chicken embryos, Dlx3 is expressed in the distal portion of the first and second pharyngeal arch mesenchyme (Pera and Kessel, 1999). In mice, Dlx3 expression in the pharyngeal arches is dependent on Endothelin signaling and is downregulated in mice deficient for downstream effectors of Edn1, Ga(q), and Ga(11) (Ivey et al., 2003). Studies of Dlx3 during newt regeneration indicate that it is expressed in cells with the ability to contribute to ventral root ganglia and spinal ganglia, but only during regeneration, and not normal development. These data suggest that Dlx3 is present in cells with NC-like properties and the potential for repair (Nicolas et al., 1996).

6.6. Dlx4

A role for Dlx4 in the NC has not been reported.

6.7. Dlx5

In the mouse, early Dlx5 expression becomes localized to the anterior neural ridge, defining the rostral boundary of the neural plate, and extends caudolaterally, marking the prospective NC (Yang et al., 1998). Dlx5 is later expressed in a BMP-dependent manner in the developing skull and mandibular bones but not in the maxilla (Ferguson et al., 2000; Holleville et al., 2007). Like Dlx2, Dlx5 mandibular expression is induced by signals from the mandibular and maxillary arch epithelia (Ferguson et al., 2000). Dlx5 is also involved in initial steps of membranous differentiation of the calvaria and can induce Runx2 and osteoblast differentiation in cultured embryonic suture mesenchyme (Holleville et al., 2007). In humans, Dlx5 is primarily detected first in the mandible and then later in the maxilla, and finally is restricted to progenitor cells of the developing teeth and bones and cartilages of the mandible and maxilla. In the developing teeth, human Dlx5 is expressed in NC-derived mesenchyme and in a subset of dental epithelia (Davideau et al., 1999).

6.8. Dlx6

Dlx6 is another Dlx gene regulated by Endothelin1 signaling. Edn1 from neighboring cells acts on cranial NC-derived ectomesenchymal cells expressing Ednra to regulate expression of crucial genes such as Dlx6 and the Dlx6 downstream target Hand2 (Fukuhara et al., 2004; Ivey et al., 2003). Endothelin signaling is critical for Dlx6 and Hand2 expression in the mandibular arch mesenchyme in a short time window between approximately E8.75 and E9.0 until E9.5. Later, Dlx6 and Hand2 expression is maintained or regulated by FGF and other signals from the epithelium (Fukuhara et al., 2004).

6.9. Emx

The empty spiracles-related homeobox genes Emx1 and Emx2 are expressed in the forebrain and are essential for forebrain development (Chiba et al., 2005; Williams et al., 1997). In NT and NC-like cells derived from ES cells by RA treatment, Emx1 and Emx2 expression occurred in response to Noggin treatment, which directs NT-like structures toward a forebrain fate (Chiba et al., 2005). Overexpression of Emx1 and Emx2 in NC-derived melanocytes downregulates the melanocyte-specific differentiation genes Mitf, Tyrp1, Dct and Tyr, and constitutive expression of Emx genes alters pigment cell morphology and growth properties. Emx regulation of Mitf suggests that one normal function of Emx genes is to inhibit Mitf activation in nonmelanocyte neuroepithelial derivatives (Bordogna et al., 2005).

6.10. Gsx

The goosecoid-related homeobox gene family includes its namesake, Goosecoid (Gsc), and the related factor Gsc2. In zebrafish, Gsc exhibits two independent phases of expression: early, in cells anterior to the presumptive notochord, and later, in NC derivatives in the larval head (Schulte-Merker et al., 1994). Zebrafish Gsc expression in the pharyngeal arches is dependent on Endothelin signaling, Mef2c function, and Dlx2 expression (Miller et al., 2007; Sperber et al., 2008). Pharyngeal arch expression of Gsc is also absent in Edn1-deficient and Ednra-deficient mouse embryos, suggesting it is one of the downstream signals triggered by activation of Ednra (Clouthier et al., 1998; Clouthier et al., 2000). In mammals, Gsc marks caudal NC-derived mesenchymal cells of the mandible, which do not receive Fgf8 signals from the rostral epithelium and give rise to the distal part of the lower jaw, whereas rostral NC mesenchyme does receive Fgf8 and gives rise to odontogenic cells. As in zebrafish, Endothelin signaling also helps to maintain positional information (Tucker et al., 1999). Gsc is also involved in patterning and morphogenesis of the NC-derived mesenchyme of the middle ear, a pharyngeal arch derivative, and Gsc is critical for tympanic ring development (Mallo, 2001). In humans, the other member of the Gsx family, Gsc2, is in the region within 22q11 that is deleted most consistently in patients with DiGeorge Syndrome/VCFS, suggesting a possible link to NC development (Gottlieb et al., 1998).

6.11. Hlx

In mouse, the H2.0-like homeobox (Hlx) gene is expressed in intestinal and hepatic mesenchyme. Hlx is required for embryonic intestine and liver growth, and homozygous loss of Hlx causes embryonic lethality. Hlx function is indirectly connected to NC function in ENS development. Without Hlx, the development of the ENS, which requires interaction between the migrating NCCs and the gastrointestinal tract mesenchyme, is abnormal. In these mutant embryos, NCCs do not enter the intestine and are primarily restricted to the lateral stomach mesenchyme (Bates et al., 2006).

6.12. Hmx1

Hmx genes comprise a novel gene family. In mouse, there are three Hmx genes: Hmx1, Hmx2, and Hmx3. Hmx2 and Hmx3 are expressed in the CNS, but Hmx1 is divergent and is expressed in NC-derived DRGs, sympathetic ganglia, and vagal nerve ganglia. In addition, Hmx2 and Hmx3 are expressed in the otic vesicle, and Hmx1 is strongly expressed in the developing eye (Wang et al., 2000).

6.13. Irx

In Xenopus, the Iroquois-related homeobox gene Iro1 is one of the genes induced in the neural plate border, which includes placodal and NCC precursors, and induction is controlled by a precise level of BMP activity needed during signaling from the epidermis to the neural plate (Glavic et al., 2004a; Mayor and Aybar, 2001). Iro1, Notch, and the Notch target gene Hes4 are all expressed in the prospective NC territory, whereas Notch ligands Delta1 and Serrate are expressed in cells surrounding the prospective NCCs. An activator fusion form of Iro1 results in enlargement of the NC territory, whereas blocking Iro1 activity inhibited NC marker expression. Activation of Iro1 and Notch signaling resulted in upregulation of Hes4 and inhibition of Bmp4 transcription during NC specification. Iro1 lies upstream of the cascade regulating Delta1 transcription. During early gastrulation, Iro1, as a positive regulator, and Snail as a repressor, act to restrict Delta1 expression at the border of the prospective NC territory. Additional signals then induce the production of NCCs (Glavic et al., 2004b).

There are six iroquois homeodomain (Irx) transcription factors in the mouse (Mummenhoff et al., 2001). Irx family members are implicated in a variety of early developmental processes, including neural prepatterning, tissue differentiation, NC development, and cranial placode formation (Feijoo et al., 2009). An additional Irx family member, Iro7, is found in zebrafish. During zebrafish gastrulation, Iro7 and Iro1 are expressed in a region of the dorsal ectoderm that includes the prospective midbrain–hindbrain domain, adjacent NC, and the trigeminal placodes in the epidermis (Itoh et al., 2002; Lecaudey et al., 2001). Early expansion of Iro1 and Iro7 expression in headless and masterblind Wnt signaling mutants correlates with expansion of the midbrain–hindbrain boundary domain, the NC, and trigeminal neurons. Knockdown of Iro7 alone shows it is essential for specification of neurons in the trigeminal placode, whereas knockdown of Iro1 and Iro7 together uncovers essential roles in NC development, where they may function as transcriptional repressors (Itoh et al., 2002).

6.14. Lbx1 AND Lbx2

The ladybird homeobox (Lbx) gene family has two members in the mouse, Lbx1 and Lbx2. Lbx1 is found in hypaxial musculature, developing dorsal spinal cord neurons, and within a cardiac NC subpopulation required, along with myocardial cell proliferation and differentiation, during tubular heart formation (Schafer et al., 2003). Lbx1-null mice have heart looping defects, increased myocardial proliferation, and changes in gene expression, but no defects in NCC migration, suggesting that Lbx1 is needed for specification of this NC subpopulation. The Lbx1 promoter driving a lacZ reporter demonstrates that Lbx1 promoter activity is upregulated in hearts of Lbx1-heterozygous, Pax3Splotch1H/Splotch1H compound mutant embryos and Lbx1-null mice, indicating that Pax3 and Lbx1 participate in a negative regulatory feedback (Schafer et al., 2003). Lbx2 is expressed in the developing urogenital and nervous systems. Lbx2-null mice appear healthy and fertile. Lbx2-lacZ null mice intercrossed with Pax3Splotch heterozygous mutant mice results in embryos with reduced Lbx2 expression in DRGs and cranial nerve ganglia, but not in the genital tubercle, suggesting that Pax3 is required for Lbx2 expression in affected NC-derived tissues (Wei et al., 2007).

6.15. Msx1 AND Msx2

The muscle segment-related homeobox (Msx) gene family consists of Msx1, Msx2, and Msx3 in mammals, but expression of only Msx1 and Msx2 has been described in the NC. Msx genes are expressed in a range of vertebrate-specific tissues, including NC, cranial sensory placodes, bone, and teeth (Davidson, 1995). In the mouse, Msx1 and Msx2 are expressed during critical stages of NT, NC, and craniofacial development. Msx1 knockdown in whole embryo culture during early stages of neurulation causes reduced growth of the maxillary, mandibular, and frontonasal prominences (presumably due to mesenchymal deficiencies), as well as eye, somite, and NT abnormalities; Msx2 knockdown produces similar results. Double knockdown does not cause a more severe phenotype, suggesting that there is no redundancy or synergy between the two factors (Foerst-Potts and Sadler, 1997). An Msx1-null allele was generated by insertion of an nuclear lacZ reporter gene into the Msx1 coding region. LacZ expression from this allele in heterozygous embryos showed novel Msx1 expression in migrating NCCs. Homozygous null mice die at birth with craniofacial defects. In most regions of the face affected in these mutants, there is no overlapping expression of Msx2 (Houzelstein et al., 1997). Msx1 and Msx2 are both expressed at sites of cellular proliferation and programmed cell death, including the cranial NC. Bmp4 can regulate cell death at these sites and induce Msx1 and Msx2 expression, and Msx2 is a key regulator of apoptosis in a BMP-mediated pathway. Constitutive ectopic Msx2 expression in P19 cells results in a marked increase in aggregation-induced apoptosis but has no effect when cells are grown as a monolayer; in this system, addition of Bmp4 induces programmed cell death through Msx2 (Marazzi et al., 1997).

6.16. Pitx2

Heterozygous Pitx2 mutations cause ocular anterior segment defects and early-onset glaucoma. Pitx2 is expressed in NC- and mesoderm-derived precursors of the periocular mesenchyme. Pitx2 homozygous loss of function in mice results in severe disruption of periocular mesenchyme structures. Pitx2 is required specifically in the NC for specification of corneal endothelium, corneal stroma and sclera, and for normal development of ocular blood vessels (Evans and Gage, 2005). In the cardiac NC, Pitx2 may function as a target of canonical Wnt signaling, but it is not essential for cardiac NC development (Ai et al., 2006).

6.17. Prrx1 AND Prrx2

The paired-related homeobox genes Prrx1 and Prrx2 both play a role in epithelial–mesenchymal interactions in the pharyngeal arches. Prrx1 is expressed in the mesenchyme of facial, limb, and vertebral skeletal precursors during mouse embryogenesis and may regulate epithelial–mesenchymal interactions required for skeletal morphogenesis. Prrx1-homozygous-null mutants die soon after birth with defects of limb, vertebral, and NC-derived craniofacial skeletal structures caused by cellular defects in formation and expansion of chondrogenic and osteogenic precursors from undifferentiated mesenchyme (Martin et al., 1995). Prrx1 is one gene involved in patterning and morphogenetic processes in the NC-derived mesenchyme in the developing middle ear, and it is essential for tympanic ring development (Mallo, 2001). Prrx2 is also expressed in pharyngeal arch mesenchyme (de Jong and Meijlink, 1993) and at sites of epithelial–mesenchymal interactions, including within the cranofacial mesenchyme. During tooth development from the early bud stage to the late bell stage, Prrx2 is expressed exclusively in NC-derived ectomesenchyme and derivatives. In both first molar and incisor primordia, Prrx2 expression is highest at the late cap and early bud stages and declines at the mid-bell stage. Prrx2 is also present in first and second molar primordia cultured from E13 jaw explants (Karg et al., 1997). In Hand1; Hand2 compound mutants, Prrx2 is dysregulated in the distal craniofacial mesenchyme (Barbosa et al., 2007).

6.18. Runx1 AND Runx2

The runt homeobox gene family has three main homologs, Runx1, Runx2, and Runx3. In chicken and mouse, Runx1 is selectively expressed in NC-derived TrkA-positive sensory neurons to mediate activation of TrkA in migratory NCCs and promote axonal growth. Without Runx activity, TrkA expression is lost, leading to neuronal death. Overexpression of Runx1 is not compatible with migratory NC multipotency but does not induce expression of pan-neuronal genes on its own. Runx1-induced neuronal differentiation toward a TrkA-positive nociceptive sensory neuron fate depends on an existing Ngn2 proneural gene program (Marmigere et al., 2006). Forced expression of Runx1 in Sox10-expressing boundary cap NCSCs strongly increased survival and was sufficient to guide differentiation of boundary cap NCSCs toward a nonpeptidergic nociceptive sensory neuron fate (Aldskogius et al., 2009).

Runx2 is a master regulator of bone formation, and Runx2-null mice have a severe loss of the osteogenic skeleton. Runx2 is also expressed in the pre-hypertrophic cartilaginous skeleton of the mouse and chicken, but its function here is not clear. Unlike mouse and chicken, zebrafish and Xenopus require Runx2 function for early cartilage differentiation, and Runx2 is expressed in mesenchymal precursors of the cartilaginous skull (Deng et al., 2008; Kerney et al., 2007). Runx2, as an osteoblast marker, is ectopically expressed in Sox9-null mouse embryos, where nasal cartilages should normally be, suggesting that without Sox9, cranial NCCs may lose chondrogenic potential and adopt an osteogenic fate (Akiyama et al., 2005; Mori-Akiyama et al., 2003). Runx2a in zebrafish is also regulated by Sox9 (Yan et al., 2005), and in the absence of Fgfrl1a, both Sox9a and Runx2b are lost from the mesenchymal condensations of the pharyngeal arches (Hall et al., 2006). A number of proteins are upstream of Runx2 in the pharyngeal arch mesenchyme: Msx, Dlx5, Barx1, and Foxe1 are all positive upstream regulators of Runx2, whereas Hand2 is a negative regulator of Runx2. Msx genes are critical for Runx2 expression in frontonasal NCCs and differentiation of the osteogenic lineage (Han et al., 2007). In the chicken embryo, Dlx5 upregulates Runx2 and subsequent osteoblast differentiation in cultured embryonic suture mesenchyme. A dominant-negative Dlx interferes with the ability of the BMP pathway to activate Runx2 expression (Holleville et al., 2007). Foxe1 knockdown morphants, with craniofacial defects, have severe reduction in expression of Sox9a, Col2a1, and Runx2b (Nakada et al., 2009). Runx2a expression in zebrafish pharyngeal arches is lost upon knockdown of Barx1 (Sperber and Dawid, 2008). Hand2 and Runx2 are partially colocalized in the mandibular primordium of the pharyngeal arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice have upregulated and ectopic expression of Runx2 in the mandibular arch (Funato et al., 2009).

6.19. Tlx

The human T-cell leukemia homeobox gene Tlx1 is one of the genes involved in a chromosomal translocation occurring in 5–10% of patients with T-cell acute lymphoblastic leukemia. Accordingly, murine Tlx1 is expressed in the developing spleen and Tlx1-null mice are asplenic (Lichty et al., 1995), but Tlx1 is also expressed in cranial NC derivatives. Two additional members of the Tlx family, Tlx2 and Tlx3, are necessary for proper ANS development (Bachetti et al., 2005). In the chicken embryo, both Tlx1 and Tlx3 are expressed in placode-derived components of the cranial sensory ganglia, but only Tlx3 is expressed in NC-derived DRG and sympathetic ganglia (Logan et al., 1998). Tlx2 is expressed in the primarily vagal NC-derived ENS, and Tlx2-null mice have ENS defects resembling human intestinal neuronal dysplasia type B (Puri and Shinkai, 2004). In general, Tlx2, together with Phox2b, which is also downstream of BMP signaling and has an expression pattern similar to Tlx2, is critical for the development of NC-derived cells adopting ANS fates. Phox2b can bind a cell-specific enhancer in the 5' regulatory region of Tlx2 and activates Tlx2 in neuroblastoma cells. Tlx2 is upregulated by overexpression of Phox2b, and Phox2b protein carrying a mutation responsible for congenital central hypoventilation syndrome (CCHS) development has a severely hindered ability to activate Tlx2 expression in vitro and in vivo (Borghini et al., 2006). Tlx2 enhances the activity of the Ret promoter in a neuroblastoma cell line (Bachetti et al., 2005). Tlx proteins may be involved in differentiation and maintenance of specific neuronal populations (Logan et al., 1998).

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53140

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