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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 9Pax Genes

Vertebrate Pax genes are related to the Drosophila paired-rule gene, paired, which encodes a protein with two DNA binding domains, a paired domain and a paired-like homeodomain.

9.1. Pax3

Pax3 is the most well-characterized Pax protein in the NC. It is also one of the most well-characterized transcription factors of the NC and has been extensively studied in NC induction and cardiac NC and melanocyte lineages. Progress in understanding the function of Pax3 has been aided by a number of mutations in mice and humans.

9.1.1. Pax3 in Early Neural Crest Development and Migration

In the mouse, Pax3 is first detected at E8.5 in the dorsal neuroepithelium and adjacent to the segmented dermomyotome (Figure 9.1). Pax3 is expressed during early migration of NCCs and in somitic cells along the NCC migratory path (Serbedzija and McMahon, 1997). Between E10 and E12, Pax3 is detected in NCCs of the developing PNS, the NC-derived craniofacial mesenchyme, and the migratory cardiac NCCs (Goulding et al., 1991). Pax3 is generally extinguished in migratory NCCs as they differentiate, but maintained in the melanocyte lineage and also sustained in cultured NCSCs [such as multipotent SKPs (Zhao et al., 2009)] in later stages of development in various lineages. The timing of expression in early NC and subsequent downregulation as NC-derived cells differentiate also implicate Pax3 as an important factor for maintaining progenitor populations. In fact, persistent Pax3 misexpression in cranial NCCs resulted in cleft palate and other craniofacial defects, ocular defects, and perinatal lethality (Wu et al., 2008). One phenotype associated with persistent Pax3 expression is that BMP-induced osteogenesis is blocked via upregulation of the Pax3 target Sostdc1, a soluble BMP inhibitor. This is one example by which Pax3 plays a role in maintaining an undifferentiated state by blocking responsiveness to differentiation signals (Wu et al., 2008).

FIGURE 9.1. Whole-mount in situ hybridizations show Pax3 in mouse, chicken, fish ( pax3a), and frog embryos.

FIGURE 9.1

Whole-mount in situ hybridizations show Pax3 in mouse, chicken, fish ( pax3a), and frog embryos. Mouse: Solloway and Robertson, 1999, 12-somite embryo, dorsal view; chicken: Bothe and Dietrich, 2006, HH10 stage embryo, dorsal view; zebrafish: Minchin (more...)

One of the reasons Pax3 function is well-characterized in the NC is the availability of a series of mutations in the murine Pax3 gene. These alleles represent a range of null, severely hypomorphic, and mildly hypomorphic forms of Pax3. Embryos homozygous for the Pax3-null mutation Pax3Splotch have NC defects (Henderson et al., 1997; Tremblay et al., 1995) including reduction or loss of spinal and sympathetic ganglia, cranial ganglia defects (Franz and Kothary, 1993; Tremblay et al., 1995), pigmentation defects, and cardiac outflow tract (OFT) septation failure (Henderson et al., 1997). Pax3Splotch NCCs fail to fully colonize target tissues because of a severe reduction in the number of NCCs that emigrate from the neural tube (NT) at the vagal and rostral trunk levels and a complete loss of cells at the caudal thoracic, lumbar, and sacral levels. There are conflicting data in the literature concerning the cell autonomy of the NC phenotype caused by a loss of Pax3. Transplants of neural tissue between mouse and chicken embryos indicate that defects in Pax3Splotch embryos are not intrinsic to the NCCs but reflect inappropriate cell interactions either within the NT or between the NT and somites (Serbedzija and McMahon, 1997). Chimeric embryos generated by injecting ES cells with a LacZ knock-in in the Pax3 locus (Pax3Splotch2G ) into wild-type blastocysts demonstrate that Pax3-deficient NCCs are rescued when surrounded by a wild-type environment (Mansouri et al., 2001). However, rescue of Pax3Splotch defects by expression of Pax3 under the control of an NT and NC-specific 1.6-kb minimal promoter demonstrates that normal NC migration and function can occur adjacent to Pax3-deficient somites, suggesting that Pax3 functions cell autonomously in the NC (Li et al., 1999).

In addition to NC defects, Pax3Splotch and Pax3Splotch2H mutants display neurulation defects and altered somitogenesis. One link between these three tissue types (NC, NT, and somites) is the importance of extracellular matrix (ECM) composition and expression of cell adhesion molecules. Versican, a chondroitin sulfate proteoglycan nonpermissive for NCC migration in vitro, is upregulated in the NCC migration path of Pax3Splotch2H embryos. Pax3 and versican normally have mutually exclusive expression; and when Pax3 is lost, the versican expression domain is expanded, correlating with an absence of migrating NCCs lateral to the NT and in the lower pharyngeal arches (Henderson et al., 1997). Ncam, another Pax3 target, depends on the attachment of polysialic acid (PSA) for its adhesive properties. Pax3Splotch2H homozygotes have a reduction of PSA-Ncam starting around E12.5, which could contribute to decreased NC migration (Glogarova and Buckiova, 2004), and the St8sia2 (a-2,8-polysialyltransferase) gene (also known as Stx in humans) is also a Pax3 target (Mayanil et al., 2001).

A wealth of knowledge regarding NC induction has been gained from studies in frog and chicken. In Xenopus, Msx1 can induce multiple early NC genes including Pax3 and Zic1 (Monsoro-Burq et al., 2005), which are among the first genes expressed in response to neural plate border-inducing signals (Davidson and Keller, 1999). Where Pax3 and Zic1 expression overlap, they act together (before other early NC marker genes like Foxd3 and Slug are expressed) to specify the neuroectoderm to adopt a NC fate (Hong and Saint-Jeannet, 2007; Sato et al., 2005) and activate Slug expression in a Wnt-dependent manner (Monsoro-Burq et al., 2005). Multiple experiments demonstrate that Pax3 and Zic1 together are both necessary and sufficient to specify NC (Hong and Saint-Jeannet, 2007; Sato et al., 2005). This process requires Fgf (Monsoro-Burq et al., 2005) and Wnt signaling (Bang et al., 1999; Sato et al., 2005); a dominant-negative version of Wnt8 blocks expression of both Pax3 and Msx1, and injection of Wnt8 results in an expansion of Pax3 expression in the lateral neural plate (Bang et al., 1999). In addition, expression of eIF4AII in Xenopus animal cap explants causes upregulation of Pax3 and other neural plate border genes such as Snail and Slug (Morgan and Sargent, 1997). In the chicken embryo, Pax3 is expressed in the dorsal NT and plays a role in specification of NCC (Osorio et al., 2009). Pax3 is upregulated in the dorsal NT in response to Wnt signaling but also depends on BMP signaling. By the start of NC migration, Pax3 begins to be downregulated (Burstyn-Cohen et al., 2004; Taneyhill and Bronner-Fraser, 2005).

Exploring the lineage and function of Pax3-expressing cells has been aided by the generation of genetic tools in the mouse. A 1.6-kb NT/NC minimal promoter fragment directs NC expression of Cre recombinase in transgenic mice (Li et al., 2000), allowing for fate-mapping of NC derivatives in combination with the R26RlacZ reporter mouse. Pax3Cre, a knock-in of Cre recombinase into the Pax3 locus, resulted in a new Pax3-null allele with homozygous defects indistinguishable from Pax3Splotch homozygotes. Pax3Cre/+ mice were used to fate-map all derivatives of endogenous Pax3-expressing cells, confirming known NC and somitic derivatives in addition to uncovering a few previously undetected lineages (Engleka et al., 2005). A hypomorphic allele, Pax3neo, results in an 80% reduction of Pax3 protein in homozygotes, and reduced limb and tongue musculature due to increased apoptosis within somites, and postnatal mortality due to an inability to suckle. However, the heart, diaphragm, trunk musculature, various NC-derived lineages, and NT were all unaffected by these partially reduced Pax3 levels. Elevated levels of the related Pax7 protein were present in unaffected neural tube and epaxial somatic components, suggesting redundancy in protein function and links in gene regulation (Zhou et al., 2008a).

9.1.2. Pax3 in Neural and Glial Lineages

After Pax3 expression is extinguished in migratory NCCs, it becomes expressed once more as they condense to form DRGs and a subset of the cranial ganglia (Baker et al., 2002). While Pax3 is detected in placodal-derived cranial neurons, Pax3 is not detected in NC-derived neurons in these ganglia, suggesting either that Pax3 is only initially expressed in glial cells or that Pax3 expression is turned off completely before neuronal differentiation proceeds (Baker et al., 2002). Pax3 is also expressed in NC-derived Schwann cell (SC) precursors when they migrate to the PNS (Kioussi et al., 1995). During SC development, Pax3 function is modulated by Sox10, a key regulator of glial development (Kuhlbrodt et al., 1998a). In developing sciatic nerves, there is an inverse correlation between expression of Pax3 and myelin basic protein (Mbp) both in vivo and in SC primary cultures. Injection of Pax3 expression vector causes a decrease in Mbp and Pax3 represses a 1.3-kb Mbp promoter fragment in cotransfection assays, suggesting that it represses Mbp transcription directly (Kioussi et al., 1995) as one way of preventing differentiation of SC precursors to myelinating SCs (Jessen and Mirsky, 1998; Kioussi et al., 1995).

In the absence of functional Pax3 (such as in Pax3Splotch mutants), the NCCs migrating to the PNS undergo premature neurogenesis (evidenced by increased Brn3 positive staining in neural tube explants), perhaps due to a change in the regulation of genes such as Hes1 and Ngn2 (needed for differentiation and proliferation) by directly binding to their promoters. In this role, Pax3 may couple migration with NCSC maintenance and neurogenesis (Nakazaki et al., 2008). In vitro, a different phenomenon is seen. Pax3 is initially expressed in all NCCs from culture but is subsequently only retained in neurons. Pax3Splotch homozygous NC cultures had an 80% reduction in the capacity to generate sensory-like neurons. Downregulation of Pax3 in DRG cultures inhibited 80–90% of newly generated sensory neurons but had no effect on survival of sensory neurons or precursors (Koblar et al., 1999).

9.1.3. Pax3 in the Cardiac Neural Crest

Pax3 expression marks migrating cardiac NCCs in the mouse embryo, but Pax3 expression is extinguished before NC population of the heart. Cardiac NCCs that populate the heart are indeed generated from Pax3-expressing precursors (Engleka et al., 2005; Epstein et al., 2000; Li et al., 2000). Mutation of Pax3, as occurs in Pax3Splotch2H homozygotes, results in development of heart defects including persistent truncus arteriosus (PTA), signs of cardiac failure (Conway et al., 1997a; Conway et al., 1997b; Conway et al., 1997c), and defects of the aortic arches, in addition to thymus, thyroid and parathyroid defects, ultimately leading to embryonic lethality. In the developing thymus and parathyroid, Pax3-expressing NCCs contribute to patterning of the endoderm of the third pouch into appropriately fated thymus vs. parathyroid domains (Conway et al., 1997b; Griffith et al., 2009). Between 60% and 85% of Pax3Splotch2H homozygotes die at E13.5–E14.5 (Conway et al., 1997a; Conway et al., 1997c). Closely related Pax3Splotch homozygotes also die by E13.5 with approximately the same spectrum of cardiac defects (Epstein et al., 2000). Pax3-expressing NCCs emigrate from the NT of both Pax3Splotch and Pax3Splotch2H homozygotes, but there is a reduction of total NCCs (as indicated by multiple markers, including AP-2) that pass through the pharyngeal arches and into the cardiac OFT (Conway et al., 2000; Conway et al., 1997b; Epstein et al., 2000). NC abnormalities in Pax3Splotch2H homozygotes result in defective excitation-contraction coupling, as indicated by a significant reduction in Ca2+ transients. There is a correlation between the presence of OFT septation defects (~60–85%) and defects in other NC lineages, such as reduction or absence of DRGs (Conway et al., 1997c). Embryos without a PTA survive until birth but then they die of neural tube defects (NTDs). These embryos do not have excitation–contraction coupling defects (Conway et al., 1997a) and have less severe defects in other NC lineages like the DRG, indicating that excitation coupling and earlier lethality were secondary to an underlying NC-related heart defect (Conway et al., 1997a; Conway et al., 1997c). The reduction in NCC number in Pax3Splotch2H homozygotes is not due to impaired migration or effects on proliferation and apoptosis in migrating NCCs but rather due to a failure of the NCCs to proliferate while still in the neural folds (before the onset of migration), which corresponds with a decrease in Wnt1 levels (Conway et al., 2000). In Pax3Splotch homozygotes, defects are again not due to disruption of cardiac NC migration per se but rather to a reduction in the number of initial progenitors and an alteration of more finely tuned migratory behavior in mutant cardiac NCCs (Epstein et al., 2000). The reduction in the initial pool of cardiac NCC progenitors is due, at least in part, to Pax3 regulation of p53-dependent apoptosis. Pax3 inhibits p53-dependent apoptosis in the dorsal NT to regulate neural tube closure (Pani et al., 2002) and is also important for normal development of the cardiac NC. In Pax3Splotch nulls, there were fewer cardiac NCCs due to an increase in cell death as migration occurred. Loss or suppression of p53 prevents defective CNC migration and apoptosis in Pax3Splotch homozygotes and restored proper OFT septation (Morgan et al., 2008a).

Expression of Pax3 in the NT and NC is also affected by environmental conditions. Oxidative stress (such as that induced by hyperglycemia in diabetic mothers) can somewhat inhibit the expression of Pax3 in the neuroepithelium (Kumar et al., 2007; Morgan et al., 2008b), and this correlates with an increased risk of congenital heart and neural tube defects observed with gestational diabetes (Kumar et al., 2007; Morgan et al., 2008b). In these embryos, few cardiac NCCs migrated to the heart and p53-mediated apoptosis of progenitor cells increases along the normal migration path (Chappell et al., 2009; Morgan et al., 2008b). Along with reduced proliferation (Kumar et al., 2007), this resulted in a significant increase in OFT and other defects (Kumar et al., 2007; Morgan et al., 2008b). These defects could be ameliorated by addition of antioxidants before Pax3 expression (Morgan et al., 2008b). Folic acid deficiencies, an independent environmental stress, are associated with increased risk for congenital heart defects, and impaired folic acid transport results in extensive apoptosis in the OFT and interventricular septum correlating with a significant reduction in Pax3 expression in the presumptive migrating cardiac NC (Tang et al., 2004). Similar to the reduction of Pax3 expression due to hyperglycemic oxidative stress, cardiac NC defects likely arise due to a reduction in the size of an initial progenitor pool. Similarly, administration of nitrofen in rats causes a reduction in Pax3 expression, changes in NC signaling, and heart defects, among other effects, and both Pax3 expression and the heart defects can be mostly recovered by administration of vitamin A (Gonzalez-Reyes et al., 2005; Gonzalez-Reyes et al., 2006). Pax3 is also one of the genes upregulated in the NT in response to arsenate treatment associated with an increase in NT closure and NCC migration defects (Wlodraczyk et al., 1996).

9.1.4. Pax3 in the Melanocyte Lineage

A subset of the NC-derived cells that emigrate from the NT travels through the dorsolateral pathway and becomes committed to the melanoblast lineage; these cells maintain Pax3 expression. Pax3 expression in the melanocyte lineage is conserved in mammals and birds (Lacosta et al., 2005). Pax3 is one of the factors in a network (along with Sox10 and Mitf) crucial for melanocyte stem cell survival and regulation of differentiation and melanin synthesis (Boissy and Nordlund, 1997; Sommer, 2005). Pax3 directly binds to and activates the Mitf and Trp1 promoters (Corry and Underhill, 2005; Lang and Epstein, 2003). When Sox10 is independently bound to the Mitf promoter, Mitf transcription is synergistically activated. The downstream genes Tyr, Tyrp1, and Dct, essential for melanin synthesis, have binding sites for Pax3, Mitf, and Sox10 (Corry and Underhill, 2005; Murisier and Beermann, 2006), indicating that a complex interaction network maintains the balance between melanocyte stem cells and differentiated melanocytes. Indeed, although Pax3 activates Mitf expression, it also competes with Mitf for occupancy of the Dct promoter and represses Dct expression. Pax3 thus maintains the undifferentiated state of these cells until repression by Pax3 is relieved through activation of the Wnt signaling pathway (Lang et al., 2005). Analysis of Pax3Splotch embryos also reveals a significant reduction in the number of melanoblasts indicating a role for Pax3 in expanding the pool of restricted progenitor cells by regulating Mitf, which is necessary for melanoblast survival (Hornyak et al., 2001). Interestingly, Pax3 is one of the genes greatly reduced in white or graying hairs, further reinforcing its role in melanocyte stem cell populations (Choi et al., 2008). This role for Pax3 in pigment cell development is conserved in zebrafish. Pax3 MO knockdown results in defective fate specification of xanthophores, but the other two pigment lineages, melanophores and iridophores, are specified and differentiate normally. Loss of xanthophores is likely due to a Pax3-driven fate switch within a pigment cell precursor population (Minchin and Hughes, 2008). Foxd3 can act to inhibit Pax3 activation of Mitf by directly binding the Pax3 protein and sequestering it from binding to the Mitf promoter (Thomas and Erickson, 2009).

Another mechanism for regulation by Pax3 is differential expression of isoforms. In mice and humans, seven alternatively spliced isoforms (a, b, c, d, e, g, and h) of Pax3 have been described, with Pax3c being the most well-characterized isoform. In mouse melanocytes in vitro, the effect of Pax3 on proliferation, migration, survival, and transformation varies depending on the isoform, with some isoforms having opposite effects (Wang et al., 2006). Microarray experiments suggest that these varying effects are due to differential regulation of distinct yet overlapping sets of genes involved in cell differentiation, proliferation, migration, adhesion, apoptosis, and angiogenesis by the various isoforms (Wang et al., 2007).

9.1.5. Pax3 in Waardenburg Syndrome, Tumors, and Other Neurocristopathies

Waardenburg syndrome (WS) patients have an autosomal dominant combination of auditory and pigment defects. This is due to a loss of melanocytes from the skin, hair, eyes, and cochlea of the inner ear, reminiscent of an NC deficiency (Read and Newton, 1997) and remarkably similar in phenotype to the Pax3Splotch heterozygous mutant mouse. Multiple studies identified Pax3 mutations in families with either type 1 or type 3 WS (Karaman and Aliagaoglu, 2006; Ptok and Morlot, 2006; Read and Newton, 1997; Tassabehji et al., 1993; Tassabehji et al., 1994; Tassabehji et al., 1995; Van Camp et al., 1995). These mutations typically alter conserved amino acids in either of the DNA binding domains or cause a loss-of-function via deletion, nonsense, splice-site, or frame-shift mutations (Tassabehji et al., 1993; Tassabehji et al., 1994; Tassabehji et al., 1995), and there is usually a close correspondence in humans and mice between the type of Pax3 molecular lesion, gene dosage, and strength of observed phenotype (Corry et al., 2008; Tassabehji et al., 1994). A subset of type 2 WS cases has been linked to mutations in Mitf (Lalwani et al., 1998; Potterf et al., 2000) and Pax3 enhances activation of Mitf by Sox10 (Potterf et al., 2000). Misregulation of Mitf due to mutation of Pax3 in type 1 and type 2 WS may also cause at least part of the WS phenotype (Potterf et al., 2000). Some patients with WS also have characteristics of Hirschsprung disease, and Pax3 is required for normal enteric ganglia formation. Pax3 can bind to and activate expression of Ret in coordination with Sox10, and mutations in both have been associated with Waardenburg–Hirschsprung syndrome (Lang et al., 2000; Lang and Epstein, 2003). This is reflected in the role for Pax3 in both pigment cells (xanthophores) and ENS in zebrafish (Minchin and Hughes, 2008). Interestingly, Pax3 mutations were typically not found in families with other neurocristopathies (Tassabehji et al., 1995).

Because of its critical role during development, especially in progenitor cell populations, it is not surprising that Pax3 also plays a role in tumor formation. Many human neuroectodermal tumors express Pax3 (Gershon et al., 2005; Parker et al., 2004; Scholl et al., 2001; Schulte et al., 1997). Pax3 was detected in combination with Pax7 in poorly differentiated tumors and tumors with malignant potential (Gershon et al., 2005). Pax3 is specifically detected in most NC-derived Ewing’s family tumors (Schulte et al., 1997) and in most peripheral neuroectodermal tumors (Schulte et al., 1997), small cell lung cancer (Parker et al., 2004), and in most primary cultured melanomas (He et al., 2005; Parker et al., 2004; Scholl et al., 2001). Pax3 expression was specific to tumor cells and not detected in surrounding normal tissue or in benign lesions, and downregulation of Pax3 expression in these tumors resulted in apoptosis of primary melanoma cells (He et al., 2005; Scholl et al., 2001), suggesting a role for Pax3 in melanoma cell survival.

9.1.6. Pax3 Regulation and Interaction with Other Genes

In addition to genetic interactions that have been discussed in previous sections, a few direct upstream regulators of Pax3 have been identified. Tead2 and its coactivator Yap1 are coexpressed with Pax3 in the dorsal NT. Tead2 binds an NC-specific enhancer in the Pax3 locus and activates Pax3 expression, and mutation of the Tead2 binding site prevents neural expression of Pax3. Further supporting the role of Tead2 as an activator of Pax3, a Tead2-Engrailed fusion protein represses retinoic acid-induced Pax3 expression in P19 embryonal carcinoma cells and in vivo (Milewski et al., 2004). A minimal promoter element for NT and NC expression in the Pax3 upstream regulatory sequence contains putative interaction sites that have been tested in P19 cells induced to express Pax3 by retinoic acid (RA) treatment. Two sites interact with the neural-specific genes Brn1 and Brn2, whereas other sites interact with Hox/Pbx heterodimers and Meis monomers, and all of these sites are important for normal Pax3 expression. Ectopic expression of both Brn2 and HoxA1 together induces Pax3 expression (Pruitt et al., 2004). Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac NCCs that ultimately specifies cardiac NCC function for OFT development but does not regulate NCC migration to the heart. Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2-null; Pbx1-null embryos display significant rescue of OFT septation, demonstrating that disruption of the Pbx1–Pax3–Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null embryos (Chang et al., 2008). In the neural tube, Nf1 modifies Pax3Splotch mutant defects through a genetic interaction, and both of these factors play important roles in development of NC-derived structures (Lakkis et al., 1999).

Genetic interactions in Pax3Splotch embryos and interactions assayed in vitro have identified some downstream effectors of Pax3. The promoter activity of Lbx1, a gene expressed in cardiac NC during tubular heart formation, is upregulated in Pax3Splotch mutants. Lbx1 nulls have defects in heart looping and myocardial hyperplasia, and Lbx1 may be required for specification of a subpopulation of cardiac NC subsequent to migration (Schafer et al., 2003). Pax3Splotch mutant embryos have reduced Lbx2 expression, as indicated by an Lbx2lacZ knock-in allele, in DRGs and cranial nerve ganglia, but not in the urogenital system, where Lbx2 is also expressed, suggesting that Pax3 is required for Lbx2 expression in NC-derived tissues (Wei et al., 2007). Foxd3 is genetically downstream of Pax3 and is not expressed in regions of Pax3 mutant embryos lacking NC (Dottori et al., 2001). In addition, direct targets of Pax3 have been identified in several NC lineages. Wnt1 is expressed in premigratory NC, and Pax3 specifically binds the Wnt1 promoter to regulate expression in a dose-dependent manner in the developing embryo (Fenby et al., 2008). Pax3 regulates TGFb2, a modifier of NC migration and differentiation, by directly binding its promoter (Nakazaki et al., 2009), and loss of one allele of Pax3, such as in Pax3Splotch heterozygotes, can reverse the TGFb2-null NTD phenotype (Nakazaki et al., 2009). Aberrant upregulation of Msx2, a regulator of BMP signaling, in Pax3Splotch mutant mice may lead in part to deficient migration of cardiac NC and OFT septation failure, and Pax3 represses Msx2 expression directly through a conserved Pax3 binding site in the Msx2 promoter (Kwang et al., 2002). A direct, physical protein–protein interaction has been shown between Pax3 and Hira, a transcriptional corepressor expressed in the NC that also physically interacts with core histones. This suggests that at certain targets, Pax3 may recruit Hira and exert a repressive effect on transcription via chromatin modifications. Hira mutants display OFT defects and Hira maps to the DiGeorge/velocardiofacial syndrome critical region 22q11. Pax3Splotch homozygotes have many of the hallmark defects of DiGeorge syndrome (Magnaghi et al., 1998).

9.2. Pax6

The Pax6 gene is disrupted in both mouse and rat small eye (Sey) mutants. Homozygous null Pax6Sey rodents have eye defects similar to a group of human developmental disorders such as Peters’ anomaly, Axenfeld–Rieger anomaly, and aniridia (Cvekl and Tamm, 2004), as well as craniofacial defects of the ocular and frontonasal regions (Kanakubo et al., 2006; Matsuo et al., 1993; Osumi-Yamashita et al., 1997) due to defective NCC migration (Matsuo et al., 1993; Nagase et al., 2001; Osumi-Yamashita et al., 1997). In the frontonasal region, Pax6 is strongly expressed primarily in the frontonasal ectoderm and is required non-cell-autonomously to guide NC migration (Osumi-Yamashita et al., 1997). In the eye, Pax6 appears to be a key factor in the interaction between NC-derived cells and placodal-derived cells (Cvekl and Tamm, 2004; Song et al., 2007). Pax6 is strongly expressed in the non-NC-derived epithelial cells of the anterior segment where it is required for lens induction and corneal and retinal development (Kanakubo et al., 2006), and it is also required non-cell-autonomously for migration of the NCCs that contribute extensively to the ocular mesenchyme (Kanakubo et al., 2006). The main result of Pax6 loss in the eye is failure of NCC migration through the Pax6-null surrounding epithelial cells, but two studies have shown that even though Pax6 is most predominantly expressed in the epithelial cells critical for anterior eye development (lens, cornea, retina), it is also expressed at low levels in the NC-derived ocular mesenchyme (corneal stroma, ciliary body, iridocorneal angle, and trabecular meshwork progenitors) (Baulmann et al., 2002; Collinson et al., 2003). Weak expression of Pax6 in NC-derived cells of the developing eye is seen from P1 to P5 and becomes weaker at later stages, with no NC Pax6 detected by P14 (Baulmann et al., 2002). In the TM progenitor cells, Pax6 is downregulated upon differentiation to trabecular meshwork cells, consistent with a role for Pax6 in maintaining cells in a progenitor state.

Pax6 is also expressed in NC-derived mesenchymal cells of the submandibular gland (SMG), otherwise known as the salivary gland. In Pax6Sey mutants, the NC-derived SMG mesenchyme exhibits defective migration (Jaskoll et al., 2002). Surprisingly, Pax6 is not detected in the epithelium, suggesting that here Pax6 has a predominantly cell-autonomous function in contrast to anterior eye development. After SMG branching morphogenesis occurs, Pax6 mesenchymal expression can no longer be detected (Jaskoll et al., 2002). Pax6 is involved in FGF-mediated branching morphogenesis of other tissues, so it may play a similar role in the SMG (Makarenkova et al., 2000).

The role of Pax6 in the eye is also likely conserved among nonmammalian vertebrates. In the chicken embryo, a population of periocular mesenchyme cells expresses Pax6 along with Lmx1b and Pitx2. This population seems to be under the control of Six3, also expressed in a subset of periocular mesenchymal cells and in differentiating anterior segment tissues. Misexpression of Six3 causes a reduction of the Pax6/Lmx1b/Pitx2-expressing mesenchymal population and disrupts the integrity of the corneal endothelium and the expression of ECM components critical for corneal transparency, leading to anterior eye defects (Hsieh et al., 2002). In Xenopus, administration of VPA causes a decrease in Pax6 expression in the eye and perturbation of NC migration and abnormal retinal development (Pennati et al., 2001). In zebrafish, morpholino knockdown of Olfm2 (olfactomedin-2 or OM2) results in alteration of Pax6 expression pattern in the eye and coincident disruption of ocular development (Lee et al., 2008a). Olfm2 knockdown also affects development of caudal pharyngeal arches resulting in defects in cartilaginous structures, indicating cranial NC-related defects (Lee et al., 2008a).

9.3. Pax7

In the mouse, Pax7 is expressed in the cranial NC and its derivatives during embryogenesis in addition to skeletal muscle and the CNS (Lang et al., 2003; Mansouri et al., 1996). Maintenance of Pax7 expression in the adult has also been described in NC-derived Schwann cells in the inner and outer capsules of neuromuscular spindles of hindlimb skeletal muscle (Rodger et al., 1999). Pax7 homozygous-null mice die of NC-related craniofacial defects shortly after weaning. Because Pax7 and Pax3 are highly homologous and have overlapping expression patterns, there is functional redundancy between the two genes (Mansouri et al., 1996). Complex gene regulatory interactions between Pax3 and Pax7 may also allow one of these proteins to compensate for the loss of the other; in Pax3neo hypomorphic mice, Pax3 protein is reduced by 80%, but where Pax3 and Pax7 expression overlap, Pax7 is upregulated and no NC defects are observed (Zhou et al., 2008a).

Pax7 in the chicken embryo is expressed in NCCs, muscle precursor cells, and the developing CNS (Kawakami et al., 1997). Pax7 is one of the earliest indicators of NC precursors, along with Pax3, Snail2, Foxd3, and Sox9, with expression beginning in the dorsal neural tube immediately after cavitation (Lacosta et al., 2005; Osorio et al., 2009) and indicates a region of early NC induction. Pax7 is necessary for NC formation and coincident induction of the markers Slug, Sox9, Sox10, and HNK-1 (Basch et al., 2006). Pax7 expression is maintained in migrating trunk NC and is then sequentially downregulated first in neuronal, then glial, then melanocyte precursors (Lacosta et al., 2005). In avian species, both Pax3 and Pax7 are expressed in melanocytes, but in the mouse, only Pax3 is expressed (Lacosta et al., 2005).

The expression pattern of zebrafish Pax7 within the NC also differs slightly from that of mouse and chicken (Seo et al., 1998). There are three additional zebrafish Pax7 truncated isoforms (Seo et al., 1998). During early zebrafish embryogenesis, both cranial and trunk NC express Pax7. As in other species, Pax7 is broadly expressed in cranial NCCs. Pax7 is also expressed in the trunk NC and includes both premigratory and migratory NCCs. Pax7 is expressed in pigment precursor cells (melanophore, xanthophore, and iridophore precursors). In melanophore precursors, Pax7 expression overlaps with early melanin pigment, one difference between zebrafish and the chicken embryo (Lacosta et al., 2007). Pigment stem cells present during the larva to adult transition also express Pax7 (Lacosta et al., 2007). Pax7 is one marker of the xanthophore lineage in zebrafish, and a reduction in Pax7 is seen after morpholino knockdown of Pax3, which causes defects in xanthophore fate specification, whereas melanophores and iridophores are specified and differentiate properly (Minchin and Hughes, 2008).

Although human Pax7 is normally restricted to mesoderm and not detected in the NC (Gershon et al., 2005.), Pax7 expression was detected along with Pax3 in human neuroectodermal tumors (Gershon et al., 2005). Pax7 is disrupted by a translocation specific for alveolar rhabdomyosarcoma, similar to Pax3/Foxo1(Fkhr) translocations (Floris et al., 2007). Not much is known about regulation of Pax7 expression in any vertebrate species. Distinct upstream and intronic enhancer elements in the Pax7 locus have been identified that can direct expression of Pax7 in the cranial NC, facial mesenchyme, mesencephalon, and pontine reticular nucleus (Lang et al., 2003). Likewise, only minimal progress has been made in identifying functional NC targets of Pax7. One study identified 34 candidate target genes by an unbiased ChIP assay and verified Pax7 occupation and activation of sites within Gbx1, Eya4, CntfR, Kcnk2, and Camk1d (Hong et al., 2008a).

9.4. Pax9

Most of the investigation into the role of Pax9 in the NC has been carried out in the mouse. Mouse Pax9 is expressed in the cranial NC, the midbrain, somites, limb mesenchyme, and in foregut endoderm derivatives (Kist et al., 2007; Peters et al., 1998). Mesenchymal cells expressing Pax9 in the nose, palate, and teeth are derived from NC (Kist et al., 2007). In the mandibular arch mesenchyme, Pax9 marks the prospective sites of tooth development before morphological indicators appear. This expression is maintained in the mesenchyme of the developing teeth, and Pax9 is necessary for tooth development to proceed beyond the bud stage (Amano et al., 1999; Peters et al., 1998). Pax9 homozygous-null mice die at birth, but NC-specific deletion of a floxed allele of Pax9 using Wnt1-Cre results in mice with cleft secondary palate and lack of tooth development (Kist et al., 2007). Pax9 in the arch mesenchyme may be regulated (directly or indirectly) by the transcription factors Hand1 and Hand2. Hand1; Hand2 compound-null mutant embryos have misregulated levels of mesenchymal Pax9 expression at E12.5 and display later mandible and tooth defects (Barbosa et al., 2007). Unlike the developing tooth, where mesenchyme originates from both NC and non-NC cells, the mesenchyme of the SMG is exclusively NC-derived (Jaskoll et al., 2002). During SMG branching morphogenesis, before budding, Pax9 is expressed in both the mandibular oral epithelium and the adjacent NC-derived mesenchyme. As the SMG undergoes epithelial thickening, Pax9 is predominantly epithelial, but some expression of Pax9 remains in the mesenchyme. A functional role for Pax9 in SMG branching morphogenesis has not been defined but likely shares some characteristics with tooth development (Jaskoll et al., 2002).

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53138

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