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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 13Sox Genes

The Sry-related HMG box (Sox) family consists of transcription factors that contain a conserved HMG box DNA-binding domain. Sox genes are often associated with regulation of fate specification and cellular differentiation during development and often function as transcriptional activators. These genes have been separated into classes from SoxA to SoxE based primarily on DNA-binding domain similarity (for phylogeny, see Bowles et al., 2000).

13.1. Sox2

Of the SoxB genes (Sox1, 2, 3, 14, 19, and 21), only Sox2 has been reported to play a cell-autonomous role in NC development. Sox2 is one of the early genes activated in the developing neural plate (Rogers et al., 2009), but it also has a broader role in NC lineages and in maintaining multipotency in other stem cell types. In Xenopus, induction of NC is always accompanied by neural plate induction, as marked by Sox2 (Bastidas et al., 2004). Although Sox2 is commonly used as a neural plate or early neural marker, neural plate expression of avian Sox2 is reduced as the NC segregates from the dorsal neural tube and migrating NCCs maintain a low Sox2 expression level. Expression of Sox2 in a subset of these cells that contribute to the developing PNS is subsequently upregulated and gradually becomes restricted to NC-derived glial sublineages (Aquino et al., 2006; Wakamatsu et al., 2004). Misexpression of Sox2 in embryonic ectoderm and in neural plate explants reveals that Sox2 inhibits NC formation, whereas later in the NC lineage (migratory and postmigratory NCCs, particularly those of the PNS), Sox2 plays a role in proliferation and differentiation (Wakamatsu et al., 2004). Conserved enhancers driving expression of chicken Sox2 in the NC have been identified (Uchikawa et al., 2003), and another study implicates the Nk-1-related gene Nbx as playing an upstream role in negatively regulating Sox2 expression and positively regulating the NC-inducing transcription factor Slug (Kurata and Ueno, 2003).

Sox2 is one of a select group of factors able to induce somatic cells to adopt a pluripotent state, as demonstrated in numerous induced pluripotent stem cell publications (Park et al., 2008; Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2009). Multiple NCSC lines express Sox2 (Aquino et al., 2006; Techawattanawisal et al., 2007; Thomas et al., 2008; Widera et al., 2009; Zhao et al., 2009). For instance, boundary cap NCSCs (bcNCSCs) are capable of differentiation into mature, functional Schwann cells (SCs) in the presence of neuregulins and express Sox2 in addition to other NC markers (Aquino et al., 2006). Multipotent NCSCs derived from rat periodontal ligament (PDL) and cultured as neurospheres express Twist, Slug, Sox2, and Sox9 (Techawattanawisal et al., 2007). A common theme recently uncovered by several studies of adult-derived NCSCs is the coexpression of a set of more NC-specific genes with a set of “reprogramming” genes, and Sox2 seems to be one factor that overlaps between the two groups. NCSCs capable of neuronal and glial differentiation derived from the rat palate (pNCSCs) and cultured as neurospheres express the NCSC markers Nestin, Sox2, and p75 as well as Klf4, Pou5f1/Oct4, and Myc and differentiated efficiently into neuronal and glial cells. This expression pattern seems to be conserved: the human palate expresses Nestin, Sox2, Pou5f1/Oct4, Klf4, and Myc (Widera et al., 2009). Multipotent skin-derived progenitors isolated from the pig (pSKPs), another adult-derived NCSC type, coexpress the pluripotency genes Pou5f1/Oct4, Sox2, Nanog, and Stat3, in addition to the NC markers p75, Twist1, Pax3, Snail2, Sox9, and Sox10, and these patterns likely apply to mouse and human SKPs (Zhao et al., 2009). Finally, self-renewing human NCC (hNCC) lines derived from pharyngulas have a transcript profile more similar to pluripotent ES cells than that of hNCC derivatives, and this includes the pluripotency markers Nanog, Pou5f1/Oct4, and Sox2 (Thomas et al., 2008).

13.2. Sox4

Of the SoxC genes (Sox4, 11, 12, 22, and 24 ), only Sox4 has a demonstrated role in the NC. The expression pattern of Sox4 in endocardially derived tissue of the OFT and AV canal and tissues of NC origin such as the pharyngeal arches and craniofacial mesenchyme is similar between chicken and mouse embryos (Maschhoff et al., 2003; Ya et al., 1998). Sox4-null mouse embryos die at embryonic day 14, presumably due to cardiac failure. Observed heart defects are only in the arterial pole of the heart, suggesting that Sox4 is involved in interactions between NC-derived myofibroblasts and the endocardial components of the OFT (Ya et al., 1998). Consistent with this role, Sox4 and Nfatc transcription factors are associated with human forms of heart defects including rare truncus malformations (Restivo et al., 2006). Lithium exposure, a causative agent for certain teratogenic heart defects, reduces levels of both Sox4 and Nfats (Chen et al., 2008).

13.3. Sox5

Sox5 is the only member of the SoxD family (which includes Sox5, 6, 13, and 23) described in the NC. The long isoform of Sox5, LSox5, has a well-known role in chondrogenesis but, in addition to its expression in cartilage, is found in neuronal, glial, early NC, and other lineages, where it also functions. Sox5-null mice die neonatally due to respiration defects (Dy et al., 2008; Smits et al., 2001). During chondrogenesis, which includes the development of the NC-derived craniofacial skeleton, LSox5 coordinates with Sox6 and Sox9 to promote cartilage development by activating expression of cartilage-specific ECM molecules (Lefebvre et al., 1998; Perez-Alcala et al., 2004). Because Sox5 and Sox6 are highly similar, they have redundant function during chondrogenesis. Conditional deletion of floxed alleles of both Sox5 and Sox6 by Prrx1-Cre results in mice with chondrodysplasia (Dy et al., 2008), illustrating the importance of both of these SoxD members in cartilage formation.

In both chicken and mouse, LSox5 is also expressed in early NCCs; LSox5 expression initiates after the early NC markers Slug and Foxd3 and is maintained in the cranial glial lineage (Morales et al., 2007; Perez-Alcala et al., 2004; Stolt et al., 2008). LSox5 has NC-inducing properties; misexpression of LSox5 in the NT activates the migratory NC marker RhoB, and prolonged LSox5 expression expands the premigratory NC domain leading to overproduction of cranial NCCs (Perez-Alcala et al., 2004). In the chicken embryo, in addition to LSox5 expression in glia, a subpopulation of NC-derived differentiating neurons transiently expresses LSox5 (Morales et al., 2007). In the mouse embryo, Sox5 expression is observed in the melanocyte lineage but is not critical for melanocyte development; there is no observed melanocyte defect in Sox5-null mice (Stolt et al., 2008). However, the loss of LSox5 somewhat rescues the melanocyte defect of Sox10 heterozygous mutants. This genetic interaction led to the finding that Sox5 modulates Sox10 in the melanocyte lineage by recruiting the chromatin modifiers Ctbp2 and HDAC1 to the regulatory regions of target genes shared with Sox10, resulting in direct inhibition of Sox10-dependent activation (Stolt et al., 2008).

13.4. Sox8

All three members of the SoxE subgroup (Sox8, Sox9, and Sox10) are expressed and function in the NC and its derivatives, with some overlapping expression domains and functions (O’Donnell et al., 2006). Sox8 is highly similar to Sox9 and Sox10 and the protein has two separate transactivation regions. Sox8 is expressed in the NC, central nervous system, limbs, muscles, kidneys, adrenal glands, gonads, and craniofacial structures during mouse embryo development (Schepers et al., 2000; Sock et al., 2001). Homozygous null Sox8 mice have lower body weights than controls, but no other detectable defects in any of the Sox8-expressing lineages (Sock et al., 2001), and this is likely due to functional redundancy shared with Sox9 and Sox10. Two studies in particular demonstrate a genetic interaction between Sox8 and Sox10, suggesting that these highly similar proteins modulate expression or function of each other (Maka et al., 2005; Reiprich et al., 2008). In adrenal development, Sox8 (along with Sox10) is expressed in NCCs that migrate to the adrenal gland and is then downregulated in differentiating catecholaminergic adrenal cells. The NCCs of Sox10-deficient mice fail to colonize the developing adrenal medulla because of improper specification at the dorsal aorta and subsequent apoptosis during migration, and they do not express Sox8. Replacement of Sox10 with Sox8 significantly rescues the adrenal phenotype, suggesting functional redundancy. Sox8-null mice have only a minimal adrenal phenotype, but the phenotype in compound mutant Sox8-homozygous, Sox10-heterozygous embryos is much worse (Reiprich et al., 2008). These same compound mutants have an increased penetrance and severity of ENS defects (increased vagal NCC apoptosis and a resulting decrease in colonization of the gut), but Sox8 nulls have no ENS defects. This demonstrates that in ENS development, Sox8 functions as a modifier of Sox10. Like Sox10, Sox8 is also expressed in vagal and enteric NCCs and is later restricted to enteric glia (Maka et al., 2005), and these two SoxE factors may act together to maintain a pool of undifferentiated vagal NC stem cells (Maka et al., 2005). Xenopus Sox8, unlike in the mouse and chicken embryo, precedes Sox9 and Sox10 in NC progenitors. Sox8 expression persists in migrating cranial and trunk NCCs, in a pattern similar to Sox9 and Sox10. Sox8 knockdown in Xenopus does not inhibit NC formation but affects the timing of NC induction, impacting the later development of multiple NC lineages due to the inability of NCCs to migrate into the periphery (O’Donnell et al., 2006).

13.5. Sox9

13.5.1. Sox9 during Neural Crest Specification

Sox9 directs the development of NC, otic placodes, cartilage, and bone. In zebrafish, there are two Sox9 orthologs, Sox9a and Sox9b, which together perform the functions of the single-copy tetrapod Sox9 (Rau et al., 2006). Zebrafish Sox9b is expressed first in the prospective NC and then in cranial and trunk NC progenitors. Sox9b expression is extinguished in migrating NCCs, but some NC derivatives reactivate Sox9b expression at later stages (Li et al., 2002). Xenopus Sox9 is expressed in the prospective NC, where it has a role in regulating NC formation, and persists in migrating cranial NCCs as they populate the pharyngeal arches (Spokony et al., 2002). Knockdown of Sox9 causes dramatic loss of NC progenitors, resulting in reduction or loss of the NC-derived craniofacial skeleton (Lee et al., 2004b; Spokony et al., 2002). Wnt- and BMP-regulated NC induction in Xenopus and chicken embryos depends on Sox9 transcriptional activator function (Lee et al., 2004b; Osorio et al., 2009; Sakai et al., 2006). Sox9 expression precedes expression of premigratory NC markers and establishes competence for NCCs to undergo an EMT, partly by directly activating the Slug promoter, but is not required for migration. Sox9 is, however, required for survival of trunk NCCs, which undergo apoptosis around the time of delamination if Sox9 is absent (Cheung et al., 2005; Sakai et al., 2006). Forced expression of Sox9 promotes NC-like properties in neural tube progenitors at the expense of CNS neuronal differentiation (Cheung and Briscoe, 2003). Sox9, together with Notch signaling, can induce ectopic Sox10 expression (Dutton et al., 2008).

13.5.2. The Role of Sox9 in Chondrogenesis

Sox9 is expressed in all cartilage progenitors, has an essential role in chondrogenesis, and marks commitment to chondrogenic differentiation (Sahar et al., 2005; Thomas et al., 1997). Heterozygous mutations in human Sox9 result in campomelic dysplasia, a lethal disorder with skeletal malformations and craniofacial defects (Spokony et al., 2002). Sox9 regulates other early NC marker genes, the cartilage-specific gene Col2a1 and the bone-specific gene Runx2a (Akiyama et al., 2005). Col2a1 is directly activated by Sox9 and Sox10 and mediated by Sox9 and Sox10 cross-regulation and cAMP-dependent PKA signaling (Suzuki et al., 2006). Analysis of Sox9Cre knock-in mice demonstrates that Sox9 is expressed before Runx2, an early osteoblast marker gene, and that all osteochondrogenic cells are derived from Sox9-expressing progenitors (Akiyama et al., 2005). The zebrafish Sox9a;Sox9b double-mutant phenotype is additive; chondrocytes do not stack in Sox9a mutants, and in Sox9b, mutants do not expand properly, whereas compound mutants fail to do either, resulting in more severe craniofacial defects (Yan et al., 2002; Yan et al., 2005). Inactivation of Sox9 causes cranial NCCs to lose chondrogenic potential, converting to an osteoblast fate, marked by ectopic expression of osteogenic marker genes such as Runx2, Osterix, and Col1a1 in the nasal cartilage region (Mori-Akiyama et al., 2003). Sox9 and Msx2 are coexpressed in a subpopulation of cranial NCC during migration that will form the mandible. Sox9 expression indicates chondrogenic lineage determination, but Msx2 represses chondrogenic differentiation until cranial NCC migration is completed (Takahashi et al., 2001). Bmp4 induces chondrogenesis at sites where Sox9 expression is high relative to that of Msx2, and ectopic chondrogenesis is associated with Sox9 and Msx2 upregulation adjacent to Bmp4 signals (Semba et al., 2000). During cranial suture closure, Sox9 is upregulated along with cartilage markers, and haploinsufficiency of Sox9 results in delayed suture closure (Sahar et al., 2005).

13.5.3. Sox9 in NCSCs

In the chicken embryo, high levels of Sox9 (or Sox10 or Sox8) expression in the NT induce a migratory NC-like phenotype and maintains these cells in an undifferentiated state (McKeown et al., 2005). Sox9 is one of the early NC transcription factors often expressed in multipotent NC-derived progenitors cells. Sox9 expression is maintained in multipotent NCSCs derived from rat PDLs (Techawattanawisal et al., 2007), multipotent dental NC-derived progenitor cells (Degistirici et al., 2008), and multipotent skin-derived precursors (Zhao et al., 2009). Sox9 is also overexpressed with another NCSC marker, Twist1, in NC-derived malignant peripheral nerve sheath tumors (MPNSTs) (Miller et al., 2006).

13.5.4. Sox9 in the Melanocyte Lineage

Sox9 is expressed by melanocytes in neonatal and adult human skin and activates transcription of Mitf, Dct, and Tyr. Within the melanocyte lineage, Sox9 is upregulated by cAMP and PKA signaling in response to UVB exposure and is downregulated by the secreted factor Agouti signal protein, resulting in decreased pigmentation (Passeron et al., 2007).

13.6. Sox10

Sox10 is an extremely important regulator at multiple steps of NC development and has been implicated in interactions with many other transcription factors in the exquisite control of multiple cell fates (Figure 13.1).

FIGURE 13.1. Whole-mount in situ hybridizations show Sox10 in mouse, chicken, fish, and frog embryos.


Whole-mount in situ hybridizations show Sox10 in mouse, chicken, fish, and frog embryos. Mouse: Bennetts et al., 2007, 9.5-dpc embryo, lateral view; chicken: Basch et al., 2006, stage 8 embryo, dorsal view; zebrafish: Rau et al., 2006, three-somite embryo, (more...)

13.6.1. Sox10 Expression during NC Induction and in Migratory NCCs

During NC development, Sox10 is first expressed in the prospective NC in the dorsal NT, and continues in multipotent migratory NCCs (Ishii et al., 2005; Young et al., 2004), in NC-derived ENS and PNS progenitors, and in the melanocyte lineage (Nonaka et al., 2008). In Xenopus, Sox10 is expressed in prospective NC and otic placode regions from the earliest stages of NC specification in a Wnt and FGF-dependent manner, and in migrating cranial and trunk NCCs (Aoki et al., 2003; Honore et al., 2003). Sox10 is initially expressed in all NCCs, colocalizing with Slug and Sox9, major regulators of NC formation, but is then downregulated in the cranial NC and persists mostly in NCCs from the trunk region (Aoki et al., 2003). In the chicken embryo, Sox10 is expressed in migrating NCCs just after Slug but is lost as cells undergo differentiation in ganglia of the PNS and ENS. It is also expressed in the developing otic vesicle, the developing CNS, and the pineal gland (Cheng et al., 2000). In the mouse, where a Sox10βGeoBAC transgene closely approximates Sox10 endogenous expression, Sox10-driven lacZ expression can be detected in the anterior dorsal NT at E8.5 and in cranial ganglia, otic vesicle, developing DRGs, thyroid parafollicular cells, thymus, and salivary, adrenal, and lacrimal glands (Deal et al., 2006; Muller et al., 2008). Sox10 expression is generally present in migratory NCCs that will give rise to nonmesenchymal NC derivatives, such as melanocytes, glia, and neurons. This includes ANS neurons such as those contributing to the lung ganglia and NC-derived pancreatic innervation (Burns and Delalande, 2005; Nekrep et al., 2008). Sox10-expressing cells have been fate-mapped by vital dye injections in Sox10::eGFP transgenic zebrafish (Wada et al., 2005), showing that some Sox10-expressing cells contribute progeny to the paired trabeculae and ethmoid bones (in zebrafish) (Wada et al., 2005).

Sox10 functions at many stages of NC development: NC formation; maintenance of multipotent migratory NCCs; and survival, specification, and differentiation of nonmesenchymal NC derivatives including melanophores and peripheral or enteric glia (Drerup et al., 2009; Elworthy et al., 2003; Kelsh, 2006; Kim et al., 2003). The effect of Sox10 loss on later stages of NC derivatives will be discussed later in this chapter (see Section 13.6.4). In Xenopus, Sox10 knockdown causes loss of NC precursors and Slug and Foxd3 expression, enlargement of non-NC domains, and increased apoptosis and decreased proliferation (Honore et al., 2003). Knockdown of Sox10 blocks induction of melanocytes and ganglia in vivo and in vitro (Honore et al., 2003). Overexpression of Sox10 causes a large expansion of the Slug expression domain in Xenopus and induces expression of HNK-1 in a broad neuroepithelial domain in the chicken embryo (Aoki et al., 2003; McKeown et al., 2005). Xenopus Sox10-injected embryos show a later increase in Trp2-expressing pigment cells, suggesting a role for Sox10 in melanocyte lineage specification (Aoki et al., 2003). Many more cells emigrate from the NT in chicken embryos with Sox10 overexpression, but these cells never express differentiated markers, indicating Sox10 maintains an undifferentiated state or inhibits terminal differentiation (McKeown et al., 2005). Addition of Sox10 to cultured cells inhibits neuronal and glial differentiation of multilineage ENS progenitor cells without interfering with neurogenic commitment (Bondurand et al., 2006).

Sox10 and Sox8 are expressed in NCCs migrating to the adrenal gland and downregulated in catecholaminergic cells (Gut et al., 2005; Reiprich et al., 2008). Sox10-deficient NCCs are not properly specified; they undergo apoptosis and do not colonize the adrenal anlage, resulting in loss of the adrenal medulla. Sox10 and Sox8 may share functional redundancy, as Sox8 homozygous-null mutations alone have only minimal effect on adrenal gland development, but defects are seen in Sox8 homozygous-null, Sox10 heterozygous-null compound mutants, and extra Sox8 can partially rescue Sox10-null adrenal defects. Also, Sox8 expression is regulated by Sox10; Sox10-null NCCs do not express Sox8 (Reiprich et al., 2008). Sox10 and Sox9 regulate each other and directly activate Col2a1 expression during cartilage and NC differentiation, and activation is further enhanced by cAMP-dependent PKA signaling (Suzuki et al., 2006). In homozygous Erbb3msp1 embryos, Sox10LacZ expression is absent in cranial ganglia and sympathetic chains, but development of other Sox10-expressing cells appears unaffected (Buac et al., 2008). Sox10 activates Krox20 by binding an NC-specific enhancer in the Krox20 locus in synergy with Krox20 itself. Inactivation of Sox10 prevents maintenance of Krox20 expression in the migrating NC (Ghislain et al., 2003).

13.6.2. Transcription Factors Upstream of Sox10 in NC Development

In chicken embryos, Pax7 is required for NC formation in vivo; blocking its translation inhibits expression of NC markers including Sox10 (Basch et al., 2006). Sox9 regulates other early NC genes including Sox10 (Yan et al., 2005). The zebrafish Foxd3mother superior mutation (which causes loss of Foxd3 NC expression) leads to a depletion of NC derivatives preceded by reduction in NC-expressed transcription factors including Sox10 (Montero-Balaguer et al., 2006). Zebrafish Foxd3sym1 (a putative null allele) mutants have normal numbers of premigratory NCCs but reduced levels of Sox10 and Snail (Stewart et al., 2006). Snail, but not Slug, controls Sox10 expression in Xenopus (Honore et al., 2003). Zic4 is involved in induction of NC markers Sox10 and Slug (Fujimi et al., 2006). Disc1 mutant cranial NCC migration defects correlate with enhanced expression of Foxd3 and Sox10, leading to a loss of craniofacial cartilage and expansion of peripheral cranial glia. Disc1 functions in transcriptional repression of Foxd3 and Sox10, mediating cranial NCC migration and differentiation (Drerup et al., 2009).

13.6.3. Regulation of Sox10

Three clusters of highly conserved sequences in the Sox10 promoter, one of which shows strong enhancer potential in cultured melanocytes, are disrupted in the transgene-insertion mutant mouse line (Sox10 Hry ) that has a large deletion in a distal upstream enhancer region in the Sox10 locus, resulting in decreased Sox10 expression, aganglionosis, and melanocyte loss in homozygous mutants (Antonellis et al., 2006). Multiple conserved enhancers of Sox10 containing Sox, Pax, AP-2, and Tcf/Lef binding sites show distinct but overlapping activities including expression in several NC derivatives such as the developing PNS, Schwann cells, melanocytes, and adrenal gland (Antonellis et al., 2008; Werner et al., 2007). Some enhancers seem to direct pan-NC regulatory control (Antonellis et al., 2008). Characterization of Sox10βGeoBAC expression confirms presence of essential regulatory regions for the PNS lineage (Deal et al., 2006). The 3' end of the conserved first intron is required for proper spatial expression of Sox10 and contains conserved binding sites for transcription factors known to be essential in early NC induction, including Tcf/Lef, Sox and Foxd3; and b-catenin, Notch signaling, and Sox9 can all induce ectopic Sox10 expression in early embryos (Dutton et al., 2008).

The activities of individual SoxE factors are regulated by sumoylation, a posttranslational modification affecting protein stability, activity, and localization. Sumoylated forms mediate some specific activities, whereas nonsumoylated forms mediate a separate set of activities (Taylor and Labonne, 2005). Sox10 protein has three sumoylation consensus sites that modulate Sox10 activity. Sox10 sumoylation represses its transcriptional activity on the target genes Gjb1 and Mitf by modulating its synergistic interaction with cofactors Krox20 and Pax3 at these promoters (Girard and Goossens, 2006). Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus, but Sox10 is an active nucleocytoplasmic shuttle protein. Sox10 has a functional Rev-type nuclear export signal (NES) within its DNA-binding domain. Mutational inactivation of this NES or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited shuttling of Sox10 from the nucleus to the cytoplasm. Inhibition of Sox10 nuclear export decreased transactivation of transfected reporters and endogenous target genes (Rehberg et al., 2002). Consistent with a role as a shuttling transcription factor, Sox10 interacts with Armcx3, an integral membrane protein of the mitochondrial outer membrane containing an armadillo repeat, and consequently is associated with the mitochondrial outer membrane when it is in the cytoplasm. Armcx3 does not possess transcriptional activity but enhances Sox10 transactivation of nAChR α3 and β4 subunit genes (Liu et al., 1999; Mou et al., 2009). Many target gene promoters have multiple Sox10-binding sites. Sox10 can bind target DNA as a monomer or dimer. Dimers generally bind through two heptameric binding sites in a specific orientation and spacing, mediated by an N-terminal DNA-dependent Sox10 dimerization domain. Dimers have a higher binding affinity and increase the angle of DNA bending (Peirano and Wegner, 2000). Specific amino acid residues in a conserved region immediately preceding the HMG domain of Sox10 are required for cooperative binding with the HMG domain during dimeric binding. Maintenance of cooperativity is essential for full activation of target promoters such as the myelin protein zero (Mpz) gene, but dimer-dependent conformational changes such as bending angle introduced into the promoter are less important (Schlierf et al., 2002). The Sox10 dimerization and transactivation domains are needed in melanocyte and ENS development, but not in early NC development. The transactivation domain is required for satellite glia differentiation and Schwann cell myelination, whereas the DNA-dependent dimerization domain is required for the transition of immature Schwann cells to the promyelinating stage (Schreiner et al., 2007).

13.6.4. Sox10 Mutants as a Model for Waardenburg Syndrome 4

Waardenburg syndrome type 4 (WS4), also known as Waardenburg–Shah or Hirschsprung–Waardenburg syndromes, encompasses a range of phenotypes with characteristics of both WS (pigment deficiencies and deafness) and Hirschsprung disease (HSCR) (aganglionic megacolon). Haploinsufficiency of Sox10 causes pigmentation and megacolon defects, also observed in Sox10 Dom/+ mice and WS4 patients with heterozygous SOX10 mutations (Britsch et al., 2001). The white spotting and aganglionic megacolon of Sox10 Dom, Ednrb piebald-lethal (sl), and Edn3 lethal spotting (ls) mouse mutants are similar to the WS4 phenotype (Pingault et al., 1998). Sox10 Dom/+ mice have pigmentation deficiency or dysganglionosis 93% of the time (dysganglionosis 79% and pigmentation deficiency 90%), and both defects are seen in 68% of mice (Brizzolara et al., 2004). Mutations in the Ret gene are responsible for approximately half of familial HSCR cases and some sporadic cases. Mutations in genes encoding Ret ligands (Gdnf and Ntn), components of the Endothelin signaling pathway (Ednrb, Edn3, Ece1), and Zeb1 and Sox10 have also been identified in patients with HSCR (Benailly et al., 2003; Parisi and Kapur, 2000). WS4 patients have mutations in Sox10, but Sox10 mutations are usually not detected in patients with HSCR alone (Pingault et al., 1998). Haploinsufficiency of Sox10 is most often associated with disorders beyond HSCR. Human Sox10 is essentially expressed in NC derivatives that form the PNS, and in the adult CNS, but is more widely expressed than in rodents, with weak transcriptional activator activity (Bondurand et al., 1998; Kuhlbrodt et al., 1998b). Because of genetic background differences, unknown modifiers, and different types of Sox10 lesions, the human WS4 phenotype among patients with Sox10 mutations covers a wide range, from chronic intestinal pseudoobstruction alone to classic WS4 to WS4 with severe peripheral neuropathies like Charcot–Marie–Tooth neuropathy type 1 (CMT1) or PCWH (peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, WS, and HSCR), and dysfunction of Sox10 may extend to the CNS, resulting in brain phenotypes (Inoue et al., 1999; Pingault et al., 2000; Southard-Smith et al., 1999; Sznajer et al., 2008; Touraine et al., 2000; Verheij et al., 2006).

Sox10 plays a cell-autonomous role in NCCs migrating into the GI tract and is essential for proper ENS development. Sox10Dom/+ mice have deficiencies of NC-derived enteric ganglia in the distal colon, whereas Sox10Dom/Dom embryos have total enteric aganglionosis and die late in embryogenesis or perinatally (Herbarth et al., 1998; Southard-Smith et al., 1998). In Sox10Dom / Dom embryos, apoptosis was increased in sites of early NCC development before these cells enter the gut (Herbarth et al., 1998; Kapur, 1999). Cell death is also increased before lineage segregation in undifferentiated, postmigratory NCCs lacking Sox10. Surviving Sox10-null NCCs do not adopt a glial fate, even in gliogenic conditions. In Sox10 heterozygous mutant NCCs, survival is normal, but fate specification is still drastically affected (Paratore et al., 2001; Paratore et al., 2002b). Mutant enteric NC-derived cells do not maintain a progenitor state and acquire preganglia traits, resulting in a reduction of the progenitor pool size (Paratore et al., 2002b).

Zebrafish Sox10 colourless mutant NCCs, another model of WS4, form only mesenchymal NC fates, whereas NCCs that would normally adopt nonmesenchymal NC fates fail to migrate and differentiate and instead undergo apoptosis. Mitf expression is disrupted in Sox10 colourless mutants, giving rise to melanophore defects (Dutton et al., 2001). ENS progenitor fate specification, marked by Phox2b, is defective in these mutants, with most NCCs failing to migrate to the GI tract primordium (Elworthy et al., 2005).

In addition to Sox10, mutations in Edn3 and Ednrb have been associated with WS4 (Karaman and Aliagaoglu, 2006; Matsushima et al., 2002). The Ednrb ENS enhancer has Sox10 binding sites, and Sox10 and Ednrb interact genetically. Sox10;Ednrb (and Sox10;Edn3) compound mutants have a drastic increase in white spotting, absence of melanocytes within the inner ear, and more severe ENS defects. In the GI tract of double heterozygous mutants, no apoptosis, proliferation, or differentiation defects in NCCs were detected, but apoptosis was increased in vagal NCCs outside of the GI tract (Cantrell et al., 2004; Stanchina et al., 2006). Sox10-expressing enteric NCC progenitors are reduced in Edn3-deficient embryos, suggesting endothelin signaling is necessary for maintenance and proliferation of Sox10-expressing ENS progenitors (Bondurand et al., 2006). Pax3, required for normal enteric ganglia formation, functions with Sox10 to activate transcription of Ret (Lang et al., 2000). Sox10 expression in enteric NCCs is decreased in Pofut1-null embryos that have defective Notch signaling, whereas enteric NCCs expressing Ascl1, a strong repressor of Sox10 suppressed by Notch signaling, are increased. Notch signaling is required for maintenance of ENS progenitors and Sox10 expression by attenuating a cell-autonomous neuronal differentiation program, at least in part by suppressing Ascl1 (Okamura and Saga, 2008). Sox8 is expressed with Sox10 in migrating vagal and enteric NCCs and is later confined to enteric glia. Loss of Sox8 alone had no effect on the ENS, but loss of Sox8 alleles in Sox10 heterozygous mice increased the penetrance and severity of Sox10 ENS defects and impaired early colonization of the GI tract by enteric NCCs, drastically increasing apoptosis in vagal NCCs outside the GI tract. The defects in ENS development of mice with Sox10 and Sox8 mutations may be caused by a reduction of the pool of undifferentiated vagal NCCs (Maka et al., 2005). Binding sites for Sox10 exist in the Ednrb ENS enhancer, suggesting that Sox10 may have multiple roles in regulating Ednrb in the ENS (Zhu et al., 2004).

13.6.5. Sox10 in Glia of the PNS

Sox10 first appears in developing NC and continues as NCCs contribute to the developing PNS, where it functions in satellite glia and Schwann cells and is a marker of Schwann cell differentiation (Kuhlbrodt et al., 1998a; Miller et al., 2006; Schreiner et al., 2007). Sox10 is also expressed in the CNS, first in glial precursors and later in myelin-forming oligodendrocytes of the adult brain (Kuhlbrodt et al., 1998a; Stolt et al., 2002). In the PNS, Sox10 is a key regulator of specification and differentiation of peripheral glial cells (Schwann cells) (Britsch et al., 2001; Svaren and Meijer, 2008). In Sox10-null mutants, neuronal cells form in DRGs, but Schwann cells and satellite glia are lost, resulting in later degeneration of sensory and motor neurons (Britsch et al., 2001; Stolt et al., 2002). During Schwann cell development, Sox10 functions synergistically with Oct6, and modulates the functions of Pax3 and Krox20, conferring specificity to these transcription factors in developing and mature glia (Kuhlbrodt et al., 1998a). Sox10 partners synergistically with NFATc4 to activate Krox20, regulating myelination genes (Kao et al., 2009). In glial cells, Sox10 associates with the N-myc interactor (Nmi) protein, which modulates Sox10 transcriptional activity in reporter assays, perhaps in a promoter-specific manner (Schlierf et al., 2005). Among the primary targets, Sox10 controls expression of Erbb3, a Neuregulin receptor, in NCCs. Downregulation of Erbb3 accounts for many defects seen in mutants, but Sox10 has functions not mediated by Erbb3, such as in the melanocyte lineage (Britsch et al., 2001). Sox10 directly regulates myelin protein zero (Mpz or P0) in Schwann cells (Peirano and Wegner, 2000). Other direct target genes of Sox10 are the myelin proteolipid protein (Plp1), extracellular superoxide dismutase (Sod3), and pleiotrophin (Ptn), and Sox10 itself (Lee et al., 2008b).

13.6.6. Sox10 in Neural Derivatives

In migrating multipotent NCCs, Sox10 preserves glial and neuronal potential. Sox10 is needed in vivo for induction of the neurogenic factors Ascl1 and Phox2b and inhibition or delay of neuronal differentiation at higher dosages (Kim et al., 2003). Undifferentiated enteric NCCs express Sox10, Phox2b, p75, and Ret. At E10.5, between 10% and 15% of these NC-derived cells in the small intestine have started to differentiate into neurons. By E12.5, 25% of Phox2b-expressing cells in the small intestine express neuronal markers such as ubiquitin carboxy-terminal hydrolase (PGP9.5), and this fraction increases to 47% at E14.5 (Young et al., 2003). Although Sox10 is maintained in glial precursors along with p75 and Fabp7 and low Ret expression, differentiating enteric neurons no longer express Sox10 and have low p75, but high Ret, expression (Young et al., 2003). Sox10 expression in undifferentiated multipotent NCCs is mutually exclusive to Foxs1, which marks emerging DRG sensory neurons. Foxs1-negative, Sox10-positive migrating NCCs with a high proliferative activity surround Foxs1-positive, Sox10-negative pioneering NCC neuronal progenitors with limited proliferation (Montelius et al., 2007). These data suggest that Sox10 may play an early role in maintaining neuronal differentiation potential of NCCs.

Sox10 mutants have a complete absence of glial differentiation but apparently normal initial neurogenesis. However, without glial cells, motor neurons and sensory neurons degenerate later in development. As neuronal and glial precursors are generated and segregated from NCCs in the DRGs, these NCCs have increased apoptosis and decreased proliferation (Sonnenberg-Riethmacher et al., 2001). In addition to affecting progenitors, there is some evidence that Sox10 may play a direct role in NC-derived sensory neurons, but this is not easy to demonstrate in mammalian embryos because of interdependence of closely associated neurons and glia. However, in zebrafish, early DRG sensory neuron survival may be independent of glia. Sox10 is expressed transiently in the sensory neuron lineage and specifies sensory neuron precursors by regulating Neurog1 (Carney et al., 2006). In zebrafish, glial cells and their target axons coalesce at an early stage and are coupled throughout migration, with axons providing instructive cues necessary to direct glial migration. Genetic ablation of glia in Sox10 mutants, uncoupling axon and glial migration, shows Sox10 has an important role in nerve fasciculation (Gilmour et al., 2002). Inactivation of zebrafish Sox10 or Neurog1 leads to more than twice the normal number of neuromasts along the posterior lateral line. Development of intercalary extra neuromasts may occur because of the absence of NC-derived peripheral glia, which may inhibit the assembly of interneuromast cells into neuromasts (Lopez-Schier and Hudspeth, 2005).

13.6.7. Sox10 in Melanocytes and Regulation of Mitf

As evident from analysis of Sox10Dom mice and WS4 phenotypes, Sox10 is a critical factor for melanocyte development. Sox10Dom/Dom embryos lack NC-derived cells expressing Mitf, Dct, and Kit, and NCC primary cultures from these embryos do not give rise to pigmented cells. In Sox10Dom/+ heterozygous embryos, melanoblasts expressing Kit and Mitf are present in reduced numbers, and pigmented cells eventually develop in nearly normal numbers both in vitro and in vivo (Potterf et al., 2001). Sox10 directly transactivates the melanocyte “master regulator” gene Mitf 100-fold through a conserved binding site and this transactivation is further stimulated by Pax3 (Potterf et al., 2000), much like the synergistic activation by Sox10 and Pax3 on the Ret promoter (Lang and Epstein, 2003). In the context of Ret, Sox10 mutants that cannot bind DNA retain the ability to activate the enhancer in the presence of Pax3, but in the context of Mitf, Pax3 and Sox10 must each bind independently to the DNA (Lang and Epstein, 2003). A Sox10 mutant with a C-terminal truncation acting as a dominant-negative reduces Mitf induction (Potterf et al., 2000). Mitf is also activated by cAMP signaling through Creb1, but the direct Creb1 activation of Mitf requires Sox10 to bind a second DNA element. In melanoma and neuroblastoma cells, activation of Mitf by either Sox10 or cAMP is mutually dependent, and ectopic Sox10 with cAMP signaling is sufficient to activate the Mitf promoter in neuroblastoma cells (Huber et al., 2003). Sox10-null zebrafish mutants also lack expression of Mitfa, the equivalent of Mitf-M. Reintroduction of Mitfa expression in NCCs can rescue melanophore development in Sox10-null zebrafish embryos, suggesting that the essential function of Sox10 during melanophore development is activation of Mitfa (Elworthy et al., 2003). Sox10 and Mitf-M are also expressed in melanoblasts migrating toward the prospective inner ear of mouse embryos but are later separately expressed in different cell types of the newborn cochlea (Watanabe et al., 2002a).

13.6.8. Coregulation of Mitf Target Genes by Sox10

Mitf controls a set of genes critical for pigment cell development and pigmentation, including Dct, Tyr, and Tyrp1, but Sox10 also augments activation of these genes (Passeron et al., 2007). The Tyr, Tyrp1, and Dct promoters all contain an E-box bound by Mitf and binding sites for RPE-specific factors such as Otx2 or for melanocyte-specific factors such as Sox10 or Pax3 (Murisier and Beermann, 2006). Sox10 colocalizes with Dct in early melanoblasts before Tyr or Tyrp1 expression, Sox10Dom/+ melanoblasts have a transient loss of Dct expression, and Sox10 transactivates the Dct promoter in vitro (Jiao et al., 2004; Potterf et al., 2001). Critical melanocyte-specific enhancers in the Dct promoter contain Sox10 and Mitf binding sites. Sox10 and Mitf on their own directly activate Dct transcription, but together activate Dct expression synergistically (Jiao et al., 2004; Jiao et al., 2006). Mitf in melanoma cells is modified by sumoylation, and an Mitf substitution mutation affecting sumoylation has enhanced synergy with Sox10 on the Dct promoter (Murakami and Arnheiter, 2005). A BAC transgene containing the Tyrp1 gene and surrounding sequences that recapitulates endogenous expression has a conserved melanocyte-specific enhancer activated by Sox10 (Murisier et al., 2006).

13.6.9. Additional Sox10 Regulation

Sox10 activity in the melanocyte lineage may be modulated by Sox5, which competes with Sox10 for shared target binding sites and recruits the corepressors Ctbp2 and Hdac1 to inhibit Sox10 target genes. Loss of Sox5 partially rescues reduced melanoblast generation and gene expression in Sox10 heterozygous mice (Stolt et al., 2008). Gli3Mos1, a truncation mutation identified as a modifier that increases the severity of Sox10LacZ/+ defects, causes more drastic reduction of pigmentation when present as a heterozygous allele in combination with Sox10 LacZ/+. Gli3 Mos1/Mos1 embryos have reduced transcripts of Sox10 and early melanoblast markers Mitf, Dct, and Si, suggesting disrupted melanoblast specification (Matera et al., 2008). Ednrb is expressed with Sox10 in melanoblasts. Ednrb-null mice maintain Sox10 expression in melanoblasts; unlike in the ENS, Sox10 does not directly activate Ednrb transcription in the melanocyte lineage, suggesting context-dependent regulation by an unknown mechanism (Hakami et al., 2006). In zebrafish, Sox10 and leukocyte tyrosine kinase (Ltk) are required for iridophore specification from NCC, and like Sox10 mutants, zebrafish Ltkshady mutants lack iridophores. Sox10 heterozygous mutants have an increase in Ltk-expressing cells, but cells also retaining Sox10 expression do not express other NCSC markers and may represent lineage-restricted progenitors or incompletely specified multipotent progenitors (Lopes et al., 2008).

13.6.10. Sox10 in Melanocyte Stem Cells

The bulge region of the adult hair follicle contains a niche for melanocyte stem cells. Development of melanocyte stem cells is controlled by Pax3, Sox10, and Mitf, and extracellular cues such as Wnt (Choi et al., 2008; Sommer, 2005). Many white spotting genes (Sox10, Pax3, Mitf, Slug, Ednrb, Edn3, Kit, Kitl) are associated with hypopigmentary disorders and deafness as a result of NCSC-derived melanocyte deficiency (Hou and Pavan, 2008). Graying hair is typically caused by defective cell migration into the bulb of the hair, and reduction of Sox10, Pax3, Mitf, and their target genes Tyr and Tyrp1 (Choi et al., 2008).

13.6.11. Sox10 in NCSCs and Cancer

Sox10 is one of the NCSC markers expressed in several different adult-derived NCSC types. Multipotent stem cell-like SKPs from facial skin of adult mice, pigs, and humans express Sox10 and p75 in addition to pluripotency-related genes (Wong et al., 2006; Zhao et al., 2009). A boundary cap is a structure composed of late migrating NC at the dorsal root entry zone and motor neuron exit points. It contains multipotent stem cells and can generate neurons and peripheral glia during embryogenesis. Sox10 marks multipotent progenitors in boundary caps and in cultured bcNCSCs (Aldskogius et al., 2009; Aquino et al., 2006).

Genetic programs essential to NC development are often activated in neuroectodermal tumors, which express Sox10, AP-2a, and Pax3. Both Sox10 and AP-2a are generally expressed in relatively differentiated neoplasms (Gershon et al., 2005). Sox10 is expressed in some neuroblastoma cell line subtypes (Acosta et al., 2009), almost all melanomas and about half of all MPNSTs, and diffusely in some schwannomas and neurofibromas (Nonaka et al., 2008). Sox10 and its target Erbb3 are both overexpressed in pilocytic astrocytoma relative to other pediatric brain tumors (Addo-Yobo et al., 2006).

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53136
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