Chapter 15Other Miscellaneous Genes

Publication Details

15.1. Atf1

Clear cell sarcoma of tendons and aponeuroses, also referred to as malignant melanoma of soft parts, is a rare NC-derived cancer. Most cases have a translocation that creates a unique chimeric Ewsr1/activating transcription factor 1 (Atf1) fusion gene transcript (Dim et al., 2007).

15.2. Cited2 AND Cited4

The Crebbp/Ep300-interacting transactivator with Glu(E)/Asp(D)-rich carboxy-terminal domain 2 (Cited2) transcription factor binds Ep300 and its paralog Creb-binding protein (Crebbp), ubiquitously expressed transcriptional coactivators and histone acetyl transferases, with high affinity to regulate transcription. Cited2 is required for NC and NT development (Bamforth et al., 2001; Weninger et al., 2005). Cited2-null embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. Cardiac defects include some typical NC-related defects such as double outlet right ventricle, persistent truncus arteriosus, and right-sided aortic arches (Bamforth et al., 2001). Cited2 interacts with and coactivates all isoforms of AP-2, and Cited2-null embryos have increased apoptosis in the midbrain region and a marked reduction in cardiac NCCs expressing the AP-2 target Erbb3 (Bamforth et al., 2001; Weninger et al., 2005). Cited2 is also expressed in the AV canal and cardiac septa, and there are some NC-independent cardiac defects such as ventricular septal defects and hypoplasia of the atrioventricular endocardial cushions evident in Cited2-null embryos (Weninger et al., 2005). Heterozygous human Cited2 mutations are also associated with congenital heart disease (MacDonald et al., 2008).

Human Cited4 and Ep300/Crebbp are present in endogenous interaction complexes. Cited4 functions as a transcriptional activator and physically interacts with all AP-2 isoforms as a coactivator in vitro but is slightly weaker than Cited2 for coactivation of AP-2g. Cell-type and AP-2 isoform-specific coactivation by different Cited proteins may be a mechanism for differential modulation of AP-2 function (Braganca et al., 2002).

15.3. Creb1

Creb1 (cyclic AMP-responsive element binding protein 1) is a basic leucine zipper transcription factor that mediates cAMP signaling by binding downstream targets at specific conserved cAMP response element (CRE) sites. In NC cultures, cAMP signaling induces Mitf and melanogenesis. A dominant-negative Creb1 inhibits Mitf expression and melanogenesis, supporting the notion that Creb activation is necessary for melanogenesis. However, constitutively active Creb1 alone is not sufficient for Mitf expression and melanogenesis, which requires simultaneous PKA signaling (Ji and Andrisani, 2005). Combined Bmp2 and cAMP signaling induces the catecholaminergic lineage in NC cultures from avian and murine cells by a direct Creb1-mediated increase in expression of Phox2a, which has two CRE sites in its promoter (Benjanirut et al., 2006; Chen et al., 2005). In NC cultures, a dominant-negative Creb1 suppresses Phox2a transcription and sympathoadrenal lineage development. Constitutively active Creb1 alone can induce Phox2a transcription, but it is not sufficient for sympathoadrenal lineage development, which also requires activation of Phox2a by phosphorylation through PKA (Chen et al., 2005). In primary NCSC culture, Gata3 transactivates the Th promoter, which does not contain a Gata3 binding site but does have a Creb1 binding site required for Gata3 transactivation. Gata3 physically interacts with Creb1 in vitro and in vivo (Hong et al., 2006). Creb1, along with Oct6, Krox20, Pax3, and Sox10, is also hypothesized to be involved in the developmental transitions of Schwann cells (Jessen and Mirsky, 1998).

15.4. Ctbp1 AND Ctbp2

In Xenopus NC, C-terminal binding protein 1 (Ctbp1) forms a complex with either Zeb1 or Zeb2, acting as a corepressor for the targets of these proteins (van Grunsven et al., 2006). Ctbp2, along with Hdac1, is recruited by Sox5, which functions in the melanocyte lineage to modulate Sox10 activity by binding the regulatory regions of melanocytic Sox10 target genes, thus directly competing against Sox10 and recruiting corepressors such as Ctbp2 and Hdac1 (Stolt et al., 2008).

15.5. Dawg

In Xenopus, the Dawg (Dachshund-like with gill expression) protein, a member of the Ski/Sno/Dac family containing a putative Ski DNA-binding domain, is expressed in the brain, sensory vesicles, and cranial NC of neurula and tailbud embryos (Seufert et al., 2005). Dawg NC function has not been tested.

15.6. Mafb

Mafb is a basic domain leucine zipper transcription factor (Eichmann et al., 1997) involved in hindbrain segmentation and anterior–posterior patterning (Barrow et al., 2000; Marin and Charnay, 2000). In the mouse, zebrafish, and chicken embryo, Mafb is expressed in r5 and r6 and associated NCCs (Eichmann et al., 1997; Grapin-Botton et al., 1998; Marin and Charnay, 2000) and acts with Krox20, Hoxa1, Hoxa2, and Hoxb2 to pattern these cells (Barrow et al., 2000; McGonnell et al., 2001). Mafb is important for subdivision of the presumptive r5 and r6 territory into definitive rhombomeres and plays an important role in regulating Hox gene transcription (Cooke et al., 2001; Prince et al., 1998).

Homozygous Maf bkreisler mutant embryos have defects in hindbrain segmentation and inner ear development (Eichmann et al., 1997). In chickens and mice, Mafb is expressed in non-NC tissues, such as vestibular and acoustic nuclei, spinal cord and brain stem neurons, and the mesonephros, but Mafb expression is not lost in some of these tissues in the Maf bkreisler homozygous mice, suggesting this allele is not a null (Eichmann et al., 1997). The zebrafish loss-of-function Maf bvalentine mutant has inappropriately specified NCCs (Prince et al., 1998) and a mixed-identity region one rhombomere in length between r4 and r7. Normally, Ephb4a is expressed with Mafb in r5 and r6, whereas the ligand Efnb2a is expressed on either side, in r4 and r7. In Maf bvalentine loss-of-function mutant embryos, Ephb4a expression is downregulated and Efnb2a expression is upregulated between r4 and r7, suggesting Mafb sets up mutually exclusive expression domains, establishing the boundaries of the r5/r6 region (Cooke et al., 2001).

Transplantation experiments in chicken embryos demonstrate that signals from somites 7–10 are important for Mafb expression in the hindbrain and corresponding NCCs. Mafb expression is maintained when r5 and r6 are grafted into the r3 and r4 region but repressed when grafted into the r7 and r8 region (Grapin-Botton et al., 1998). RA beads mimic the effect produced by the somites in repressing Mafb in r5/6 and progressively inducing it more rostrally as RA concentration increases (Grapin-Botton et al., 1998). RA signaling, in part by restricting Mafb and Krox20 expression domains, is essential for specifying rhombomere identity and caudal hindbrain segmentation (Dupe et al., 1999). Inactivation of RARa and RARb results in expansion of the Mafb expression domain to twice normal size, enlargement of r5, disappearance of the r5/r6 boundary, and profound alterations of rhombomere identities (Dupe et al., 1999). Raldh2-null mice, which have RA deficiency and die at midgestation, have a severe reduction of Mafb expression (Niederreither et al., 2000). In chicken embryos, application of exogenous FGFs to the NT leads to ectopic expression of Mafb and Krox20 in the NC of the somitic hindbrain (r7 and r8), whereas inhibition of FGF signaling causes downregulation of Mafb and Krox20 (Marin and Charnay, 2000).

15.7. MEDIATOR SUBUNITS Med12 AND Med24

Mediator is a coactivator complex that assists the interaction of DNA-binding transcription factors with RNA polymerase II (Rau et al., 2006). Mediator subunit 12 [Med12, also known as thyroid hormone receptor-associated protein 230 (Trap230)] and Med24 (also known as Trap100) are both important during NC development. In zebrafish, the embryonic lethal homozygous Med12kohtalo mutant embryos show defects in brain, NC, and kidney development. In these mutants, cells initiate differentiation pathways in the affected tissues, and many cell type-specific genes are expressed, but differentiation is often incomplete, associated with a failure in morphogenesis (Hong et al., 2005). Another Med12 mutation was identified in zebrafish from a screen for mutants resembling Sox9 loss-of-function. Med12 acts as a critical coactivator for Sox9, which directs development of NC, otic placodes, cartilage, and bone (Rau et al., 2006).

During development, Med24 is expressed in a pattern consistent with functions in both ENS and craniofacial skeletal development. The Med24lessen loss-of-function mutant or knockdown of Med24 has a significant reduction in ENS neurons and defects in cranial NC-derived structures. Initial specification and migration of the NC is unaffected in Med24lessen mutants, but proliferation of ENS progenitors is significantly reduced. Med24 is not required for initial steps of cranial NC development but is essential for later proliferation of ENS progenitors. Med24 acts cell autonomously in pharyngeal endoderm and indirectly influences the development of NC-derived cartilages. The intestinal endoderm is also essential for ENS development, and it is unclear if this may be where Med24 is functioning to affect ENS development (Pietsch et al., 2006).

15.8. Mef2

The four Myocyte enhancer factor 2 (Mef2) genes, Mef2a, Mef2b, Mef2c, and Mef2d, encode transcription factors belonging to the MADS (MCM1-agamous-deficiens-serum response factor) family of DNA binding proteins that also includes Srf (Lyons et al., 1995). Mef2 transcription factors bind a conserved A/T-rich sequence in many skeletal and cardiac muscle gene promoters and are some of the earliest markers for the cardiac muscle lineage. Mef2a, Mef2c, and Mef2d are expressed first in the myocardium and NCCs (E8.5–E10.5) and then later in myotomes, prospective limb muscle, and the CNS (Edmondson et al., 1994; Lyons et al., 1995). Reduced expression of Mef2c and Hand1 in Hif1a-null embryos may cause defective ventricle formation, and these mutants also have NCC migration defects leading to abnormal OFT and vessel remodeling and hypoplastic pharyngeal arches (Compernolle et al., 2003). Mef2c also interacts with Myocardin (Myocd) to regulate smooth muscle transcription of the Myocd gene (Creemers et al., 2006).

In the mouse, Mef2c is required for expression of Dlx5, Dlx6, and Hand2 in the pharyngeal arches. NC-specific deletion of Mef2c results in neonatal lethality due to severe craniofacial defects. Mice heterozygous for Mef2c, Dlx5, or Dlx6 have no defects, but double heterozygous Mef2c;Dlx5 mice exhibit defective palate development and neonatal lethality (Verzi et al., 2007). In zebrafish, Mef2c (mef2ca) is also required in cranial NC for proper pharyngeal skeletal patterning; Mef2c mutants have craniofacial defects resembling Edn1 partial loss-of-function and Mef2c interacts genetically with Edn1. Expression of Edn1-dependent target genes Hand2, Dlx4, Dlx5, Dlx6, Bap, and Gsc requires Mef2c function (Miller et al., 2007).

15.9. Mrf2

During differentiation of a multipotent NCC line (MONC-1) toward a VSMC fate, the transcription factors modulator recognition factor 2a and b (Mrf2a and Mrf2b), members of the AT-rich interaction domain (ARID) family of transcription factors known to regulate differentiation, are highly induced. In vivo, Mrf2a is expressed in adult mouse cardiac and vascular tissues. Overexpression of Mrf2a and Mrf2b in 3T3 fibroblasts induces expression of vascular smooth muscle marker genes like smooth muscle a-actin and smooth muscle 22a and slows proliferation (Watanabe et al., 2002b).

15.10. Myb AND Mybl2

The myeloblastosis protooncogene Myb (or c-Myb) and the homologous Mybl2 (Myb-like 2, also known as B-Myb) encode transcriptional regulators containing a unique Myb domain similar to a helix–turn–helix domain of eukaryotic homeodomain proteins. In the chicken embryo, Myb is expressed in neuroectoderm and participates in the regulation of trunk NCCs. Reduction of endogenous Myb in vitro and in ovo prevents formation of migratory NCCs. Moderate overexpression of Myb in naive intermediate neural plate triggers EMT and NCC migration, probably through cooperation with Bmp4 signaling. Bmp4 activates Myb expression, causing accumulation of transcripts of the Bmp4 downstream genes Msx1 and Slug, important in the early NC. Reduction of Myb prevents Bmp4-induced NC formation (Karafiat et al., 2005). Conditional expression of Myb in an NC-derived neuroblastoma cell line causes upregulation of Igf1, Igf1r, and Igfbp5 expression. The Igfbp5 promoter has two Myb binding sites bound by Myb and Mybl2 in vitro and in vivo, and these Myb proteins directly enhance transcription from an Igfbp5 reporter (Tanno et al., 2002). During Xenopus embryogenesis, Mybl2 is preferentially expressed in the developing nervous system and NCCs. Within the developing NT, Xenopus Mybl2 gene transcription occurs preferentially in proliferating, non-differentiated cells (Humbert-Lan and Pieler, 1999).

15.11. Myocd, Mkl1, Mkl2

Myocardin (Myocd) and the Myocd-related transcription factors Mkl1 [megakaryoblastic leukemia/myocardin-like 1, also known as Myocardin-related transcription factor a (Mrtfa)] and Mkl2 (Mrtfb) act as coactivators for Srf and play a key role in cardiovascular development (Li et al., 2005; Oh et al., 2005). Embryos homozygous for an Mkl2 loss-of-function mutation die perinatally from cardiac OFT defects and thin-walled myocardium, accompanied by an earlier failure in differentiation of VSMCs within the NC-derived pharyngeal arch arteries (Li et al., 2005; Oh et al., 2005). The Mkl2-null phenotype is distinct from the Myocd-null phenotype, suggesting unique roles for each of these Srf coactivators in development of different subsets of VSMCs in vivo (Oh et al., 2005), despite normal migration and initial patterning of cardiac NCCs (Li et al., 2005). Myocd is the earliest known marker specific to both cardiac and smooth muscle lineages during embryogenesis. The activity of a Myocd enhancer in the cardiovascular system requires both Mef2 and FoxO proteins, and Myocd regulates its own enhancer through Mef2 independently of Srf (Creemers et al., 2006).

15.12. Nfatc3 AND Nfatc4

Nuclear factor of activated T-cells (Nfat) transcription factors are a calcium-activated family originally identified by their role in immune response, but several of these are also expressed and function in NCCs. Nfat activity, together with calcineurin, is essential for neuregulin and ErbB signaling and plays a role in NC diversification and differentiation of Schwann cells (Kao et al., 2009). Nfatc3 and Nfatc4 are activated downstream of Calcineurin, which is activated by an increase in cytoplasmic calcium ions upon addition of neuregulin to Schwann cell precursors. Nfatc4 interacts synergistically with Sox10, an Nfat nuclear partner, to activate Krox20, which in turn regulates genes necessary for myelination. Mouse embryos lacking CalcineurinB1 in the developing NC have defects in Schwann cell differentiation and myelination (Kao et al., 2009). In undifferentiated cells from the NC-derived PC12 pheochromocytoma cell line, an Nfat-activated reporter construct is transcribed upon addition of exogenous Wnt1 and Wnt7a. However, there may be other factors interpreting signals downstream of Nfat; heterologous expression of Wnt1 enhances proliferation, but opposite effects were observed in PC12 cells expressing Wnt7a (Spinsanti et al., 2008).

15.13. Nfyb

Zebrafish Nfyb (nuclear transcription factor y-b) becomes restricted to prospective craniofacial cartilage regions and the developing notochord. Nfyb knockdown causes reduction in head size, loss of some craniofacial cartilages, and increased apoptosis in the head, with a depletion of cranial NCCs required for mandibular and pharyngeal arch formation (Chen et al., 2009).

15.14. NUCLEAR RECEPTORS Nr2f1, Nr2f2, Nr5a1, Nr5a2

Nr2f (also known as COUP-TF) genes encode orphan members of the steroid/thyroid hormone receptor superfamily highly expressed in the vertebrate developing nervous system with putative roles in neuronal development and differentiation. In the mouse, there are two homologous Nr2f genes (Nr2f1 and Nr2f2), and their expression patterns overlap extensively. Nr2f1-null animals die perinatally. Mutant embryos have altered morphogenesis of the ninth cranial ganglion, possibly due to extra cell death in the neuronal precursor cell population. At midgestation, aberrant nerve projection and branching were observed in several other regions of mutant embryos. Nr2f1 is required for proper fetal and postnatal development and has some functions distinct from Nr2f2, which has not been directly investigated in the NC (Qiu et al., 1997).

Nr5a1 (also known as steroidogenic factor 1 or Sf1) heterozygous mutant embryos have a decrease in adrenal precursors starting at E10, but after E13.5, increased cell proliferation in Nr5a1 heterozygous embryos compensates and almost normal adrenocortical size is restored. NC-derived adrenomedullary precursors migrate normally in heterozygous and null embryos, but later in development, medullary growth is compromised in both genotypes. Despite the small adrenal size in Nr5a1 heterozygotes, steroidogenic capacity per cell is elevated in their primary adult adrenocortical cells, consistent with upregulation of some Nr5a1 target genes in heterozygous Nr5a1 adrenal glands (Bland et al., 2004). Expression of Nr5a2, also known as fetoprotein transcription factor (Ftf), occurs in the yolk sac endoderm, pharyngeal arches and NCCs, and in the foregut endoderm during liver and pancreatic morphogenesis (Rausa et al., 1999).

15.15. Rela (NFkB SUBUNIT)

In Xenopus, indirect activation of NFkB by injection of Bcl2l1 mRNA enables NFkB to directly upregulate Slug and Snail, rescuing the mutant phenotype of Slug, a factor normally required for expression of mesodermal and NC markers. Slug indirectly upregulates NFkB subunits and directly downregulates the proapoptotic Caspase9 (Zhang et al., 2006a). Regulation of NFkB activity in rat DRGs and the NC-derived pheochromocytoma cell line PC12 indicates a role for NFkB in delayed rather than immediate-early responses of the PNS and related cell lines to inflammatory cytokines (Wood, 1995). Wnt1 production and NFkB activation are linked in the PC12 cell line (Sun et al., 2008). The neurosteroid dehydroepiandrosterone (DHEA) protects NC-derived PC12 cells from serum deprivation-induced apoptosis. DHEA induces sequential phosphorylation of prosurvival kinases, which then activate Creb1 and NFkB which in turn induce expression of the antiapoptotic Bcl2 genes (Charalampopoulos et al., 2008).

15.16. Srf

Like Mef2, Serum response factor (Srf) is a member of the MADS family of transcription factors and is a key regulator of the expression of smooth muscle and cardiac muscle genes (Creemers et al., 2006). Cardiac-specific response to Edn1 requires activity of Srf combined with the tissue-restricted Gata proteins. When cotransfected, Srf and Gata factors synergistically activate cardiac-specific Edn1-inducible promoters containing Gata and Srf binding sites (Morin et al., 2001). Srf is a transcriptional coactivator with Myocd; most of the Myocd target genes are dependent on Srf (Creemers et al., 2006). In Xenopus animal cap explants containing NCCs, Myocd-dependent induction of smooth muscle genes is synergistically activated by Srf but antagonized by Gata6 (Barillot et al., 2008). Inactivation of Srf, a downstream effector of the Erk cascade, produces conotruncal and craniofacial defects much like those of Erk2 deletion mutants (Newbern et al., 2008). Srf also directly regulates miR-145 and miR-143, microRNAs that promote differentiaton and repress proliferation of SMCs (including those derived from the NC) (Cordes et al., 2009).

15.17. IMMEDIATE-EARLY GENES Sp1, Sp3, AND Sp4

The Sp genes are among the immediate-early transcription factors and bind to many promoters. Because of their more ubiquitous nature, transcription mediated by these factors is very likely to be modulated by interactions with other transcription factors. Several motifs for Sp1 binding are present in the regulatory region of the cell surface glycoprotein Mcam, a cell adhesion molecule associated with tumor progression and metastasis in human malignant melanoma (Sers et al., 1993). Transcription from the Tyr promoter, able to drive expression of a reporter gene in immortalized quail NCCs, may also be mediated via an Sp1-binding motif (Ferguson and Kidson, 1996). Expression from the Ret promoter in the NC-derived TT cell line requires only 70 bp of sequence upstream of the transcription start site, which contains two Sp1/Sp3 binding sites, and expression was abrogated by removal of these Sp1/Sp3 binding sites (Andrew et al., 2000). Tlx1 transfection enhances the activity of the Ret promoter in the SK-N-MC cell line by stimulating a region of the promoter with binding sites for Sp1, altering the interaction of Sp1 with the Ret promoter (Bachetti et al., 2005). Overexpression of Gata3 causes an increase in dopamine b-hydroxylase (Dbh)-expressing neurons in primary NCSC culture through two upstream enhancers that do not contain Gata3 binding sites but do contain binding sites for Sp1 and AP4, both of which physically interact with Gata3 (Hong et al., 2008b). In an NC-derived cell line that exhibits the characteristics of immature Schwann cells, Smads may act indirectly to promote GFAP expression through induction of Sp1, which is induced by Bmp2 (Dore et al., 2009). Expression of Sp4 (HF1b) is required for specification of the cardiac conduction system, and tissue-specific deletion of Sp4 shows its requirement in both the cardiomyogenic and NC lineages. Absence of Sp4 in the NC leads to arrhythmogenesis, conduction system defects, and atrial and atrioventricular dysfunction resulting from deficiencies in Ntrk3, a neurotrophin receptor (St Amand et al., 2003; St Amand et al., 2006).

15.18. Stat3

The signal transducer and activator of transcription 3 (Stat3) gene encodes a transcription factor that is a marker of pluripotency and one of a cohort of pluripotency genes expressed in NCSCs such as multipotent skin-derived precursors (SKPs) derived from facial skin (Zhao et al., 2009). Xenopus Hes4, essential for NC progenitor survival and maintenance, induces Delta1 through Stat3 (Nichane et al., 2008). A conserved Stat binding site provides a major contribution to the expression of AP-2a in the facial prominences (Donner and Williams, 2006). Glial differentiation occurs in response to Bmp4 and is specifically blocked by a dominant-negative Stat3. Upon Bmp4 treatment, Mtor associates with Stat3 and facilitates its activation. Inhibition of Mtor prevents Stat3 activation and glial differentiation (Rajan et al., 2003). In vitro, Cntf and Lif induce nuclear translocation of Stat3 in NC-derived cells, increase neuronal or glial marker expression, and decrease expression of the NC progenitor cell marker, Nestin (Chalazonitis et al., 1998). Dysfunctional signaling in Ret mutants often results in constitutive activation of Stat3 (Plaza Menacho et al., 2005).

15.19. Tcof1

Treacher Collins syndrome is an autosomal-dominant craniofacial disorder caused by haploinsufficiency of the Tcof1 gene product Treacle (Dixon and Dixon, 2004; Shows and Shiang, 2008). Tcof1 expression is high in developing NC, but much lower in other tissues, and heterozygous knockout of Tcof1 in the mouse embryo causes craniofacial malformation and lethality; this phenotype is very sensitive to genetic background and can be rescued by reducing p53 and inhibiting apoptosis (Dixon and Dixon, 2004; Shows and Shiang, 2008).

15.20. Tead2

The transcriptional enhancer activator domain family member 2 or Tead-box 2 (Tead2) transcription factor, along with its coactivator Yap1, is coexpressed with Pax3 in the dorsal NT. Tead2 binds an essential binding site within a Pax3 NC-specific enhancer to activate Pax3 expression. A Tead2-Engrailed fusion protein represses RA-induced Pax3 expression in vivo and in P19 cells (Milewski et al., 2004). Tead2-null mice have a significantly increased risk of NT closure defects, but early expression of Pax3 is normal, suggesting a Pax3-independent early role. NTDs could be suppressed by folic acid or the p53 inhibitor pifithrin-a (Kaneko et al., 2007). Tead2 is also required later specifically for enhancer activation in NC-derived smooth muscle cells and the dorsal aorta (Creemers et al., 2006)

15.21. Tsc22d1

TGFb1 Stimulated Clone-22 domain 1 (Tsc22d1) is a leucine zipper transcription factor with ubiquitous expression during early development. Tsc22d1 expression is later upregulated mainly at sites of epithelial–mesenchymal interactions (limb bud, tooth primordiurn, hair follicle, kidney, lung, pancreas) and in many NC-derived tissues including pharyngeal arch mesenchyme, cranial ganglia, sympathetic ganglia, DRGs, and the craniofacial skeleton. It is also expressed in the heart and in osteochondrogenic regions throughout the embryo (Dohrmann et al., 1999). Specific function of this protein has not yet been evaluated in the NC.