Chapter 14Zinc Finger Genes

Publication Details

14.1. Gata2/3

The Gata family consists of zinc finger transcription factors that bind the sequence G-A-T-A. In the chicken embryo, Gata2 (Gata3 in the mouse) is an essential member of the transcription factor network controlling sympathetic neuron development, which includes Ascl1, Hand2, Phox2a, and Phox2b. Gata2 is expressed in developing sympathetic neuronal precursors, beginning after Ascl1, Phox2b, Hand2, and Phox2a but before the noradrenergic marker genes Th and Dbh (Tsarovina et al., 2004). Gata2 expression increases in response to the other factors in this network, which are all BMP-responsive factors, and is itself responsive to BMP signaling. As further evidence that Gata2 is downstream of these factors, Gata2 is not expressed in Phox2b-null mice (Tsarovina et al., 2004). Gata2 loss-of-function decreases sympathetic chain size and reduces Th expression. Ectopic expression of Gata2 in chick NC precursors results in neuronal differentiation toward a nonautonomic, non-Th-expressing phenotype, demonstrating that it is sufficient for autonomic neuron differentiation but requires coregulators within the sympathetic lineage (Tsarovina et al., 2004). In zebrafish Hand2hands off mutants, sympathetic ganglion primordia expressing Phox2a, Phox2b, and Ascl1 are formed, but these cells have significantly reduced expression of Gata2 (Lucas et al., 2006).

Mammalian Gata3 regulates development of the thymus and adrenal glands, SNS, ear, and kidney, and is implicated in the formation of autosomal dominant hypoparathyroidism, sensorineural deafness, and renal anomaly syndrome (Airik et al., 2005; Raid et al., 2009). Expression of Gata3 causes an increase in Dbh- and Th-expressing neurons in primary NCSC culture. Gata3 transactivates the norepinephrine-synthesizing Dbh gene promoter together with Sp1 and AP4 factors bound to the Dbh promoter (Hong et al., 2008b). Gata3 transactivates the Th promoter via a promoter element that contains a binding site for Creb, but not Gata3, which physically interacts with Creb both in vitro and in vivo (Hong et al., 2006). A 625-kb Gata3 YAC transgene is missing regulatory elements that confer Gata3 expression in a subset of NC-derived lineages (thymus and sympathoadrenal system) and fails to rescue embryonic lethality, suggesting that neuroendocrine deficiency in the SNS causes death (Lakshmanan et al., 1999). Gata3-null mice have reduced Th and Dbh leading to a reduction in norepinephrine, but several other SNS genes are not altered (Lim et al., 2000).

In Gata3-null embryos, norepinephrine deficiency is the immediate cause of embryonic lethality, usually between E11 and E12 (Hong et al., 2006). Treatment with sympathomimetic beta-adrenergic receptor agonist or catechol intermediates (Lim et al., 2000; Raid et al., 2009) improves survival up to E18, allowing for further analysis of heart defects. These rescued mutants have heart defects such as ventricular septal defects, double outlet right ventricle, aortic arch abnormalities, and persistent truncus arteriosus. Short OFT and insufficient rotation of the truncus arteriosus during looping may be the main causes of these malformations (Lim et al., 2000; Raid et al., 2009). A Gata3lacZ knock-in recapitulated the endogenous Gata3 and was robustly expressed in the endocardial ridges and endothelium of distal OFT. Reporter expression was also strong in the mesenchyme and epithelium of ventral pharyngeal arches and lower in the AV canal (Raid et al., 2009). In the ventricles and atria, Gata3 is not expressed after E13.5, but LacZ activity persisted, allowing lineage tracing of cells formerly expressing Gata3,with activity detected in semilunar valves, atrioventricular valves, OFT, and aortic arch arteries (Raid et al., 2009). Gata2/3 may be important for expression of key cardiovascular development genes like Hand2 (Ruest et al., 2004). The Hand2 promoter has two conserved Gata binding sites required for Hand2 expression within the right ventricle, suggesting a role for direct regulation by Gata proteins. Gata factors are not restricted to the right ventricle, so they are likely to cooperate with coregulators to achieve right ventricular-specific regulation (McFadden et al., 2000). Cardiac-specific response to Edn1 requires synergy between the mostly ubiquitous SRF and tissue-restricted Gata proteins, which bind an Edn1 response element. SRF and Gata proteins form a complex and in transient cotransfections synergistically activate other Edn1-inducible promoters that contain both Gata and SRF binding sites (Morin et al., 2001).

14.2. Gata4

Gata4 is expressed in many NCSCs. Most migratory NCCs in E9.5–E11.5 embryos are labeled by a transgenic 5-kb Gata4 promoter driving expression of a fluorescent reporter (Pilon et al., 2008). Between E10.5 and E11.5, the Gata4 reporter is more active in boundary cap cells, where the presence of cells with NCSC properties has been corroborated (Aquino et al., 2006). In vitro analysis of the properties of Gata4-expressing cells also supports the expression of Gata4 within NCSCs (Pilon et al., 2008).

14.3. Gata6

Gata6 is strongly expressed in embryonic ectoderm, NT, and NC-derived cells, closely parallel to expression of Bmp4, a direct downstream target of Gata4 and Gata6 (Nemer and Nemer, 2003). Gata6 protein is also abundant in parts of the gut, pulmonary system, myocardium of the heart, and regions of NC- and sclerotome-derived chondrogenesis, but significantly reduced within the endocardial cushions and OFT of the heart, regions expressing the highest levels of Gata6 RNA within the heart, suggesting translational regulation (Brewer et al., 2002a; Brewer et al., 2002b). During differentiation of animal cap explants containing NCCs, Myocardin-dependent expression of smooth muscle genes acts synergistically with SRF but is antagonized by Gata6 (Barillot et al., 2008). Conditional inactivation of Gata6 in VSMCs or NCCs results in perinatal mortality with a spectrum of OFT defects, demonstrating a cell-autonomous requirement for Gata6 in NC-derived VSMCs. These OFT defects are due to severe reduction of Sema3c levels, suggesting the primary function of Gata6 during cardiovascular development may be to regulate the patterning of the cardiac OFT and aortic arch (Lepore et al., 2006).

14.4. Krox20

Krox20, a C2H2 zinc finger transcription factor related to Drosophila Krüppel, has two main functions during vertebrate embryonic development. The first is patterning of the hindbrain and associated NCCs by establishing segmented r3 and r5 domains, and the second is control of Schwann cell (SC) development.

14.4.1. Krox20 in Schwann Cells

Krox20 is expressed in early NCCs, in glial components of the cranial and spinal ganglia, and in NC-derived boundary cap cells (Wilkinson et al., 1989). Developmental transitions during SC development are regulated by Krox20, Oct6, Pax3, Sox10, Creb, and some bHLH factors (Bhatheja and Field, 2006; Jessen and Mirsky, 1998; Jessen and Mirsky, 2002; Kuhlbrodt et al., 1998a; Svaren and Meijer, 2008). Krox20 is not needed during the initial activation of Myelin gene expression in developing SCs but is required, together with Oct6, for the transition of SCs from the promyelinating to myelinating stage (Chelyshev Iu and Saitkulov, 2000; Kamholz et al., 1999). Mutations in human Krox20 are associated with Charcot–Marie–Tooth neuropathy type 1 (CMT1), a peripheral nerve demyelinating disease (Kamholz et al., 1999). Krox20 and Pax3 functions are modulated by Sox10 in developing and mature glia (Kuhlbrodt et al., 1998a). Sumoylation of Sox10 modulates its binding synergy with Krox20 and Pax3 on the Gjb1 and Mitf promoters (Girard and Goossens, 2006). SC precursors migrate along growing axons toward their final location. Krox20 is a major target of promyelinating signals, and Krox20 promotes many phenotypic changes in immature SCs that characterize their transition to myelinating cells. Krox20 interacts functionally with neuregulin and TGFb, two factors implicated in myelination in postnatal ganglia (Jessen and Mirsky, 2002). Neuregulin addition to Schwann cell precursors initiates an increase in cytoplasmic Ca2+ that activates calcineurin and the downstream transcription factors Nfatc3 and Nfatc4. Sox10 is an Nfat nuclear partner and synergizes with Nfatc4 to activate Krox20, which regulates genes necessary for myelination (Kao et al., 2009). Jun is required for SC proliferation and death and is downregulated by Krox20 during myelination. Forced expression of Jun in SCs prevents myelination, and in injured nerves, Jun is required for appropriate dedifferentiation, reemergence of immature SC state, and nerve regeneration (Mirsky et al., 2008).

A lacZ knock-in allele of Krox20, initially restricted to boundary cap cells in trunk regions, can be used to trace the fate of these cells and their progeny during development. Trunk boundary cap-derived cells migrate along peripheral axons and colonize spinal root ganglia and DRGs, giving rise to all SC precursors, some neurons (mostly nociceptive), and satellite cells. Boundary cap cells are a source of PNS progenitors that arrive in a secondary wave of migration after the major ventrolateral migratory stream of NCCs (Maro et al., 2004). In vitro, gliogenesis of cultured boundary cap-derived NCSCs (bcNCSCs), monitored by expression of Krox20, Sox2, Sox19, S100, GFAP, and fibronectin, differentiates similarly to that in vivo, sequentially adopting SC precursor and immature SC fates before maturing into myelinating and nonmyelinating SCs (Aquino et al., 2006).

14.4.2. Krox20 in Patterning

Krox20 is involved in segmentation and patterning of part of the hindbrain and its associated NCCs (Nardelli et al., 2003). Krox20 expression is conserved among mice, frogs, fish, and chicken in r3 and r5 and in a stream of NCCs migrating from r5 toward the third branchial arch (Bradley et al., 1993; Ghislain et al., 2003; Nardelli et al., 2003; Nissen et al., 2003; Nonchev et al., 1996; Wilkinson et al., 1989). Expression of Krox20 is downregulated first in r3 and then in r5 (Oxtoby and Jowett, 1993). Krox20 is often used as a marker of the segmented hindbrain, particularly for odd-numbered rhombomeres (Deflorian et al., 2004; Eroglu et al., 2006; Ishii et al., 2005; Kitaguchi et al., 2001; Labosky and Kaestner, 1998; Menegola et al., 2004; Menegola et al., 2005; Nardelli et al., 2003). Expression occurs in precursors in both r5 and r6, and both rhombomeres contribute to Krox20-expressing NC, emigration occurring first from r6 and later caudally from r5. In Krox20 mutants, hindbrain segmentation is disrupted, but at least some prospective r3 and r5 cells persist (Voiculescu et al., 2001). The remaining r3 cells acquire an r2 or r4 identity, and r5 cells acquire an r6 identity. Embryonic chimeras between Krox20 homozygous-null and wild-type cells show that Krox20 is required for formation of alternating rhombomere identities by coupling segment formation, cell segregation, and specification of regional identity (Voiculescu et al., 2001). In the chicken embryo, transplantation of r6 to the level of r4 or r5 causes many Krox20-expressing cells to migrate rostral to the otic vesicle, but transplantation of r5 to the r4 position results in only a few migrating cells that express Krox20. This demonstrates that Krox20 expression in pharyngeal arch NC does not necessarily correlate with rhombomeric segmentation, and there may be intrinsic differences between the r5 and r6 Krox-20-expressing populations (Nieto et al., 1995).

Krox20 controls the segment-restricted upregulation of Hoxb2 and Hoxa2 (Nonchev et al., 1996; Wilkinson, 1993). Krox20-null embryos have a clear loss of Hoxa2 expression in r3, placing Hoxa2 downstream of Krox20. An enhancer in the Hoxa2 locus with two Krox20 sites is needed for r3/r5 activity, but not NC and mesodermal activity. Ectopic expression of Krox20 in r4 can transactivate the Hoxa2-lacZ reporter (Nonchev et al., 1996). Comparative genomic analysis of the striped bass Hoxb2a-b3a intergenic region to those from zebrafish, pufferfish, human, and mouse demonstrated the presence of a conserved Krox20 binding site-containing enhancer necessary for r3, r4, and r5 expression (Scemama et al., 2002). In addition to regulating Hoxa2 and Hoxb2, Krox20 regulates its own transcription as part of an autoregulatory loop required in expansion and maintenance of Krox20-expressing territories (Voiculescu et al., 2001) A conserved NC enhancer containing two conserved Krox20 binding sites can recapitulate Krox20 NC expression in transgenic mice. This enhancer includes essential Sox binding sites and is strongly activated by Sox10 in synergy with Krox20 to maintain Krox20 expression in the migrating NC (Ghislain et al., 2003). Interactions at three conserved long-range enhancer elements (A, B, and C) also control Krox20 expression. Element A contains Krox20-binding sites, which are required, along with Krox20 protein, for activity. B and C are activated at the earliest stage of Krox20 expression in r5 and r3-r5, respectively, independently of Krox20 binding, initiating Krox20 expression. HNF1b is a direct initiator of Krox20 expression at element B (Chomette et al., 2006).

RA signaling greatly influences Krox20-mediated hindbrain and NCC patterning. Segment-restricted expression of Krox20 responds rapidly to RA treatment and undergoes a progressive series of changes in segmental expression (Marshall et al., 1992). Mouse embryos treated with RA just before differentiation of the cranial neural plate and the start of segmentation have reduction in preotic hindbrain length, loss of rhombomeric segmentation, and NC defects. These defects correlate with changes in Krox20 and Hoxb1 distribution (the r3 domain of Krox20 is absent and the Hoxb1/Krox-20 boundary is poorly defined) (Morriss-Kay et al., 1991). Inactivation of both RARα and RARb causes expansion of the Krox20 domain into r6 and r7, and defects in structures partially derived from post-otic rhombomere-associated mesenchymal NCCs like the thymus (Dupe et al., 1999). Raldh2-null embryos do not have r3- and r5-restricted Krox20 expression but instead a single broad domain of Krox20 (Niederreither et al., 2000).

Expression of Krox20 is modulated by a complex interplay between RA, Wnts, FGFs, and BMPs (Nardelli et al., 2003). In Xenopus animal explants, Wnt1 and Wnt3a, expressed in the dorsal NT, synergize with the neural inducers Noggin and Chordin to generate the NC, marked by Krox20, AP-2a, and Slug. Overexpression of Wnt1 or Wnt3a in the neuroectoderm of whole embryos led to a dramatic increase of Slug and Krox20-expressing cells, but hindbrain expression of Krox20 remained unaffected (Saint-Jeannet et al., 1997). Adding FGFs to the chicken NT causes ectopic Krox20 expression in caudal hindbrain NC (r7 and r8) and expansion of the Krox20-expressing area in the neuroepithelium. Application of an FGF pathway inhibitor leads to downregulation of Krox20 in the hindbrain neuroepithelium and NC (Marin and Charnay, 2000).

14.5. Mecom/Evi1

Mecom / Evi1 encodes a putative protooncogenic transcription factor containing 10 zinc finger motifs and is associated with myeloid neoplasia, myeloid leukemia, and myelodysplasia (Hoyt et al., 1997; Mead et al., 2005). Mecom is expressed in embryonic mesoderm, pronephric tissue, primary head folds, and NC-derived cells associated with the PNS (Hoyt et al., 1997; Kazama et al., 1999; Mead et al., 2005). Mice lacking full-length Mecom die at approximately E10.5 with widespread hypocellularity, hemorrhaging, and disrupted paraxial mesenchyme development, defects in the heart, somites, cranial ganglia, and peripheral nervous system (Hoyt et al., 1997). Mecom may also be important for general regulation of early neuroectodermal differentiation (Kazama et al., 1999).

14.6. Nczf

Nczf (neural crest zinc finger), a target gene of Tlx2, encodes a novel Krüppel-associated box zinc finger transcriptional repressor. The Nczf consensus binding sequence is found within regulatory regions of Ednrb and Mitf loci, suggesting a possible role for Nczf as a sequence-specific regulator of NC development (Kitahashi et al., 2007).

14.7. Prdm1 AND Prdm2

The PR domain transcriptional repressors Prdm1 and Prdm2 contain SET/zinc-finger domains. Murine Prdm1, also known as B lymphocyte-induced maturation factor (Blimp1), functions as a master regulator of B-cell terminal differentiation. Prdm1-null embryos die at mid-gestation with placental and pharyngeal arch defects, but despite its role in NC induction, NC formation is unaffected (Vincent et al., 2005). Zebrafish Prdm1 is induced by BMP signaling in cells at the boundary of the neural plate and nonneural ectoderm (Hernandez-Lagunas et al., 2005; Roy and Ng, 2004). Loss of Prdm1 activity inhibits specification of the NC and primary sensory neuron development, resulting in a reduction in NCCs and complete loss of Rohon-Beard sensory neurons. Overexpression of Prdm1 in mutants rescues both phenotypes, and in wild-type zebrafish, results in the generation of supernumerary default-fate primary sensory neurons. BMP-induced Prdm1 activity is needed for cell fate specification of both NCCs and primary sensory neurons (Hernandez-Lagunas et al., 2005; Roy and Ng, 2004). Pheochromocytomas and abdominal paragangliomas are rare tumors from NC-derived chromaffin cells. The Prdm2 tumor suppressor locus is in a region of chromosome 1 frequently deleted in these tumors. Loss of heterozygosity at the Prdm2 locus was detected in most of the tumors, and Prdm2 mRNA was downregulated in tumors compared to normal adrenal control (Geli et al., 2005).

14.8. Snail

Vertebrate Snail is a zinc finger transcription factor expressed in NC and mesoderm and orthologous to Drosophila Snail. It regulates EMT during gastrulation, and this function is at least partially conserved in EMT of the NC. Early in NC development, the two main functions of Snail are to promote delamination during EMT and to suppress apoptosis (Figure 14.1).

FIGURE 14.1. Whole-mount in situ hybridizations show Snail in mouse, chicken, fish, and frog embryos.


Whole-mount in situ hybridizations show Snail in mouse, chicken, fish, and frog embryos. Mouse: Sefton et al., 1998, 10.5-dpc embryo, lateral view; chicken: Sefton et al., 1998, stage 18 embryo, lateral view; zebrafish: Rau et al., 2006, three-somite (more...)

14.8.1. Xenopus Snail

In Xenopus, Snail is expressed in the developing neural folds at the neural plate border (NPB) in what will become the NC and roof plate; this expression is maintained in migratory NCCs (Gadson et al., 1993; Linker et al., 2000; Mayor et al., 1995). Snail and its paralog Slug are among the earliest genes that respond to NC-inducing signals from the mesoderm of the dorsal or lateral marginal zone (LaBonne and Bronner-Fraser, 2000; Mayor et al., 1995). Snail expression precedes Slug expression, and Slug expression precedes expression of Twist, another early NC transcription factor (Aybar et al., 2003; Linker et al., 2000). One of the factors upstream of Snail is Msx1. Expression of dominant-negative Msx inhibits Snail and other early NC markers, and resulting NC defects can be rescued by injection of Snail or Slug (Tribulo et al., 2003). Conditional Snail gain-of-function and loss-of-function demonstrate that Snail is upstream of Slug and induces expression of Slug and other NC markers (Zic5, Foxd3, Twist and Ets1), indicating a key role for Snail in NC specification and migration (Aybar et al., 2003). At the NPB, Snail represses and therefore restricts the expression of Delta1, which interacts with Notch to activate the Bmp4 repressor Hes4a; modulation of Bmp4 levels by Snail provides permissive conditions for specification of NPB cells (Glavic et al., 2004b). Snail protein generally acts as a transcriptional repressor and complexes with Jub (an Ajuba Lim protein) through a SNAG repression domain to silence transcription of genes such as Cdh1 (E-cadherin), a molecule important for NC delamination (Langer et al., 2008).

14.8.2. Zebrafish Snail

In zebrafish, Snail is expressed in the entire NC and in mesodermal derivatives of the head region (Hammerschmidt and Nusslein-Volhard, 1993). Although Snail is an early NC marker, it is downstream of several other, earlier NC transcription factors such as Sox9 and Foxd3 (Montero-Balaguer et al., 2006; Stewart et al., 2006; Yan et al., 2005). Two zebrafish Foxd3 mutants, Foxd3sym1 and Foxd3mother superior, when homozygous, show reduced levels of Snail expression in premigratory NCCs and subsequent depletion of many NC derivatives (Montero-Balaguer et al., 2006; Stewart et al., 2006).

14.8.3. Chicken Snail

During early stages of development, many expression domains of Slug and Snail in mouse compared to chicken embryos are reversed, but in later NC development, the sites of expression of Slug and Snail are conserved between these two species (Sefton et al., 1998). In early NC development of the chicken embryo, Slug performs the main functions that Snail carries out in the mouse, and chicken Snail is not expressed in the premigratory NC. However, electroporation experiments in the chicken embryo have shown functional equivalence between both Slug and Snail genes of both species (del Barrio and Nieto, 2002).

14.8.4. Mouse Snail

Snail is expressed in the mouse premigratory NC. Mouse Snail, together with Sox9, which provides competence for NCCs to undergo EMT, is sufficient to induce an EMT in neural epithelial cells (Cheung et al., 2005). In mouse embryos, a homozygous null mutation in Snail is embryonic lethal because of the early role of Snail during gastrulation, but an NC-specific deletion of the Snail gene demonstrates that Snail is not essential for NC formation and delamination (Murray and Gridley, 2006a; Murray and Gridley, 2006b). Functional redundancy between Snail and its paralog Slug seems to account for at least part of the lack of dramatic NC phenotype. Slug-null mice are viable and fertile, and only a percentage of these exhibit cleft palate, whereas compound Snail-heterozygous, Slug-null mutant mice exhibit a completely penetrant cleft palate. Also, NC-specific deletion of Snail in a Slug-null genetic background results in multiple craniofacial defects, including cleft palate (Murray and Gridley, 2006a; Murray et al., 2007).

14.8.5. Human Snail

In humans, Snail function is highly significant because of its role in EMT during tumor progression (Okajima et al., 2001; Paznekas et al., 1999; Twigg and Wilkie, 1999). Studies of Snail in tumorigenesis of multiple tissue types, including those derived from the NC, reveal that Snail is expressed in these tumors and that Snail-induced EMT is associated with direct repression of Cdh2(E-cadherin) transcription (Blanco et al., 2002; Fendrich et al., 2009; Kuphal et al., 2005; Locascio et al., 2002). Snail expression may affect cellular differentiation (Takahashi et al., 2004), and levels of expression inversely correlate with the grade of tumor differentiation (Blanco et al., 2002). The progression of NC-derived cancers, such as melanoma, has been linked to expression of Snail (Fendrich et al., 2009; Kuphal et al., 2005; Palmer et al., 2009). Knockdown of Snail in a melanoma cell line results in downregulation of the EMT-associated genes Mmp2, Emmprin, Sparc, Timp1, Tpa, RhoA, and Notch4, and reinduction of Cdh1 (Kuphal et al., 2005). Confirmed binding sites for Ets1/ Yy1 and Sox recognition motifs are present in a conserved 3' enhancer of the Snail gene, and both of these are needed for full activity in melanoma cells. Yy1 is needed for Snail expression; it is present only in cells expressing Snail, and Yy1 knockdown leads to downregulation of Snail (Palmer et al., 2009).

14.9. Slug

Slug, also known as Snail2, is highly similar to Snail and in many instances, as described below, can substitute for Snail because of its functional redundancy. The function of Slug seems to vary greatly in vertebrates; in mouse, Slug is not expressed in the premigratory NC, and NC specification functions are carried out by Snail alone. In the chicken embryo, Slug is expressed in premigratory NC and seems to cover many of the functions of Snail (Figure 14.2).

FIGURE 14.2. Whole-mount in situ hybridizations show Slug in mouse, chicken, fish, and frog embryos.


Whole-mount in situ hybridizations show Slug in mouse, chicken, fish, and frog embryos. Mouse: Sefton et al., 1998, 10.5-dpc embryo, lateral view; chicken: Sefton et al., 1998, stage 18 embryo, lateral view; zebrafish: Eroglu et al., 2006, 12-hpf embryo, (more...)

14.9.1. Avian Slug

In the chicken embryo, Slug is expressed in the NC, and knockdown of Slug specifically inhibits NC EMT (Nieto et al., 1994). Transplantation experiments show that the expression of Slug in addition to other dorsal markers Wnt1 and Wnt3a, and subsequent formation of NCCs, can be induced by the juxtaposition of nonneural and neural ectoderm (Dickinson et al., 1995). When the avian neural folds are ablated, the NC population can be replaced by the remaining neuroepithelial cells. Slug expression is an early response in the reformation of the NC and begins shortly after midline closure and before emergence of NCCs (Buxton et al., 1997; Sechrist et al., 1995). During the course of NC formation, Slug expression, as a marker for NC, can be repressed by engraftment of notochords or Shh-secreting cells, suggesting that prospective NCCs in the open neural plate are not yet committed to an NC fate (Selleck et al., 1998). Neural folds derived from all rostrocaudal levels of the open neural plate are competent to express Slug, but in the absence of tissue interactions, Slug becomes downregulated, indicating that additional signals are needed for maintenance of Slug expression (Basch et al., 2000).

Noggin and Bmp4 in the dorsal NT may trigger delamination of specified, Slug-expressing NCCs (Sela-Donenfeld and Kalcheim, 1999). During NC formation, quail Deltex2 is expressed throughout the ectoderm. Misexpression of a dominant-negative form of Deltex in the ectoderm inhibited expression of the NC marker Slug and NC inducer Bmp4, and cotransfection of Bmp4 can rescue Slug expression (Endo et al., 2003). In the anterior ectoderm, BMP signaling is needed for Slug expression (Sakai et al., 2005). Slug and subsequent EMT are effectively induced by BMP4, and phosphorylated Smad1 is detected in the neural plate and the neural folds (Sakai et al., 2005). Gain-of-function and loss-of-function experiments reveal that Sox9 is essential for BMP signal-mediated induction of Slug and subsequent EMT in avian NC. Slug can also activate its own promoter, and activation is enhanced synergistically by Sox9 directly binding to the Slug promoter (Sakai et al., 2006). Chicken Slug, in synergy with Sox9, is sufficient to induce an EMT in neural epithelial cells (Cheung et al., 2005).

A major role for Slug in the early NC is specifying competence to undergo EMT, which is associated with a decrease in cell–cell adhesion, loss of N-cadherin on the surface of NCCs, and changes in integrin activity as migration begins (Duband et al., 1995). TUNEL labeling and Slug expression were observed in a subpopulation of early migrating NCCs and presumably occur in subpopulations of both NC and neuroepithelial cells (Lawson et al., 1999). Slug marks premigratory and early migratory cranial NC (Del Barrio and Nieto, 2004). When avian Slug is knocked down in the premigratory NC, several dorsal NT genes are upregulated due to derepression of Slug targets. Cdh6, one of the target genes, contains three clustered E-box pairs that Slug binds directly to regulate Cdh6 transcription (Taneyhill et al., 2007). Cdh6 is expressed in the dorsal NT before NC emigration but is then repressed by Snail2 in premigratory and early migrating cranial NCCs. Knockdown of Cdh6 leads to premature NCC emigration, and Cdh6 overexpression disrupts migration (Coles et al., 2007). Slug cooperates with Ets1, specifically expressed in cranial NCCs, which is responsible for mobilization of premigratory NCCs (Theveneau et al., 2007).

14.9.2. Xenopus Slug and Upstream Regulation

A large body of research in Xenopus has helped to reveal the complex regulatory network involved in NC induction and specification, identifying many factors upstream of Slug. In Xenopus, Slug, along with its ortholog Snail, is one of the earliest markers of the developing NC (LaBonne and Bronner-Fraser, 1998; LaBonne and Bronner-Fraser, 2000). Slug expression, along with the transcription factors AP-2 and Krox20, marks the prospective NC at the dorsal most part of the NT, and expression of these genes seems to be controlled by Wnt1, Wnt3a, noggin, and chordin (Saint-Jeannet et al., 1997). However, Slug overexpression is not sufficient to induce NC. In addition to Slug, inhibition of BMP signaling seems necessary, but this requirement can be bypassed in the presence of Wnt or FGF signaling, allowing Slug-expressing ectoderm to form NC (LaBonne and Bronner-Fraser, 1998). During NC specification, Slug expression can also be induced by Snail. Slug alone is unable to induce other NC markers in animal cap assays, and Snail and Slug are functionally equivalent when tested in overexpression studies, suggesting that Snail can carry out some functions previously associated with Slug. This is supported by rescue experiments in embryos injected with dominant-negative constructs, indicating that Snail lies upstream of Slug in the genetic cascade leading to NC formation and that it plays a key role in crest development (Aybar et al., 2003). Slug, Snail, and Twist all are expressed in the presumptive neural folds and mark the presumptive NC and roof plate, initiating expression sequentially, with Snail preceding Slug, which precedes Twist. At the beginning of gastrulation, Snail is present in a unique domain of expression in a lateral region of the embryo in both superficial and deep layers of the ectoderm, as are Slug and Twist (Linker et al., 2000). Slug is expressed specifically in the prospective NC region and continues in pre- and postmigratory cranial and trunk NC. Slug, like Snail, is also induced by dorsal or lateral marginal zone mesoderm. Explants of the prospective NC are competent to express Slug (Mayor et al., 1995). Slug is specified later than Snail, which is not detected until stage 12 (Mayor et al., 1995). Wnt signaling is implicated in direct Slug regulation during NC formation because two regions of the Xenopus Slug promoter, one of which contains an essential Lef/B-catenin binding site, are necessary and sufficient to drive NCC-specific expression (Vallin et al., 2001). Downstream of signaling pathways, an NC regulatory network consisting of Sox10, Foxd3, and Slug seems to be one of the driving forces that shape the early NC (Pohl and Knochel, 2001).

Slug marks the prospective NC (Bastidas et al., 2004). Msx1 and Msx2 are required for NC Slug expression (Khadka et al., 2006; Tribulo et al., 2003). In the developing neural tube, Msx1 induces Pax3 and Zic1 cell autonomously, then Pax3 combined with Zic1 activates Slug in a Wnt-dependent manner (Monsoro-Burq et al., 2005; Sato et al., 2005). Loss of either Msx1 or Msx2 leads to changes in neural and epidermal expression boundaries, but loss of one gene can be compensated for by overexpression of the other (Khadka et al., 2006).

14.9.3. Downstream of Xenopus Slug

As described for Snail, the Slug protein also contains a conserved SNAG repression domain required for the assembly of a repressor complex and interacts directly with Ajuba LIM corepressor proteins to silence targets such as Cdh2 (E-cadherin) (Langer et al., 2008). In the developing neural folds, apoptosis is more prevalent than in the rest of the neural ectoderm, and Slug, as an antiapoptotic factor, along with Msx1, which promotes cell death, have opposing effects, such that there is less apoptosis in the prospective NC than in the region of the neural folds adjacent to the NC, where Msx1 expression is maintained. Overexpressing Slug can protect cells from apoptosis, and this effect can be reversed by expressing the apoptotic factor Bax. Slug and Msx1 control transcription of Bcl2 and several Caspases required for apoptosis (Tribulo et al., 2004), and an absence of NC marker expression after Slug knockdown can be rescued by expression of the antiapoptotic protein Bcl-xL. Bcl-xL’s effects are dependent on IkB kinase-mediated activation of the bipartite transcription factor NF-kB. NF-kB, in turn, directly upregulates Slug and Snail transcription. Slug indirectly upregulates NF-kB subunits and directly downregulates transcription of proapoptotic Caspase9 (Zhang et al., 2006a). This antiapoptotic function of Slug allows it to control cell numbers among developing NC derivatives (Tribulo et al., 2004).

Experiments fusing the Slug DNA binding domain with activation or repression domains demonstrate that Slug acts as a repressor during NC generation (LaBonne and Bronner-Fraser, 2000; Mayor et al., 2000). Overexpression of Slug leads to expanded expression of NC markers and an excess of melanocytes (LaBonne and Bronner-Fraser, 2000). Slug antisense RNA did not suppress early expression of the related gene Snail but led to reduced expression of both Slug and Snail in later-stage embryos, whereas the expression of another NC marker, Twist, was not affected. Downregulation of Slug and Snail coincided with inhibition of NCC migration and reduction or loss of many NC derivatives. In particular, formation of rostral cartilages was often highly aberrant, whereas the posterior cartilages were less frequently affected (Carl et al., 1999). The effects of Slug antisense RNA on NC migration and cartilage formation defects could be rescued by the injection of Slug or Snail mRNA. Slug is required for NC migration and Snail may be functionally redundant, and both genes are required to maintain each other’s expression in NC development (Carl et al., 1999).

Slug overexpression in the NT of the chicken embryo increases Rhob expression, the number of HNK-1-positive migratory cells, and cranial NC production (del Barrio and Nieto, 2002). Rhov is an early expressed NC marker essential for NCC induction. RhoV mRNA is maternally expressed and accumulates shortly after gastrulation in the NC forming region. RhoV depletion impairs expression of the NC markers Sox9, Slug, or Twist but has no effect on Snail induction (Guemar et al., 2007). Ectopic expression of both dominant-negative and constitutively active Rho GTPase mutants after RNA or DNA microinjection disrupted the endogenous expression of Slug, Foxd3, and Snail. Constitutively active RhoA was inhibitory, whereas dominant-negative RhoA increased NC marker gene expression. Rho GTPases can regulate the expression of Slug during NC ontogeny (Broders-Bondon et al., 2007).

14.9.4. Mouse Slug

When the mouse Slug gene was identified, cloned, and characterized (Jiang et al., 1998a; Jiang et al., 1998b; Rhim et al., 1997; Savagner et al., 1998; Sefton et al., 1998), its expression pattern was strikingly different from that of other studied vertebrates. Unlike in avians and amphibians, mouse Slug is not expressed in premigratory NCCs but rather only in migratory NCCs (Jiang et al., 1998a; Savagner et al., 1998). Many of the sites of Slug and Snail are reversed when comparing rodents to birds and amphibians; however, the later NC expression of both Slug and Snail seems to be more conserved among all vertebrates (Sefton et al., 1998). In both mice and rats, the earliest significant expression of Slug is in cranial NCCs that are migrating and invading the first pharyngeal arch, and Slug expression is maintained in craniofacial NC derivatives (Savagner et al., 1998). Surprisingly, Slug-null embryos have normal NC formation, migration, and differentiation, demonstrating that neither expression pattern nor biological function of Slug are conserved (Jiang et al., 1998a; Murray and Gridley, 2006b). Slug homozygous-null mice are mostly viable and fertile (Murray and Gridley, 2006a), with only melanocyte defects, making the Slug homozygous-null mouse a model for type 2 Waardenburg syndrome (WS2) (Perez-Mancera et al., 2006; Tachibana et al., 2003), and a few mice also have cleft palate (Murray et al., 2007). Compound Snail-heterozygous, Snail-null embryos have a completely penetrant cleft palate, and NC-specific Snail deletion in a Slug-null background also results in multiple craniofacial defects, including cleft palate (Murray et al., 2007). Analysis of a lacZ knock-in into the Slug locus shows that Slug is highly expressed in the craniofacial mesenchyme and NC-derived craniofacial skeleton as well as in the OFT and endocardial cushions of the heart (Oram et al., 2003). Slug is critical for the development of many NC-derived cells. Overexpression of Slug in mice carrying a Slug transgene does not cause any apparent developmental defects, but these adult mice have a 20% incidence of sudden death as well as cardiac failure that may be associated with progression and proliferation of mesenchymal tumors (Perez-Mancera et al., 2006; Perez-Mancera et al., 2005).

In zebrafish, Slug mRNA accumulates at the neural plate border including the prospective NC, and expression later becomes restricted to the NC. Slug is one of the earliest NC-specific genes known in zebrafish (Thisse et al., 1995).

14.9.5. Slug in Humans

Patients with WS2 have been identified with homozygous deletions in Slug leading to absence of Slug protein, demonstrating the requirement of Slug in the melanocyte lineage (Hou and Pavan, 2008; Sanchez-Martin et al., 2002; Sanchez-Martin et al., 2003; Tachibana et al., 2003). This role is supported by a number of cases of human piebaldism without Kit mutations that have been attributed to Slug mutations (Tachibana et al., 2003). Mitf can transactivate the Slug promoter, and a genetic interaction exists between Slug and Kit (Sanchez-Martin et al., 2002). Slug and several other NC regulatory genes can be found in primary human melanocytes that form melanomas and is required for metastasis of transformed melanoma cells (Gupta et al., 2005). Slug duplication is associated with tetralogy of Fallot and cleft palate among other defects (Perez-Mancera et al., 2006).

14.9.6. Snail and Slug in Stem Cell Culture

During the directed differentiation of rat cortical neuron stem cells into a variety of NC lineages, Slug is one of the induced premigratory NC markers (Gajavelli et al., 2004). Multipotent stem cells derived from rat periodontal ligament and porcine skin-derived precursors (SKPs) expressed Slug as well as other NC markers (Techawattanawisal et al., 2007; Zhao et al., 2009). Snail is one of several NC markers induced in the formation of NC-like cells during the differentiation of human embryonic stem cells (hESCs) toward a neuronal lineage in the presence of mouse stromal cells (Pomp et al., 2005). Slug and Snail are both among the NC markers that are expressed in mouse dental NC-derived progenitor cells (Degistirici et al., 2008).

14.10. Zeb1 AND Zeb2

The zinc finger E-box binding homeobox (Zeb) transcriptional repressor family has two factors expressed in NC: Zeb1 (also known as deltaEF1) and Zeb2 (also known as Smad-interacting protein 1/SIP1). Zeb1 is a transcriptional corepressor of Smad target genes with functions in the patterning of NC-derived cells, CNS, and midline structures. Mutations in Zeb1 can lead to neurological disorders in addition to dysmorphic features, megacolon, and other malformations (Sztriha et al., 2003). A human Zeb1 mutation linked to Hirschsprung’s disease suggests that deficiency of Zeb1, resulting in disrupted BMP/Smad signaling, may contribute to ENS defects (Cacheux et al., 2001). Zeb2 is expressed in NC derivatives, notochord, somites, limbs, and a few domains of the CNS. Zeb2-null mice die perinatally with severe T-cell deficiency of the thymus and various skeletal defects, including defects in the NC-derived craniofacial skeleton (cleft palate, Meckel’s cartilage hyperplasia, nasal septum dysplasia, shortened mandible) (Takagi et al., 1998). Xenopus Zeb1 is initially strongly expressed in prospective neurectoderm and later continues primarily within the NT and NC (van Grunsven et al., 2000). In Xenopus, Zeb1 and Zeb2 are coexpressed in migratory cranial NC, in the retina, and in the NT. Overexpression of Zeb2, like Zeb1, reduces expression of BMP-dependent genes, but only Zeb1 induces neural markers. Zeb1 and Zeb2 both form complexes with Smad3, coactivators p300 and pCAF, and the corepressor Ctbp1, but have different repression efficiencies (van Grunsven et al., 2006).

14.11. Zfp704

Zinc finger protein 704, also known as glucocorticoid-induced gene-1 (Gig1), is a C2H2 zinc finger protein. In mouse embryos, X-gal staining of a Zfp704nuclear lacZ knock-in shows expression in several NC-derived lineages (Yamamoto et al., 2007).

14.12. Zic1

The Zic genes are vertebrate orthologs of the Drosophila pair rule gene odd-paired encoding C2H2 zinc finger transcription factors (Kuo et al., 1998). There are five Zic orthologs (Zic1–Zic5) conserved in mammals, birds, frogs, and fish (Inoue et al., 2007; Keller and Chitnis, 2007; Nakata et al., 1998; Warner et al., 2003), and zebrafish has a Zic6 gene lacking a subset of expression domains typical of other Zic family members (Keller and Chitnis, 2007). The Zic genes are generally expressed in the dorsal NT and play multiple roles during neural development: NC induction, neural differentiation, inhibition of neurogenesis, left–right asymmetry, and regulating proliferation (Elms et al., 2003; Keller and Chitnis, 2007; Merzdorf, 2007; Warner et al., 2003). Although the Zic genes have broadly overlapping expression, mutants for individual Zic genes typically have distinct phenotypes (Inoue et al., 2007). Zic1, Zic2, and Zic3 are very similar to each other (Nakata et al., 1998), whereas Zic4 is very close to Zic5 (Fujimi et al., 2006). Zic proteins can interact physically with other Zic proteins or regulate Zic transcription, indicating a large potential for crosstalk among Zic family members (Fujimi et al., 2006; Keller and Chitnis, 2007).

Zic1 encodes a transcriptional activator expressed throughout the presumptive neural plate that becomes restricted to the dorsal NT and NC (Kuo et al., 1998; Nakata et al., 1998). Zic1 plays a role in patterning the early neural plate and in formation of the NC, somites, and cerebellum (Cornish et al., 2009; Kuo et al., 1998). Zic1 is also one of the earliest molecular indicators of neural fate determination in Xenopus (Tropepe et al., 2006); it renders animal cap ectoderm competent to respond to the neural inducer Noggin and modifies the array of genes induced by Noggin. Activated Zic1 induces NC and dorsal NT markers in both animal caps and whole embryos. In more ventral ectoderm, Zic1 induces formation of loose cell aggregates suggestive of NC precursor cells as it suppresses ventral fates (Kuo et al., 1998).

In neuralized ectoderm, Msx1 induces Pax3 and Zic1, and then Pax3 and Zic1 activate Slug in a Wnt- and Foxd3-dependent manner (Monsoro-Burq et al., 2005; Sasai et al., 2001; Sato et al., 2005). Pax3 and Zic1 expression overlaps in the presumptive NC area before NC marker genes Foxd3 and Slug, and this coexpression is essential for initiating NC differentiation (Sato et al., 2005). Overexpression of Zic1 and Pax3 together induces ectopic NC differentiation in ventral ectoderm, but overexpression of either alone expands NC marker expression only within the dorsolateral ectoderm (Nakata et al., 1998; Sato et al., 2005). In Xenopus, Pax3 and Zic1 are necessary and sufficient to promote hatching gland and preplacodal fates, respectively, whereas their combined activity is essential to specify the NC (Hong and Saint-Jeannet, 2007). Inhibition of BMP signaling is critical for activation of Xenopus Zic1 expression and establishing neural identity (Tropepe et al., 2006). Zic1 is not expressed in migratory NC (Sun Rhodes and Merzdorf, 2006). In the chicken embryo, Zic1, Zic2, and Zic3 are expressed in the hindbrain, NC, periotic mesenchyme, and inner ear. Zic1 and Zic2 have very similar expression patterns and strength (Zic2 is generally slightly weaker), and Zic3 has very weak expression (Warner et al., 2003).

14.13. Zic2

Murine Zic2 is first expressed in the entire neural plate, becomes restricted to the lateral region, and is required for timing of neurulation. Knockdown of murine Zic2 delays neurulation, resulting in holoprosencephaly (HPE, also associated with human Zic2 mutations) (Houston and Wylie, 2005; Nagai et al., 2000), spina bifida, perinatal mortality, delayed differentiation of the dorsal-most neural plate (roof plate and NCCs), and impaired development of NC derivatives such as the DRGs (Nagai et al., 2000). A Zic2 hypomorphic allele or loss-of-function delays NC production resulting in a decrease of NCCs. In addition, Zic2 contributes to hindbrain patterning and is essential for normal development of r3 and r5 (Elms et al., 2003). In Xenopus, Zic2 is expressed broadly in the ectoderm (Nakata et al., 1998), then becomes restricted to stripes that alternate with regions giving rise to primary neurons, and inhibits neurogenesis and induces NC differentiation by counteracting the neurogenic inducing activity of Gli proteins (Brewster et al., 1998). RA and Shh signaling have opposite effects on Gli3 and Zic2 that prepattern the neural plate and RA cannot rescue the inhibitory effect of Zic2 on primary neurogenesis. RA treatment inhibits the expression of Xenopus Zic2 and Shh in the neural ectoderm while expanding the Gli3 expression domain, but a retinoid antagonist induces Zic2 and Shh. Shh overexpression enlarges the neural plate at the expense of the NC and upregulates Zic2 while downregulating Gli3 (Franco et al., 1999). Overexpression of Zic2 enhanced neural and NC-derived tissue formation in embryos and in animal cap explants (Nakata et al., 1998).

14.14. Zic3

Xenopus Zic3 plays a significant role in neural and NC development (Nakata et al., 1997; Zhu et al., 2007). Zic3 is induced by inhibiting Bmp4 and is first detected in the prospective neural plate following Chordin expression, earlier than most proneural genes. Overexpression of Zic3 results in hyperplastic neural and NC-derived tissue in vivo and induces expression of proneural and NC marker genes in animal cap explants (Nakata et al., 1997). Overexpression of Meis1b in animal cap explants induces Zic3 and can also induce ectopic NC-derived tissues (pigmented cell masses) in developing embryos (Maeda et al., 2001). Zic3, which contains several AP-1 binding sites, is also induced by AP-1 in a dose-dependent manner. Knockdown of the AP-1 component Jun blocks Activin-induced Zic3 expression, whereas coinjection of Jun mRNA rescues downregulated Zic3 expression (Lee et al., 2004a). Mutations in Zic3 cause situs ambiguus or isolated congenital heart defects in humans, such as transposition of the great arteries (Chhin et al., 2007; Zhu et al., 2007). Mutant Zic3 protein is expressed at lower levels, is degraded more quickly, has decreased transcriptional activation of a TK-luciferase reporter, and reduced activity in left–right asymmetry and NC induction in Xenopus embryos (Chhin et al., 2007). Zic3-null mice also have left–right asymmetry defects, associated cardiovascular, vertebra/rib, and CNS malformations, and 20% lethality but no detectable defects in NC-derived tissues, presumably because of redundancy of other Zic proteins in the NC (Zhu et al., 2007).

14.15. Zic4 AND Zic5

Xenopus Zic4 expression is detected mainly in the NPB, dorsal NT, and somites, similar to Zic1. Injection of Zic4 mRNA caused the induction of NC marker genes, excess pigment cell formation, and excess neural tissue, suggesting Zic4 can induce neural and NC tissue similar to other Zic family members. The zinc-finger domain is critical for many Zic4 functions, but the C-terminal regions of Zic4 and Zic5 are distinct from Zic1, Zic2, and Zic3 in their involvement in inducing NC genes Slug and Sox10 (Fujimi et al., 2006). Zic5, like the other Zic proteins, is expressed in the early NC and is sometimes used as an NC marker (Aybar et al., 2003).