The Fox (forkhead box) family consists of a large number of genes that contain a conserved winged helix DNA binding domain. In the mouse, there are 35 Fox genes (http://biology.pomona.edu/fox/). Fox transcription factors often are associated with control of cell differentiation, as will be demonstrated in many of the examples below. They may act as regulatory “gatekeepers” at different steps along NC differentiation pathways. Several Fox proteins have been hypothesized to act as pioneering factors, binding to DNA and perhaps required in many tissues for the recruitment of additional transcription factors and chromatin modifiers (Cuesta et al., 2007; Sekiya et al., 2009; Zaret et al., 2008).
Foxc1 plays an important role in the NC-derived periocular mesenchyme, crucial for the development of the eye. In the anterior eye of humans and mice, NCCs populate the inner layers of the cornea, the iridocorneal angle, and the anterior part of the iris (Ittner et al., 2005; Iwao et al., 2009; Sowden, 2007). Foxc1 is expressed throughout development of the eye, and mutations in human Foxc1, along with mutations in Pitx2, have been associated with Axenfeld–Rieger anomaly (ARA), an autosomal dominant form of anterior segment dysgenesis (ASD) (Lines et al., 2002; Sowden, 2007). Upon identification of Foxc1 as a candidate gene for ARA/ASD, mouse models were identified allowing for detailed expression and functional studies. One mouse model of ARA is the congenital hydrocephalus (ch) mutant mouse (Hong et al., 1999), which harbors a nonsense mutation in the Foxc1 locus. Mice heterozygous for Foxc1ch have anterior segment defects, whereas homozygous mutants have cranial NC-derived skeletal defects leading to lethal hydrocephalus among other developmental abnormalities (Hong et al., 1999).
Foxc1 and Pitx2 act together to specify primarily NC-derived mesenchymal progenitor cells as they migrate around the embryonic “optic cup.” Foxc1 and Pitx2 also regulate differentiation of these same mesenchymal cells to produce the various cell types of the anterior segment (Sowden, 2007). In culture, TGFb enhances Foxc1 and Pitx2 expression, and anterior segment morphogenesis requires TGFb signaling (Ittner et al., 2005). TGFb2 from the lens is required for Foxc1 and Pitx2 expression in the NC-derived cornea and angle (Ittner et al., 2005; Iwao et al., 2009). Inhibition of TGFb signaling (Ittner et al., 2005) or reduction of TGFb2 signaling in mouse embryonic NCCS by disruption of heparan sulfate synthesis (Iwao et al., 2009) leads to reduced phosphorylation of Smad2 and downregulation of Foxc1 and Pitx2 (Iwao et al., 2009), resulting in ARA/ASD-like ocular defects due to NCC differentiation and survival defects (Ittner et al., 2005). Posterior to the lens, inactivation of TGFb signaling specifically in NCCs causes morphogenetic and patterning defects similar to human persistent hyperplastic primary vitreous (Ittner et al., 2005). The expression and perhaps function of Foxc1 is likely conserved in the eye; in later stages of Xenopus embryogenesis, Foxc1 transcripts can be found within NCCs surrounding the eye. It is also detected in the mandibular, hyoid, and pharyngeal arches and in the heart (Koster et al., 1998).
Another FoxC gene expressed in the NC is Foxc2. In early mouse embryos, Foxc2 is expressed in cranial NC and can be found later in the dorsal aorta. Foxc2 heterozygous mice have ASD-like eye defects (Smith et al., 2000), and Foxc2-null mice have defects in the NC-derived cranial skeleton and in NC-mediated remodeling of the aortic arch resulting in embryonic or perinatal lethality (Iida et al., 1997). During heart development, Foxc1 and Foxc2 likely share some functional redundancy (Kume et al., 2001). Both genes are expressed in cardiac NCCs and the second heart field, endocardium, and proepicardium. Compound-null mutants have a wide spectrum of cardiac abnormalities including defects in the NC-mediated septation of the outflow tract, likely due to extensive cardiac NCC apoptosis during migration (Seo and Kume, 2006).
Foxd3 is the most studied Fox gene in the NC and likely the most critical for NC development. Expression in premigratory and migratory NCCs was first described in the early chicken embryo (Yamagata and Noda, 1998), and soon after in the mouse embryo (Labosky and Kaestner, 1998). In all vertebrates studied, Foxd3 expression begins in the dorsal NT as commitment to NC fate occurs, and as the cells migrate to their final destinations, Foxd3 expression diminishes (Dottori et al., 2001; Hromas et al., 1999; Yamagata and Noda, 1998) (Figure 5.1). One exception is the melanoblasts, which migrate away from the dorsal neural tube at a later stage and do not express Foxd3 (Kos et al., 2001).
The only other members of the FoxD subgroup with reported NC expression or function are Xenopus Foxd1 and Foxd2. Foxd1 is expressed in prospective NCCs during early embryonic development but is extinguished in the NC at the time of migration. Grafting experiments and overexpression of Foxd1 suggest that it represses NCC migration (Gomez-Skarmeta et al., 1999). Foxd2 expression was observed in cranial NCCs surrounding the eye, migrating into the second and third visceral pouches, and also in ethmoidal and mandibular processes of the facial skeleton (Pohl and Knochel, 2002).
5.2.1. Foxd3 in Neural Crest Progenitor Cells
In the chicken embryo, misexpression of Foxd3 within the dorsal NT causes an expansion of the NC domain and represses differentiation of interneurons (Dottori et al., 2001; Kos et al., 2001). Cells ectopically expressing Foxd3 upregulate Cadherin7, delaminate, and migrate away from the NT, and cells maintaining expression of Foxd3 do not differentiate normally (Dottori et al., 2001). Here, Foxd3 acts independently of Slug, another early NC marker, to promote the development of NCCs from the developing NT (Dottori et al., 2001). In the mouse embryo, Foxd3 is required for maintenance of multipotent NC progenitors. An NC-specific deletion of a floxed allele of Foxd3 results in a broad loss or severe reduction of NC derivatives, including the craniofacial skeleton, pharyngeal arch NCCs, sensory ganglia, DRGs, cranial ganglia, and enteric ganglia (Teng et al., 2008). The cardiac NC progenitor population also seems to be greatly reduced. Foxd3 likely plays a role in controlling NC survival because these Foxd3 conditional mutants have an increase in NC apoptosis (Teng et al., 2008). An additional role in early NC development for Foxd3 suggested by work in human cells is the maintenance of a progenitor state through prevention of terminal quiescence, possibly by inhibiting the cyclin-dependent kinase inhibitor p21. RA-induced expression of neuronal differentiation markers in pluripotent human teratocarcinoma cells was not influenced by forced overexpression of Foxd3, but an RA treatment-associated inhibition of proliferation, due to an increase in p21, was overcome by Foxd3 overexpression (Hromas et al., 1999).
In zebrafish, knockdown of Foxd3 results in an early reduction of several NC lineages, including jaw cartilage, sympathetic and enteric neurons, glia, and iridophores. Foxd3 is required for the differentiation of a subset of NC lineages but does not appear to greatly affect NC induction or initial migration away from the NT. In the DRGs, where Foxd3 expression persists, it is required cell-autonomously for DRG development, but expression is then reduced as the DRGs fully differentiate (Lister et al., 2006). Zebrafish Foxd3sym1 homozygous mutants that contain a mutation in the winged helix domain have defects very similar to those observed after Foxd3 knockdown. Again, the number of premigratory NCCs is not affected, but the levels of other early NC genes (Snail1b and Sox10) are reduced, and there is delayed migration and a reduction in the number of migratory trunk NCCs. Similar to the mouse, Foxd3 plays a role in the survival of some NCCs; the Foxd3sym1 mutants have increased apoptosis in the NC at the level of the hindbrain (Stewart et al., 2006). Zebrafish Foxd3mother superior mutants that have a disruption in a distal regulatory element likewise have similar defects and, similar to the Foxd3sym1 mutants, have a reduction of the NC transcription factors Snail, Sox9b, and Sox10. In these mutants, too, the premigratory NCCs form in normal numbers, suggesting that Foxd3 maintains NC progenitor pools (Montero-Balaguer et al., 2006). Although the study of Foxd3 has been limited primarily to the typical model organisms, Foxd3 expression is observed in the NC-derived osteogenic cells of the turtle plastron bone (Cebra-Thomas et al., 2007). Little is known about Foxd3 gene regulation, but one factor identified upstream of Foxd3 is the Disrupted in schizophrenia 1 protein (Disc1). Loss of Disc1 in zebrafish embryos results in failure of the cranial NCCs to migrate away from the midline dorsal to the NT. Disc1 functions in the transcriptional repression of Foxd3 and Sox10, mediating cranial NCC migration away from the NT and cranial NCC differentiation. Continued expression of Foxd3 and Sox10 affects cranial NCC development, leading to loss of craniofacial cartilage and expansion of cranial peripheral glia. Foxd3 and Sox10 have many functions in cranial NCCs, including maintenance of precursor pools, initiation of migration, and induction of differentiation (Drerup et al., 2009). Reduction of Foxd3 or Sox10 also results in loss or reduction of neuromodulatory GnRH cells of the terminal nerve possibly arising from cranial NC (Whitlock et al., 2005).
5.2.2. NC Induction and Regulation of Foxd3
In Xenopus, Foxd3 is initially expressed within the Spemann organizer where it functions in mesoderm induction and then later in premigratory NCCs (Pohl and Knochel, 2001). Much of the work examining the role of Foxd3 within the complex gene regulatory network that promotes NC induction has been performed in Xenopus. Foxd3 is one of a complex network of factors including Msx1, Zic1, Zic5, Hes4, Snail, Slug, Pax3, Pax7, Sox10, Sox9, and Twist1 that act to specify the NC. Foxd3 expression is downstream of Msx1, Hes4, Zic1, Pax3, Snail, Sox10, and Sox9 (Aybar et al., 2003; Honore et al., 2003; Nagatomo and Hashimoto, 2007; Osorio et al., 2009; Sato et al., 2005; Tribulo et al., 2003; Yan et al., 2005). However, these networks also include feedback mechanisms, as Foxd3 also regulates expression of several genes initially upstream of Foxd3, such as Sox10 (Cheung et al., 2005; Dutton et al., 2008). Also, many of these genes regulate expression of the other genes in the network once they are activated, and this is true for Foxd3. Chicken LSox5 is initially expressed in premigratory and then migratory NC after FoxD3 expression. However, forced expression of LSox5 enlarges the NC domain and prolongs NC segregation, resulting in overproduction of Foxd3-expressing cranial NCCs, another example of a feedback mechanism (Perez-Alcala et al., 2004). Early expression of Foxd3 in the prospective NC also requires activation by the Wnt pathway and is inhibited by BMP signaling (Pohl and Knochel, 2001; Taneyhill and Bronner-Fraser, 2005). In Xenopus, Foxd3 expression in the prospective NC is similar to expression of other early NC markers such as Slug and Zic1 and is itself required for NC induction, at least in part through regulation of Slug (Sasai et al., 2001). Overexpression of Foxd3 in the embryo or ectodermal explants results in expanded expression of NC markers. A dominant-negative Foxd3 construct inhibits NC differentiation in the embryo, but this can be rescued by coinjection of Slug. In animal cap explants, dominant-negative Foxd3 can inhibit NC differentiation even in the presence of the NC-inducers Slug and Wnt3a, and Foxd3 is necessary for the induction of Slug by Zic proteins (Sasai et al., 2001). Expression of Snail, Slug, and Foxd3 together leads to delamination from the neural tube (Tucker, 2004). Upon delamination, Foxd3 becomes downregulated in most lineages at the start of migration (Taneyhill and Bronner-Fraser, 2005).
In addition to Slug, there are a few genes that are known to lie downstream of Foxd3, but not many direct targets of this winged helix transcription factor are known. Foxd3 regulates expression of cell adhesion molecules N-cadherin, Integrinb1, Laminin, and Cadherin7, required for NC migration (Cheung et al., 2005). In Xenopus mesoderm induction, Foxd3 acts as a repressor (Steiner et al., 2006; Yaklichkin et al., 2007). Foxd3 recruitment of Groucho corepressors is essential for the transcriptional repression of target genes and induction of mesoderm in Xenopus (Yaklichkin et al., 2007). Recent evidence in embryonic stem cells (ESCs) supports the hypothesis that Foxd3 acts as a pioneer factor to open chromatin and affect downstream gene expression. Foxd3 regulates demethylation at a CpG in the Alb1 enhancer, implicating this stem cell protein in maintenance and/or generation of transcriptional competence in ESCs (Xu et al., 2009). This has not yet been investigated in NC or NCSCs.
5.2.3. Foxd3 Suppresses the Melanocyte Lineage
One well-characterized target of Foxd3 is Mitf. The first NCCs that migrate out from the NT are specified as neurons and glia. The subsequent wave of migrating NCCs will give rise to melanocyte precursors (Kos et al., 2001; Thomas and Erickson, 2009). Mitfa, a master regulator of melanogenesis, is only expressed in Foxd3-negative NCCs, and the number of cells expressing Mitfa increases in Foxd3 mutants. Foxd3 controls the lineage choice between neurons and glia versus melanocytes by sequestering Pax3 to indirectly repress Mitf expression during the early phase of NC migration. Foxd3 thus represses melanogenesis and is expressed exclusively in neural and glial precursors, whereas Mitf is expressed only in melanoblasts. Ectopic expression of Foxd3 represses Mitf in cultured NCCs and in melanoma cells (Thomas and Erickson, 2009). There is also evidence that Foxd3 can act directly on the Mitfa promoter to repress its expression (Curran et al., 2009; Ignatius et al., 2008). Misexpression of Foxd3 in late-migrating NCCs suppresses development of the melanocyte lineage, whereas knockdown of Foxd3, both in vivo and in vitro, results in expansion of the melanoblast population and perhaps loss of neuronal and glial precursors (Kos et al., 2001). In zebrafish, Hdac1 is required to suppress Foxd3 expression in a subset of the NC, thus de-repressing Mitfa and allowing melanogenesis to proceed (Ignatius et al., 2008). In zebrafish Hdac1colgate mutants, Foxd3 expression is increased and prolonged, and although normal numbers of premigratory NCCs are induced, fewer melanoblasts are specified, and there is a delay in differentiation and decreased migration of melanophores and melanophore precursors. Mitfa expression and melanophore defects in Hdac1colgate mutants are rescued by partial reduction of Foxd3 expression.
Mutations in human Foxe1 are linked to the Bamforth–Lazarus syndrome, characterized by hypothyroidism and cleft palate (Nakada et al., 2009). Zebrafish Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development (Nakada et al., 2009). Morpholino knockdown of Foxe1 function resulted in disrupted craniofacial and pharyngeal skeleton development, likely as a result of suppressed chondrocytic proliferation. The initial steps of NC migration and pharyngeal arch specification occur normally, but later chondrocyte proliferation and differentiation was perturbed, as indicated by a reduction in Sox9a, Col2a1, and Runx2b (Nakada et al., 2009).
In Xenopus, Foxf1a is expressed in cranial NC (Koster et al., 1999). In mouse, the cranial NC expression of both Foxf1 and Foxf2 is restricted to the palatal mesenchyme, where their expression levels, along with those of Osr2, are regulated by Shh-Smo signaling (Lan and Jiang, 2009). Foxf2 expression in the mesenchyme around the oral cavity is stronger than Foxf1 expression, and Foxf2 null mice have a cleft palate, the only reported defect (Ormestad et al., 2004). Foxf1-null mutants exhibit early lethality due to extraembryonic- and lateral plate mesoderm-related defects (Ormestad et al., 2004).
In the mouse, Foxj3 is expressed in the neuroectoderm at early stages (starting at E8.5) and continues in the NC and NC-derived structures, such as the facioacoustic, trigeminal, and DRGs (Landgren and Carlsson, 2004).
Xenopus Foxn2 is detected in the early eye field and later in the pharyngeal arches, vagal ganglion, and developing retina (Schuff et al., 2006). Xenopus Foxn3 is expressed in NCCs and the early eye field and in pharyngeal arches (Schuff et al., 2006; Schuff et al., 2007). Knockdown of Foxn3 results in defects of the cranial NC-derived jaw cartilage, the cranial nerves, and the eye (Schuff et al., 2007). In the developing jaw, NC migration occurs normally, but NC differentiation is disrupted. In the eye, loss of Foxn3 results in increased apoptosis. Foxn3 may regulate craniofacial and eye development in part by the recruitment of two HDAC proteins Sin3 and Rpd3 that were identified as potential binding partners of Foxn3 (Schuff et al., 2007).
Genes of the FoxO subgroup are often important for cell cycle regulation. Both Foxo1 and Foxo3a have been demonstrated to play a role in rat NC-derived enteric nervous system precursor survival and neurite extension, acting as substrates of Akt in a PI3K-dependent pathway (Srinivasan et al., 2005). The Gdnf protein in ENS precursors induces phosphorylation of Akt leading to a decrease of Foxo1 and Foxo3a. Misexpression of active Foxo1 induces ENS precursor death, whereas a dominant negative represses ENS precursor cell death (Srinivasan et al., 2005). In Xenopus, Foxo1 is expressed in head mesenchyme anterior to the eye and within the pharyngeal arches. Foxo3 is expressed in NCCs at the late neurula stage and later in the head and pharyngeal arches (Pohl et al., 2004).
The murine Foxs1 gene is expressed in NC-derived cells such as cranial ganglia, DRGs, and neurons of the enteric ganglia, as revealed by a knock-in allele placing the lacZ gene into the Foxs1 locus (Heglind et al., 2005). Foxs1 mutant mice have affected motor function and body weight, but this may be due solely to the function of Foxs1 in the CNS (Heglind et al., 2005). Foxs1 specifically marks early sensory neurons and sensory neuron precursors of the trunk, and is expressed exclusively in both NC-derived and ectodermal placode-derived peripheral sensory neurons, but not in nonneuronal NC-derived cell types (Montelius et al., 2007). Expression of Foxs1 does not overlap with Sox10 expression, suggesting that acquisition of Foxs1 expression represents an important lineage restriction in the differentiation of multipotent NCCs toward a sensory neuron fate. Migrating NC-derived sensory neuron precursors that express Foxs1 form clusters in the developing ganglion and exhibit reduced proliferation compared to surrounding Sox10-positive cells, and begin to express Ngn1 and Brn3 as the DRG condenses (Montelius et al., 2007).
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Nelms BL, Labosky PA. Transcriptional Control of Neural Crest Development. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. Chapter 5, Fox Genes.