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PAX3 and neural crest

Studies on the mouse Splotch mutation, a deletion in the transcription factor Pax-3, have revealed that Pax-3 is essential for normal development of the neural crest. The defect in neural crest development was investigated using a Wnt-l/LacZ reporter construct to mark neural crest cells. Staining embryos for beta-galactosidase activity at different developmental stages reveals a severe reduction in the number of neural crest cells that emigrate from the neural tube at the vagal and rostral trunk levels. At the caudal thoracic, lumbar, and sacral levels there is a complete loss of neural crest cell emigration. No evidence was seen for any delay in the onset of neural crest cell migration at anterior levels. Pax-3 is expressed in the dorsal neural tube (where the neural crest cells originate), in migrating neural crest cells and also in somitic cells along the migratory pathway. Hence, it is not clear which aspect of the Pax-3 expression accounts for the observed phenotype. This problem was addressed by transplanting neural tissue between mouse and chick embryos. These studies indicate that the defect in the Splotch mutation is not intrinsic to the neural crest cells themselves, but appears to reflect inappropriate cell interactions either within the neural tube or between the neural tube and the somite. Transplanted neural tubes from Splotch mutants, when fused with normal neural tubes, give rise to normal neural crest cells migrating along the appropriate ventral neural crest cell migration pathway and colonizing both sympathetic ganglia and dorsal root ganglia. Apparently, an extracellular signal acting downstream of Splotch can make up for the Splotch deficiency, and allows cells to adopt a normal phenotype (Serbedzija, 1997).

Splotch mice, which harbour mutations in the Pax3 gene, exhibit neural crest-related abnormalities, including pigmentation defects, reduced or absent dorsal root ganglia and failure of cardiac outflow tract septation in homozygotes. Although splotch neural crest cells fail to colonize target tissues, they initiate migration in vivo and appear to migrate as well as wild type neural crest cells in vitro, suggesting that the neural crest abnormality in splotch may reside not in the neural crest cells themselves, but rather in the extracellular environment through which they migrate. The expression of genes encoding extracellular matrix molecules has been examined in Sp2H homozygous embryos and a marked over-expression of transcripts for the chondroitin sulphate proteoglycan versican has been found in the pathways of neural crest cell migration. Use of cadherin-6 expression as a marker for neural crest demonstrates a striking correlation between up-regulation of versican expression and absence of migrating neural crest cells, both in the mesenchyme lateral to the neural tube and in the lower branchial arches of Sp2H homozygotes. Pax3 and versican have mutually exclusive expression patterns in normal embryos whereas, in Sp2H homozygotes, versican is generally over-expressed with `infilling' in regions that would normally express functional Pax3. Versican, like other chondroitin sulphate proteoglycans, is non-permissive for migration of neural crest cells in vitro. It is suggested that over-expression of this molecule leads to the arrest of neural crest cell migration in splotch embryos. Pax3 may serve to negatively regulate versican expression during normal development, thereby guiding neural crest cells into their pathways of migration (Henderson, 1997).

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. The deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. Evidence is provided that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development (Kwang, 2002).

Three lines of evidence support the hypothesis that Pax3 regulates Msx2 through a direct effect on its promoter. (1) Msx2 lacZ transgenes are upregulated in the dorsal neural tube of Pax3Sp/Sp embryos in a manner similar to the endogenous Msx2 gene. (2) The 560 bp Pax3-responsive region of the Msx2 promoter includes a 520 bp stretch that is highly conserved (87%) in 5' flanking DNA of the human MSX2 gene. Within this stretch is a single conserved Pax3 consensus site that Pax3 binds with high affinity. (3) Mutation of this element, designated Pax site 1M, causes upregulation of Msx2 lacZ transgene expression in the dorsal neural tube. This upregulation is similar in spatial pattern to that of the Msx2 lacZ transgenes in the Splotch mutant background. These data strongly suggest that Pax3 regulates Msx2 lacZ transgenes through a direct interaction with Pax site 1. Whether Pax site 1 is functional in the context of the endogenous Msx2 promoter is unclear, though in situ hybridization data show that in the Splotch mutant background, the changes in the pattern of endogenous Msx2 expression are strikingly similar to those of the Delta4Msx2-hsplacZ transgene bearing a mutation in Pax site 1. An analysis of approximately 13 kb of genomic sequence flanking the Msx2 gene has thus far failed to identify additional elements capable of driving hsp68-lacZ expression in the neural tube and neural crest; thus Pax site 1 may be of crucial importance in the context of the endogenous Msx2 promoter (Kwang. 2002).

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk was examined. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells (Baker, 2002).

Pax3 is a transcription factor that is required by Pre-migratory neural crest cells give rise to the peripheral nervous system, melanocytes, some vascular smooth muscle, and numerous other derivatives. These cells require the transcription factor Pax3, and both mice and humans with Pax3 deficiency exhibit neural crest-related developmental defects. Pax3 is also expressed in the dorsal neural tube, and by myogenic progenitors in the presomitic mesoderm and the hypaxial somites. Molecular pathways that regulate Pax3 expression in the roof plate probably represent early upstream signals in neural crest induction. An enhancer region in the Pax3 genomic locus has been identified that is sufficient to recapitulate expression in neural crest precursors in transgenic mice. Tead2 (Drosophila homolog: Scalloped), a member of the Tead box family of transcription factors, binds to a neural crest enhancer and activates Pax3 expression. Tead2, and its co-activator YAP65, are co-expressed with Pax3 in the dorsal neural tube, and mutation of the Tead2 binding site in the context of Pax3 transgenic constructs abolishes neural expression. In addition, a Tead2-Engrailed fusion protein is able to repress retinoic acid-induced Pax3 expression in P19 cells and in vivo. These results suggest that Tead2 is an endogenous activator of Pax3 in neural crest (Milewski, 2004).

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. Focal cell labelling and orthotopic grafting, followed by whole embryo culture have been used to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonization of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resemble wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, reciprocal grafts were performed of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrate normally in the +/+ environment, and +/+ cells migrate normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurs when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. It is concluded that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment (Chan, 2004).

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it has been show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2004).

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it was shown that Msx1 and Pax3 are both required for neural crest formation; they display overlapping but nonidentical activities, and Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2005).

A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. The cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm (Sato, 2005).

Wnt signalling is required for neural crest (NC) induction; however, the direct targets of the Wnt pathway during NC induction remain unknown. This study shows that the homeobox gene Gbx2 is essential in this process and is directly activated by Wnt/beta-catenin signalling. By ChIP and transgenesis analysis it was shown that Gbx2 regulatory elements that drive expression in the NC respond directly to Wnt/beta-catenin signalling. Gbx2 has previously been implicated in posteriorization of the neural plate. This study unveils a new role for this gene in neural fold patterning. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. The NC specifier activity of Gbx2 is dependent on the interaction with Zic1 and the inhibition of preplacodal genes such as Six1. In addition, that Gbx2 is upstream of the neural fold specifiers Pax3 and Msx1. These results place Gbx2 as the earliest factor in the NC genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC specification. A new genetic cascade is proposed that operates in the distinction between anterior placodal and NC territories (Li, 2009).

Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Through a transcriptome analysis of frog microdissected neural border, this study identified an extended gene signature for the premigratory neural crest, and novel potential members of the regulatory network were defined. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, 25 Pax3 and Zic1 direct targets within this signature were uncovered. The neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). It was also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network (Plouhinec, 2014).

PAX3 and neural differention

A P19 embryonal carcinoma stem cell line has been identified that carries an insertion of the E. coli LacZ gene in an endogenous copy of the Pax-3 gene. Expression of the Pax-3/LacZ fusion gene in neuroectodermal and mesodermal lineages following induction of differentiation by chemical treatments (retinoic acid and dimethylsulfoxide) has been characterized using this line and is consistent with the previous localization of Pax-3 expression in the embryo to mitotically active cells of the dorsal neuroectoderm and the adjacent segmented dermomyotome. Pax-3/LacZ marked stem cells were also utilized as target cells in mixing experiments with unmarked P19 cells that had been differentiated by pretreatment with chemical inducers. Induction of beta-galactosidase and neuroectodermal markers in the target cells demonstrates that (1) some differentiated P19 cell derivatives transiently express endogenous Pax-3- and neuroectoderm-inducing activities; (2) undifferentiated target stem cells respond to these activities even in the presence of leukemia inhibitory factor, and (3) the endogenous activities can be distinguished from, and are more potent than, retinoic acid treatment in inducing neuroectoderm. These observations demonstrate that P19 embryonal carcinoma cells provide a useful in vitro system for analysis of the cellular interactions responsible for neuroectoderm induction in mammals (Pruitt, 1992).

During early patterning of the vertebrate neuraxis, the expression of the paired-domain transcription factor Pax-3 is induced in the lateral portions of the posterior neural plate via posteriorizing signals emanating from the late organizer and posterior nonaxial mesoderm. Using a dominant-negative approach, in explant assays it has been shown that Pax-3 inductive activities from the organizer do not depend on FGF, retinoic acid, or XWnt-8, either alone or in combination, suggesting that the organizer may produce an unknown posteriorizing factor. However, Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. Pax-3 expression in the lateral neural plate expands in XWnt-8-injected embryos and is blocked by dominant-negative XWnt-8. Similarly, the homeodomain transcription factor Msx-1, which like Pax-3 is an early marker of the lateral neural plate, is induced by posterior nonaxial mesoderm and blocked by dominant-negative XWnt-8. Rohon-Beard primary neurons, a cell type that develops within the lateral neural plate, are also blocked in vivo by dominant-negative Xwnt-8. Together these data support a model in which patterning of the lateral neural plate by Wnt-mediated signals is an early event that establishes a posteriolateral domain, marked by Pax-3 and Msx-1 expression, from which Rohon-Beard cells and neural crest will subsequently arise (Bang, 1999).

Pax-3 is a paired-type homeobox gene specifically expressed in the dorsal and posterior neural tube. Pax-3 expression is initiated by signals that posteriorize the neuraxis, and then secondarily restrict dorsal neural tube development in response to dorsal-ventral patterning signals. In chick and Xenopus gastrulae the onset of Pax-3 expression occurs in regions fated to become posterior CNS. Hensen's node and posterior non-axial mesoderm (which underlies the neural plate) induce Pax-3 expression when combined with presumptive anterior neural plate explants. In contrast, presumptive anterior neural plate explants are not competent to express Pax-3 in response to dorsalizing signals from epidermal-ectoderm. In a heterospecies explant recombinant assay with Xenopus animal caps (ectoderm) as a responding tissue, late, but not early, Hensen's node induces Pax-3 expression. Chick posterior non-axial mesoderm also induces Pax-3, provided that the animal caps are neuralized by treatment with noggin. The putative posteriorizing factors, retinoic acid and bFGF, induce Pax-3 in neuralized animal caps. However, blocking experiments with a dominant-inhibitory FGF receptor and a dominant-inhibitory retinoic acid receptor suggest that Pax-3 inductive activities arising from Hensen's node and posterior non-axial mesoderm do not strictly depend on FGF or retinoic acid. Although retinoic acid is an attractive candidate for an endogenous inducing factor, a dominant-negative form of the xRAF-gamma1 receptor fails to block induction of Pax-3. In addition, ectopic expression of a dominant-negative RAR-alpha1 in Xenopus embryos enhances anterior expression and suppresses expression of posterior neural markers, but it does not alter the Pax-3 expression pattern (Bang, 1997 and Blumberg, 1997).

The transcription start site and DNA sequence elements required for the induction of murine Pax3 expression in differentiating P19 embryonal carcinoma cells have been localized. These elements consist of a promoter and additional elements located within 1.6 kbp 5' to the transcription start site. Sequence elements within this 1.6 kbp region are also sufficient to mediate the induction and dorsal restriction of Pax3 of transgenic mice in the neural tube and somites throughout the hindbrain and trunk. Additional elements required for expression anterior to the hindbrain and in migrating myoblasts are located within 14 kbp 5' to the transcription start site. This region also contains element(s) that repress Pax3 expression in the ventral body wall mesoderm of the tail bud (Natoli, 1997).

Members of the paired box (Pax) gene family are expressed in discrete regions of the developing central nervous system, suggesting a role in neural patterning. In this study, the chicken homologs of Pax-3 and Pax-6 have been isolated: both genes are very highly conserved and share extensive homology with the mouse Pax-3 and Pax-6 genes. Pax-3 is expressed in the primitive streak and in two bands of cells at the lateral extremity of the neural plate. In the spinal cord, Pax-6 is expressed later than Pax-3, with the first detectable expression preceding closure of the neural tube. Once the neural tube closes, transcripts of both genes become dorsoventrally restricted in the undifferentiated mitotic neuroepithelium. The removal of the notochord, or implantation of an additional notochord, dramatically alters the dorsoventral (DV) expression patterns of Pax-3 and Pax-6 (Drosophila homolog: Eyeless). These manipulations suggest that signals from the notochord and floor plate regulate the establishment of the dorsoventrally restricted expression domains of Pax-3 and Pax-6 in the spinal cord. The rapid changes to Pax gene expression that occur in neural progenitor cells following the grafting of an ectopic notochord suggest that changes to Pax gene expression are an early effect of the notochord on spinal cord patterning (Goulding, 1993).

Pax3 and Pax7 are transcription factors sharing high sequence identity and overlapping patterns of expression in particular in the dorsal spinal cord. Analysis of Pax3 and Pax7 double mutant mice demonstrates that both genes share redundant functions to restrict ventral neuronal identity in the spinal cord. In their absence, the En1 expression domain is expanded dorsally but that of Evx1 is not affected. At embryonic day 9.5, Wnt4 normally starts to be expressed in the dorsal spinal cord. In the Double Pax mutants, Wnt4 is not expressed in the dorsal spinal cord, while the expression in the ventral spinal cord is only reduced. Thus Pax3 and Pax7 are necessary for the initiation and/or maintenance of Wnt4 expression in the dorsal spinal cord. The expression of En1 is extended dorsally into the Pax7 expression domain in double mutants. Since En1 expression defines V1 interneurons, the expansion of En1 suggests that some dorsally located cells might have acquired the ventral cell fate of the V1 interneuron type. Therefore, one possible role of Pax3 and Pax7 might be to restrict ventral neuronal identity. In addition, Pax3 and Pax7 are expressed in commissural neurons and double mutant embryos exhibit highly reduced ventral commissures. These findings reveal two distinct regulatory pathways for spinal cord neurogenesis, only one of which is dependent on Pax3/7 and 6 (Mansouri, 1998).

Cranial sensory ganglia in vertebrates develop either from the ectodermal placodes, the neural crest, or both. Although much is known about the neural crest contribution to cranial ganglia, relatively little is known about how placode cells form, invaginate and migrate to their targets. Pax-3 is identified as a molecular marker for placode cells which contribute to the ophthalmic branch of the trigeminal ganglion. Pax-3 expression in the ophthalmic placode is observed as early as the 4-somite stage in a narrow band of ectoderm contiguous to the midbrain neural folds. Its expression broadens to a patch of ectoderm adjacent to the midbrain and the rostral hindbrain at the 8- to 10-somite stage. Invagination of the first Pax-3-positive cells begins at the 13-somite stage. Placodal invagination continues through the 35-somite stage, by which time condensation of the trigeminal ganglion has begun. To challenge the normal tissue interactions leading to placode formation, the cranial neural crest cells were ablated or barriers were implanted between the neural tube and the ectoderm. The results demonstrate that although the presence of neural crest cells is not mandatory for Pax-3 expression in the forming placode, a diffusible signal from the neuroectoderm (perhaps a Wnt or TGFbeta family member) is required for induction and/or maintenance of the ophthalmic placode (Stark, 1997).

Placodes are discrete regions of thickened ectoderm that contribute extensively to the peripheral nervous system in the vertebrate head. The adenohypophseal placode forms the anterior pituitary gland, while the lens placode forms the crystallin-secreting lens cells of the eye. The olfactory, otic and lateral line placodes give rise to the nose, ear and lateral line system, respectively. These three placodes form a wide variety of cell types, including ciliated sensory receptors, sensory neurons, neuroendocrine cells, glia and other supporting cells. The trigeminal and epibranchial (geniculate, petrosal and nodose) placodes give rise only to sensory neurons in cranial sensory ganglia. The paired-domain transcription factor Pax-3 is an early molecular marker for the avian ophthalmic trigeminal (opV) placode, which forms sensory neurons in the ophthalmic lobe of the trigeminal ganglion. Using ablation and barrier implantation experiments it has been shown that a diffusible signal from the midbrain/rostral hindbrain is necessary for Pax-3 induction or maintenance in the opV placode. Collagen gel cultures and heterotopic quail-chick grafts have been used to examine the competence, specification and induction of Pax-3 in the opV placode. At the 3-somite stage, the whole head ectoderm rostral to the first somite is competent to express Pax-3 when grafted to the opV placode region, though competence is rapidly lost thereafter in otic-level ectoderm. Pax-3 specification in presumptive opV placode ectoderm occurs by the 8-somite stage, concomitant with robust Pax-3 expression. From the 8-somite stage onward, significant numbers of cells are committed to express Pax-3. The entire length of the neural tube has the ability to induce Pax-3 expression in competent head ectoderm and the inductive interaction is direct. The caudal border of Pax-3 expression in the opV placode may be defined by an inhibitor at the r2,3 level. (Baker, 1999).

Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. The role that Pax-3 plays in neuronal cell development was investigated by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, the addition of dexamethasone leads to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponds to a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibit increased cell to substrate adhesion, extend long neurite processes and express genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression is followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation induced in the cell lines lacking Pax-3 can be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor is downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells: in its absence, the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation (Reeves, 1999).

Neural tube defects are common and serious human congenital anomalies. These malformations have a multifactorial etiology and can be reproduced in mouse models by mutations of numerous individual genes and by perturbation of multiple environmental factors. The identification of specific genetic interactions affecting neural tube closure will facilitate an understanding of molecular pathways regulating normal neural development and will enhance the ability to predict and modify the incidence of spina bifida and other neural tube defects. A genetic interaction is reported between Nf1, encoding the intracellular signal transduction protein neurofibromin, and Pax3, a transcription factor gene mutated in the Splotch mouse. Both Pax3 and Nf1 are important for the development of neural crest-derived structures and the central nervous system. Splotch is an established model of folate-sensitive neural tube defects, and homozygous mutant embryos develop spina bifida and sometimes exencephaly. Neural development is grossly normal in heterozygotes and neural tube defects are not seen. In contrast, a low incidence of neural tube defects is found in heterozygous Splotch mice that also harbor a mutation in one Nf1 allele. All compound homozygotes have severe neural tube defects and die earlier in embryogenesis than either Nf1(-/-) or Sp(-/-) embryos. Occasional exencephaly occurs in Nf1(-/-) mice and more subtle CNS abnormalities are identified in normal-appearing Nf1(-/-) embryos. Though other genetic loci and environmental factors affect the incidence of neural tube defects in Splotch mice, these results establish Nf1 as the first known gene to act as a modifier of neural tube defects in Splotch. It seems most likely that Pax3 and Nf1 function in parallel pathways where both contribute to normal neural tube closure (Lakkis, 1999).

In the chick, Pax3/7 is expressed in the alar plate of the mesencephalon. The optic tectum differentiates from the alar plate of the mesencephalon, and expression of Pax3/7 is well correlated to the tectum development. To explore the function of Pax3 and Pax7 in the tectum development, Pax3 and Pax7 were misexpressed in the diencephalon and ventral mesencephalon. Morphological and molecular marker gene analysis indicate that Pax3 and Pax7 misexpression causes fate change of the alar plate of the presumptive diencephalon to that of the mesencephalon, that is, a tectum and a torus semicircularis are formed ectopically. Ectopic tectum in the diencephalon appears to be generated through sequential induction of Fgf8, En2 and Pax3/7. In ventral mesencephalon, which expresses En but does not differentiate to the tectum in normal development, Pax3 and Pax7 misexpression induces ectopic tectum. In normal development, Pax3 and Pax7 expression in the mesencephalon commences after Otx2, En and Pax2/5 expression. In addition, the expression domains of Pax3 and Pax7 are consistent with the presumptive tectum region in a dorsoventral axis. Taken together with normal expression pattern of Pax3 and Pax7, results of misexpression experiments suggest that Pax3 and Pax7 define the tectum region subsequent to the function of Otx2 and En (Matsunaga, 2001).

Pax3 in rhabdomyosarcoma

To investigate the role of the translocation-associated gene Pax3:Fkhr in alveolar rhabdomyosarcomas, a Cre-mediated conditional knock-in was generated of Pax3:Fkhr into the mouse Pax3 locus. Exploring embryonic tumor cell origins, a Pax3 allele was replaced with Pax3:Fkhr throughout its expression domain, causing dominant-negative effects on Pax3 and paradoxical activation of the Pax3 target gene, c-Met. Ectopic neuroprogenitor cell proliferation also occurs. In contrast, activation later in embryogenesis in cells that express Pax7 results in viable animals with a postnatal growth defect and a moderately decreased Pax7+ muscle satellite cell pool, phenocopying Pax7 deficiency but remarkably not leading to tumors (Keller, 2004a).

Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. The first mouse model of alveolar rhabdomyosarcoma is reported, using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing (Keller, 2004b).

Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, Alveolar-RMS (A-RMS), is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene to FOXO1 (homolog of Drosophila Foxo). The molecular underpinnings of PAX-FOXO1-mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. This study reports a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, a comprehensive deletion screen was performed against the Drosophila autosomes, and mutation of Mef2, a myogenesis lynchpin in both flies and mammals, was demonstrated to dominantly suppresses PAX7-FOXO1 pathogenicity and act as a PAX7-FOXO1 gene target. Additionally, mutation of mastermind, a gene encoding a MEF2 transcriptional co-activator, was shown to similarly suppress PAX7-FOXO1, further pointing towards MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease (Galindo, 2014: PubMed).

A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR

Alveolar rhabdomyosarcoma (ARMS) is an aggressive myogenic-type tumor and a gain-of-function disease, caused by misexpression of the PAX3-FKHR or PAX7-FKHR fusion oncoprotein from structurally rearranged chromosomes. PAX3-FKHR misexpressed in terminally differentiating mouse myofibers can cause rhabdomyosarcoma at a low frequency, suggesting that skeletal muscle is an ARMS tissue of origin. Because patterned muscle is widely viewed as irreversibly syncytial, questions persist, however, regarding this potential pathogenetic mechanism for ARMS tumor initiation. To further explore this issue, transgenic Drosophila lines were generated that conditionally express human PAX-FKHR. PAX7-FKHR causes nucleated cells to form and separate from syncytial myofibers, which then spread to nonmuscular tissue compartments, including the central nervous system, and that wild-type PAX3 demonstrates similar potential. It is further shown that Ras, which is known to interfere with the differentiation of myogenic cells, genetically interacts with PAX7-FKHR: constitutively activated Ras enhances PAX7-FKHR phenotypes, whereas loss-of-function ras alleles dominantly suppress PAX7-FKHR activity, including rescue of lethality. These results show that PAX-FKHR can drive the generation of discrete nucleated cells from differentiated myofibers in vivo, argue for syncytial muscle as an ARMS tissue of origin, and demonstrate that Drosophila provides a powerful system to screen for genetic modifiers of PAX-FKHR (Galindo, 2006; full text of article).

Temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. This study demonstrates how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. These studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; this study proposes that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs (Ohyama, 2008).

This analysis makes a number of key points. First, it provides a novel insight into how cellular diversity can be achieved within the embryo in response to a limited repertoire of signalling molecules. In addition to the well-accepted view that BMP signal opposes Shh activity in a spatial manner, ventrally derived Bmp7 signalling can oppose Shh signalling in a temporal manner to specify ventral progenitors within the hypothalamus. The deployment of the two signals in this versatile temporal manner in turn leads to novel modules of transcription factor expression, in order to achieve elaborate cellular diversity. This work adds to the growing body of data suggesting that cell fate in the neural tube is governed through the temporal integration of, and adaptation to, signalling ligands (Ohyama, 2008)

Table of contents

paired continued: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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