Phenotypic Mutation 'Widget'

• Allele: Widget
• Mutation Type: critical splice donor site (1 bp from exon)
• Chromosome: 1
• Coordinate: 78,099,227 bp (GRCm39)
• Base Change: C ⇒ T (forward strand)
• Gene: Pax3
• Gene Name: paired box 3
• Synonym(s): Splchl2, Pax-3
• Chromosomal Location: 78,077,904-78,173,771 bp (-) (GRCm39)
• MGI Phenotype: FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene is a member of the paired box (PAX) family of transcription factors. Members of the PAX family typically contain a paired box domain and a paired-type homeodomain. These genes play critical roles during fetal development. Mutations in paired box gene 3 are associated with Waardenburg syndrome, craniofacial-deafness-hand syndrome, and alveolar rhabdomyosarcoma. The translocation t(2;13)(q35;q14), which represents a fusion between PAX3 and the forkhead gene, is a frequent finding in alveolar rhabdomyosarcoma. Alternative splicing results in transcripts encoding isoforms with different C-termini. [provided by RefSeq, Jul 2008] ; PHENOTYPE: Effects on homozygotes for mutations in this gene vary in severity and include embryonic to perinatal death, malformations of neural tube, spinal ganglia, heart, vertebral column, hindbrain and limb musculature. Heterozygotes have white belly spots and variable spotting on the back and extremeties. [provided by MGI curators]
• Mapped: No
• Institutional Source: Beutler Lab
• Gene Model: predicted gene model for protein(s): [ENSMUSP00000004994 ^†] [ENSMUSP00000084320 ^†] † probably from a misspliced transcript
• AlphaFold: P24610
• SMART Domains: Protein: ENSMUSP00000004994; Gene: ENSMUSG00000004872

Domain	Start	End	E-Value	Type
PAX	34	159	1.99e-91	SMART
low complexity region	164	185	N/A	INTRINSIC
HOX	219	281	6.6e-27	SMART
Pfam:Pax7	347	391	5.9e-29	PFAM

• Predicted Effect: probably null
• SMART Domains: Protein: ENSMUSP00000084320; Gene: ENSMUSG00000004872

Domain	Start	End	E-Value	Type
PAX	34	159	1.99e-91	SMART
low complexity region	164	185	N/A	INTRINSIC
HOX	219	281	6.6e-27	SMART
Pfam:Pax7	346	391	5.3e-27	PFAM

• Predicted Effect: probably null
• Meta Mutation Damage Score: 0.9480
• Is this an essential gene?: Probably essential (E-score: 0.822)
• Phenotypic Category: Unknown
• Mode of Inheritance: Unknown
• Local Stock: Live Mice
• MMRRC Submission: 038196-MU
• Last Updated: 2016-05-13 3:09 PM by Peter Jurek
• Record Created: 2015-01-14 3:38 PM by Kimberly Hawkins
• Record Posted: 2015-05-15
• Science Writers: Anne Murray
• Illustrators: Peter Jurek
• Authors: Kimberly Hawkins and Jamie Russell

Accession Number

NCBI RefSeq: NM_008781, NM_001159520; MGI:97487

Alleles Listed at MGI

All mutations/alleles(35) : Chemically and radiation induced(1) Chemically induced (ENU)(4) Radiation induced(4) Spontaneous(5) Targeted(21)

Phenotypic Description

Figure 1. The Widget phenotype.

The Widget phenotype was identified among N-ethyl-N-nitrosourea (ENU)-induced mice of the pedigree R2473, some of which exhibited variable white spotting of the fur (Figure 1).

Nature of Mutation

Whole exome HiSeq sequencing of the G1 grandsire identified 62 mutations. A mutation in Pax3 was presumed to be causative because the Widget belted phenotype mirrors that of other Pax3 alleles (see MGI). The mutation in Pax3 is a G to A transition at base pair 78,122,590 (v38) on chromosome 1, or base pair 74,927 in the GenBank genomic region NC_000067 within the donor splice site of intron 6. The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in skipping of the 166-nucleotide exon 6 (out of 9 total exons), resulting in a frame-shift and coding of 60 aberrant amino acids followed by a premature stop codon.

<--exon 5         <--exon 6 intron 6-->        exon 7--> 65275 ……GAGGCCCGAGTGCAG……ACGTCTATTCCACAAG gtatctaggaagagaaa……CCGTGTCAGATCCCA……CCTCCAACCCCATGA 260 ……-E--A--R--V--Q-……-T--S--I--P--Q-- -P--C--E--I--P-……-P--P--T--P--*            correct           deleted                                                                                  aberrant

Genomic numbering corresponds to NC_000067. The donor splice site of intron 6, which is destroyed by the Widget mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red. 

Protein Prediction

Figure 2. The domain structure of mouse PAX3. The Widget mutation is indicated. Abbreviations: PD, paired domain; OCT, octapeptide motif, HD, homeodomain; TA, transactivation domain.

Figure 3. Crystal structure of human PAX3 HD in complex with DNA. The human HD encompasses amino acids 219-278. Figure was generated with UCSF Chimera and is based on PDB:3CMY. The image is interactive; click to rotate and to view the space-filled model. The light blue portion in the space-filled model is the DNA strand, while the dark turquoise portion is the PAX3 HD.

Figure 4. Human PAX3 encodes multiple isoforms. For more information see the text and Table 1 . Abbreviations: PD, paired domain; OCT, octapeptide motif, HD, homeodomain; TA, transactivation domain.

Table 1. Human PAX3 generates multiple isoforms Splice Variant Molecular description Function Tissue expression Expression in cancers References PAX3A Transcript contains exons 1-4; truncated prematurely in intron 4; protein lacks the HD and the TA domain Reduces differentiated melanocyte proliferation and migration Cerebellum, esophagus, skeletal muscle Reduced in melanoma compared to normal melanocytes (14;20;21) PAX3B Transcript contains exons 1-4; truncated prematurely in intron 4; protein lacks the HD and the TA domain Reduces differentiated melanocyte proliferation and migration Expressed in most tissues including esophagus, stomach, cerebellum, liver, and pancreas Reduced in melanoma compared to normal melanocytes PAX3C Transcript retains intron 8 and translation continues from exon 8 for five codons into intron 8 before termination; protein has 10 unique C-terminal 10 amino acids Increases differentiated melanocyte proliferation and migration, and survival; functions in anchorage-independent growth Low levels in undifferentiated myogenic cell lines, but increased at later stages of differentiation Predominant in melanoma and small-cell lung cancer (15-17;21-23) PAX3D Transcript lacks intron 8 and translation proceeds from exon 8 to 9; protein does not have a portion of the TA domain PAX3E Transcript contains exons 8, 9, and 10 but lacks introns 8 and 9; protein is similar to PAX3D Decreases differentiated melanocyte proliferation and migration Untested Untested (21;22) PAX3G Transcript is a truncated form of PAX3D; protein lacks a portion of the TA domain encoded by exon 8 No affect on melanocyte proliferation or apoptosis, but reduced migration; functions in anchorage-independent growth Untested Predominant in neuroblastoma (21;22) PAX3H Transcript is a truncated form of PAX3E; protein lacks a portion of the TA domain encoded by exon 8 Increases differentiated melanocyte proliferation, migration, and survival; functions in anchorage-independent growth Untested Predominant in neuroblastoma (21) PAX3I Transcript: alternative use of the CAG codon as a 3’ splice acceptor site of intron 2; protein does not contain a Gln between amino acids 74 and 75 Distinct DNA-binding properties Untested Rhabdomyosarcoma (24-26)

Paired box (PAX) 3 [PAX3; alternatively, melanocyte specific factor (MSF)] is a member of the PAX family of transcription factors (1). The paired domain (PD; amino acids 34-159) and homeodomain (HD; amino acids 219-281) of PAX3 are both DNA-binding domains that can act independently or together to bind target genes [Figure 2; (2-6;6-8)]. For example, the PD is required for PAX3 binding to the Tyrp1 and Dct promoters, while both the HD and PD are required for binding to the Mitf promoter (5). The structure of the highly conserved human PAX3 HD in complex with DNA has been solved [Figure 3; PDB:3CMY; (9)]. The PAX3 HD folds into a prototypical HD fold of three α-helices (α1, α2, and α3), connected by short loops, and an extended N-terminal arm which is disordered in the absence of DNA (9). Upon interaction with DNA, the arm inserts into the major groove to interact with bases in both strands of the DNA as well as with the sugar-phosphate backbone. Serine 50 is essential for specifying the DNA sequence PAX3 will bind. Ser50 binds DNA that has two inverted TAAT motifs separated by either two (P2) or three (P3) base pairs (10). Two HDs from adjacent PAX3 molecules bind to DNA in a head-to-head arrangement. Helices α1 and α2 are arranged in an antiparallel fashion relative to each other and perpendicular to α3, which fits into the major groove. PAX3 also has an octapeptide motif (HSIDGILS; amino acids 186-193) and a C-terminal transcription activation (TA) domain (amino acids 282-484). The octapeptide may function as a binding motif for a DNA methylation or demethylation factor (7;8). The TA domain modulates the DNA binding specificity of PAX3 (11). PAX3 function may be regulated by phosphorylation and ubiquitination; Ser205 was identified as a site of phosphorylation on PAX3 (12;13).

Human PAX3 undergoes alternative splicing to generate multiple transcripts [Figure 4 and Table 1; (6;14-17)]. The multiple protein isoforms differ in structure and the activities of the paired, homeodomain, and transactivation domains (14;18;19). Mouse Pax3 only has one documented isoform.

Expression/Localization

Pax3 is expressed during early embryogenesis and development (6). Pax3 is expressed starting at approximately embryonic day (E)8.5 in the dorsal neural tube, developing hindbrain, mesencephalon, and prosencephalon (6;27;28). Pax3 is expressed in the presomitic mesoderm and throughout the epithelial somite, before expression is restricted to the dermomyotome (29). Pax3 expression persists as melanoblasts develop and migrate from the neural crest and as the melanocytes localize in developing hair follicles (30). Pax3 expression is lost in neurons when maturing neurons exit the ventricular zone and migrate through the intermediate zone. Pax3 has not been detected in the adult mouse. Pax3 downregulation during differentiation of neural crest precursors is essential for proper cranial development. Persistent Pax3 expression in neural crest derivatives results in cleft palate, ocular defect, malformation of the sphenoid bone, and perinatal lethality (31).

Background

Table 2. Select Pax3 targets Target Brief description of function PAX3-associated affect References Met Receptor tyrosine kinase Promotes cell migration during limb muscle and melanocyte development (50) Ret Promotes cell migration during enteric ganglia formation (51;52) Mbp Factors involved in myelination of nerves in the peripheral nervous system Maintains a nonmyelinated axonal phenotype for Schwann cells (4;32;53;54)   Gfap Inhibits neural stem cell differentiation towards astrocytes Ngfr Enhances nerve cell survival and differentiation L1cam Enhances L1 expression to support Schwann cell precursor migration to the peripheral nervous system Ncam1 Pax3 represses expression; cell-cell adhesion Neurog2 Transcription factor Initiates neuronal lineage specification (55) Tgfa and Tgfb Members of the epidermal growth factor (EGF) family of lignds Neural crest migration; TGFβ signaling regulates cell-cell adhesion, growth, differentiation, and migration (56;57) Mitf Transcription factor Melanocyte development; Mitf binding to Dct, Tyr, Tyrp1, and c-Kit (see the record for Pretty2) initiates the melanogenic cascade (42;46;58) Dct Pigment enzyme Maintains the “stemness” of migratory neural cells to inhibit melanocyte differentiation Tyrp1 Pigment enzyme; see the record for chi Promotes melanogenic cascade (59;60). Hes1 Transcription factor that suppresses transcription Maintains the “stemness” of migratory neural cells and embryonic melanoblasts and melanocyte stem cells in the bulge area of adult mouse hair follicles (55;61) Trp53 (alternatively, p53), Pten, Bcl2l1 (alternatively, Bcl-XL) Antiapoptotic factors (see lentil for information about Trp53bp1) Promotes survival of melanocytes (and possibly melanoma cells) (62-64) Six1, Eya2, Myod1, Myf5, Myog, Sostdc1 Myogenic regulatory factors Skeletal myogenesis (31;40;65) Abbreviations: Met, Met proto-oncogene; Ret, Ret proto-oncogene; Mbp, myelin basic protein; Gfap, glial fibrillary acidic protein; Ngfr, nerve growth factor receptor; L1cam, L1 cell adhesion molecule; Ncam1, neural cell adhesion molecule 1; St8sia2, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2; Tgfa/b, transforming growth factor alpha/beta; Mitf, microphthalmia-associated transcription factor; Dct, dopachrome tautomerase; Tyrp1, tyrosinase-related protein 1; Hes1, hairy and enhancer of split 1 (Drosophila); Neurog2, neurogenin 2; Trp53, transformation related protein 53; Pten, phosphatase and tensin homolog; Bcl2l1, BCL2-like 1; Six1, sine oculis-related homeobox 1; Eya2, eyes absent 2 homolog (Drosophila); Myod1, myogenic differentiation 1; Myf5, myogenic factor 5; Myog, myogenin; Sostdc1, sclerostin domain containing 1

Figure 5. PAX3 regulates several genes necessary for melanoblast survival during development. The Wnt/β-catenin, MC1R, Kit, and ET-3/ET B R signaling pathways regulate the transcription of microphthalmia-associated transcription factor ( Mitf ) in NC-derived melanocyte precursors as well as regulate the phosphorylation of the melanocyte-specific MITF isoform (MITF-M). The Mitf promoter is regulated by the transcription factors PAX3, SOX10, Lef1/TCF, and CREB during melanocyte development. MITF-M regulates several target genes to mediate melanocyte survival ( Bcl2 and Met ), proliferation ( e.g., Cdk2 and Tbx2 ), and differentiation [ e.g., Tyr, Tyrp1, Slc45a2, Dct, Pmel, and Mc1r ). In the Wnt signaling pathway, binding of the Wnt ligand to a Frizzled/LRP-5/6 receptor complex leads to the activation of the cytosolic protein, Dishevelled. Dishevelled inhibits the β-catenin degradation complex containing APC, Axin, and GSK3. Stabilized hypophosphorylated β-catenin subsequently interacts with TCF/Lef1 in the nucleus to activate transcription. In MC1R signaling, αMSH activates MC1R, leading to GDP/GTP exchange on the G-protein. The GTP-bound G α subunit is released and activates adenylyl cyclase. Adenylyl cyclase catalyzes the production of cAMP, the activation of PKA and PKA-induced activation of the CREB family of transcription factors. The SCF/c-Kit signaling pathway modifies MITF post-translationally by phosphorylating Ser73 by the mitogen-activated protein kinase (Ras/Raf/MEK/ERK) pathway and Ser409 through RSK. RSK activation also results in CREB phosphorylation/activation. The ET-3/ET B R signaling pathway induces the phosphorylation of Ser298 on MITF through activation of the phospholipase C/PIP 2 /DAG/PKC pathway; the ET-3/ET B R merges with the ERK signaling pathway downstream of c-Kit. For a more comprehensive view of ET B R signaling please see the record for gus-gus . Abbreviations: PKC, protein kinase C; GSK, glycogen synthase kinase; APC, adenomatosis polyposis coli; cAMP, cyclic AMP; MITF, microphthalmia-associated transcription factor; MC1R, melanocortin 1 receptor; αMSH, alpha melanocyte stimulating hormone; LRP, low-density lipoprotein receptor-related protein; MEK, mitogen-activated protein kinase kinase 1/2; H-Ras, v-Ha-ras Harvey rat sarcoma viral oncogene homolog; c-Raf1, v-raf-1 murine leukemia viral oncogene homolog 1; RSK, ribosomal protein S6 kinase 90kDa; CREB, cAMP responsive element binding protein 1; PKA, protein kinase A; SCF, stem cell factor. Some protein structures are modeled after existing crystal structures: β-catenin, PDB: 1I7W ; MITF, PDB: 4ATH ; PKC, PDB: 1CPK .

The PAX protein family regulates pattern formation, morphogenesis, cellular differentiation, and organogenesis by activating (or repressing) genes that encode secreted proteins, cell surface receptors, cell cycle regulators, and transcription factors.A list of select PAX3 targets and there respective functions in the above-described developmental processes are described in Table 2 (49). PAX3 is essential for normal development of the peripheral nervous system, the trigeminal (cranial nerve V), superior (IX), and jugular (X) ganglia as well as the frontal, ophthalmic, and spinal accessory nerves (32;33).

PAX3 controls cardiovascular development. PAX3 is essential for cardiac outflow tract septation in the mouse (34). Mice with homozygous mutations in Pax3 exhibit persistent truncus arteriosus whereby a single vessel emerging from the embryonic heart does not divide into the proximal aorta and the pulmonary artery. Double outlet right ventricle, a less-severe cardiovascular disorder, is also sometimes observed in which the truncus arteriosus is septated, but the vessels emerge from the right ventricle and a ventribular septal defect allows for outlet of blood from the left ventricle (35). The Pax3 mutant mice can also display thinning of the compact layer of the embryonic heart and defects in excitation-contraction coupling due to decreased inward calcium current (34). Defects in cardiovascular development are proposed to be due abnormal cardiac neural crest cell migration or function.

In the muscular system, PAX3 is essential for limb muscle precursor migration, but not for differentiation (36); PAX3 is not necessary for trunk muscle development (37). Newly formed somites are a grouping of epithelial cells that differentiate into sclerotome and dermomyotome upon signals from the ventral midline (37;38). The dermamyotome subsequently forms the dermis and muscle. Skeletal muscles in vertebrates are derived from two bilateral rows of somites on either side of the neural tube. Axial muscles are derived from the medial portion of the somite, while the limb muscles develop from the lateral somite (39). Precursors of limb muscles are derived from the lateral portion of the dermomytomes immediately adjacent to the limb buds. The cells in the limb bud subsequently differentiate and fuse to form primary myotubes of limb muscles. PAX3 and PAX7 are upstream regulators of myogenesis (36;37;40). PAX3 is expressed in myogenic precursor cells that will migrate from the somite to become the musculature of the abdominal walls and limbs (37). PAX3 expression marks migrating myogenic progenitor cells that have not activated myogenic determination genes.

During melanocyte development in the embryo, PAX3 and SOX10 (mutated in Dalmatian) are both required for the expression of Mitf, which is required for melanoblast survival after migration from the dorsal neural tube to the migration staging area [Figure 5; (41-43)]. The melanoblast population is derived from a much larger number of neural crest progenitors with melanoblasts migrating axially as well as laterally (44). Most melanoblasts migrate from the cervical region of the embryo and different cell dispersion mechanisms may operate in the head and trunk regions. Three distinct phases occur in trunk melanoblast development: a primary proliferative phase, followed by migration, leading to a second phase of proliferation by the dispersed cells. PAX3, SOX10, MITF, and LEF1 bind directly to the Dct promoter, which encodes an enzyme necessary for melanin pigment production in melanocytes (45-48). PAX3 and MITF share the same binding site within the Dct promoter and compete for occupancy (46). Expression of Tyr (see the record for ghost) and Tyrp1 (see the record for chi), additional melanin-generated enzymes in pigment cells, is induced a few days later.
 

Mutations in PAX3 are linked to craniofacial-deafness-hand syndrome [OMIM: #122880; (66;67)] and alveolar rhabdomyosarcoma 2 [OMIM: #268220; (68)] as well as Waardenburg syndrome (WS) type 1 [WS1; OMIM: #193500; (69)] and WS type 3 (WS3; alternatively, Klein-Waardenburg syndrome; OMIM: #148820) (70;71)]. Patients with craniofacial-deafness-hand syndrome exhibit absence or hypoplasia of the nasal bones, sensorineural deafness, small or short noses with slit-like nares, hypertelorism, short palpebral fissures, and reduced movement at the wrist. Alveolar rhapdomyosarcomas are caused by chromosomal translocations that encode either a PAX3-FKHR(FOXO1A) or PAX7-FKHR fusion protein that subsequently acts as a strong transcriptional activator of transcription factors and signaling components that affect myogenesis and transform myoblast and fibroblast cell lines in culture [reviewed in (29;72)]. The PAX3/FKHR(FOXO1A) fusion protein is able to activate expression of Pdgfar (platlet-derived growth factor alpha receptor); PAX3 alone does not regulate expression of Pdgfar (73). WS is an autosomal dominant condition causes variable degrees of deafness, dystopia canthorum (i.e., lateral displacement of the eyes), pigment defects (e.g., white forelock and differently colored eyes), and limb muscle hypoplasia (WS3 only).

Putative Mechanism

White-spotting mutants are a subclass of coat color mutants in mice. These mutants exhibit white spots that include belly spots, head spots, belts spanning the caudal trunk region, piebald spotting and peppering. Unlike color variations in mice which are typically due to alterations in melanin production, distribution, or deposition into hair and/or skin, white-spotting mutants are often the result of improper melanoblast development or survival. The lack of pigment in the adult reflects the absence of mature melanocytes in that area due to defects at various stages of melanocyte development including proliferation, survival, migration, invasion of the integument, hair follicle entry and melanocyte stem cell renewal (74;75). White-spotted mutants have been ascribed to a variety of genes including ones encoding the transcription factors MITF and SOX10, the KIT receptor tyrosine kinase (mutated in Casper and Pretty2), a G-protein coupled receptor and its ligand (EDNRB (mutated in gus-gus) and endothelin-3 respectively), a transmembrane protein (Mucolipin 3) as well as ADAMTS-20 (mutated in splotch2), an ECM associated metalloprotease (74;75).  

A spontaneous white belly spot phenotype was observed in a mouse, designated as Splotch, and attributed to a spontaneous splice acceptor site mutation in Pax3 (76;77). Homozygous Splotch mice are embryonic lethal by embryonic day (E) 14 due to neural tube closure defects in the lumbo-sacral area (i.e., spina bifida), absence (or reduction in size) of the dorsal root ganglia, exencephaly, abnormal formation of the cardiac outflow tract, and absent limb musculature (37). Heterozygous Splotch mice are viable and exhibit variable white belly spotting. Another Splotch allele, Splotch-delayed, results in a missense mutation within the paired domain and results in a less severe phenotype than observed in Splotch mice (i.e., only the spina bifida phenotype) and the mice survive until shortly after birth (78-80). Several other Splotch alleles have also been identified including Splotch^4H (a large deletion in Pax3) and Splotch^2H (an intragenic deletion) (77;81-83). Each of these mutants disply variable white belly spotting in the heterozygote. Two ENU-induced Pax3 mutants (Pax3^wbs and Pax3^Sp-1Xzg) exhibit variable white belly spotting in heterozygous mice (58;84). The Pax3^wbs phenotype was linked to a missense mutation at base pair 319 in exon 2, which caused a premature stop codon at amino acid 107 (58). Homozygous Pax3^wbs mice were embryonic lethal by E14.5 (58), similar to the Splotch mice (76). Similar to other Pax3 mutants, heterozygous Widget mice exhibit white belly spotting; the phenotype(s) in homozygous mice were not examined. Pigment defects in Pax3 mutants are attributed to reduced melanoblast numbers, not in defects through the migratory pathway (85).

Primers

Primers cannot be located by automatic search.

References

  1. Schafer, B. W. (1998) Emerging Roles for PAX Transcription Factors in Cancer Biology. Gen Physiol Biophys. 17, 211-224.

  2. Epstein, D. J., Vogan, K. J., Trasler, D. G., and Gros, P. (1993) A Mutation within Intron 3 of the Pax-3 Gene Produces Aberrantly Spliced mRNA Transcripts in the Splotch (Sp) Mouse Mutant. Proc Natl Acad Sci U S A. 90, 532-536.

  3. Chalepakis, G., and Gruss, P. (1995) Identification of DNA Recognition Sequences for the Pax3 Paired Domain. Gene. 162, 267-270.

  4. Chalepakis, G., Jones, F. S., Edelman, G. M., and Gruss, P. (1994) Pax-3 Contains Domains for Transcription Activation and Transcription Inhibition. Proc Natl Acad Sci U S A. 91, 12745-12749.

  5. Corry, G. N., and Underhill, D. A. (2005) Pax3 Target Gene Recognition Occurs through Distinct Modes that are Differentially Affected by Disease-Associated Mutations. Pigment Cell Res. 18, 427-438.

  6. Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, J. R., and Gruss, P. (1991) Pax-3, a Novel Murine DNA Binding Protein Expressed during Early Neurogenesis. EMBO J. 10, 1135-1147.

  7. Ziman, M. R., and Kay, P. H. (1998) A Conserved TN8TCCT Motif in the Octapeptide-Encoding Region of Pax Genes which has the Potential to Direct Cytosine Methylation. Gene. 223, 303-308.

  8. Kay, P. H., Harmon, D., Fletcher, S., Ziman, M., Jacobsen, P. F., and Papadimitriou, J. M. (1997) Variation in the Methylation Profile and Structure of Pax3 and Pax7 among Different Mouse Strains and during Expression. Gene. 184, 45-53.

  9. Birrane, G., Soni, A., and Ladias, J. A. (2009) Structural Basis for DNA Recognition by the Human PAX3 Homeodomain. Biochemistry. 48, 1148-1155.

  10. Wilson, D., Sheng, G., Lecuit, T., Dostatni, N., and Desplan, C. (1993) Cooperative Dimerization of Paired Class Homeo Domains on DNA. Genes Dev. 7, 2120-2134.

  11. Cao, Y., and Wang, C. (2000) The COOH-Terminal Transactivation Domain Plays a Key Role in Regulating the in Vitro and in Vivo Function of Pax3 Homeodomain. J Biol Chem. 275, 9854-9862.

  12. Miller, P. J., Dietz, K. N., and Hollenbach, A. D. (2008) Identification of Serine 205 as a Site of Phosphorylation on Pax3 in Proliferating but Not Differentiating Primary Myoblasts. Protein Sci. 17, 1979-1986.

  13. Boutet, S. C., Disatnik, M. H., Chan, L. S., Iori, K., and Rando, T. A. (2007) Regulation of Pax3 by Proteasomal Degradation of Monoubiquitinated Protein in Skeletal Muscle Progenitors. Cell. 130, 349-362.

  14. Tsukamoto, K., Nakamura, Y., and Niikawa, N. (1994) Isolation of Two Isoforms of the PAX3 Gene Transcripts and their Tissue-Specific Alternative Expression in Human Adult Tissues. Hum Genet. 93, 270-274.

  15. Barber, T. D., Barber, M. C., Cloutier, T. E., and Friedman, T. B. (1999) PAX3 Gene Structure, Alternative Splicing and Evolution. Gene. 237, 311-319.

  16. Parker, C. J., Shawcross, S. G., Li, H., Wang, Q. Y., Herrington, C. S., Kumar, S., MacKie, R. M., Prime, W., Rennie, I. G., Sisley, K., and Kumar, P. (2004) Expression of PAX 3 Alternatively Spliced Transcripts and Identification of Two New Isoforms in Human Tumors of Neural Crest Origin. Int J Cancer. 108, 314-320.

  17. Pritchard, C., Grosveld, G., and Hollenbach, A. D. (2003) Alternative Splicing of Pax3 Produces a Transcriptionally Inactive Protein. Gene. 305, 61-69.

  18. Underhill, D. A., and Gros, P. (1997) The Paired-Domain Regulates DNA Binding by the Homeodomain within the Intact Pax-3 Protein. J Biol Chem. 272, 14175-14182.

  19. Seo, H. C., Saetre, B. O., Havik, B., Ellingsen, S., and Fjose, A. (1998) The Zebrafish Pax3 and Pax7 Homologues are Highly Conserved, Encode Multiple Isoforms and show Dynamic Segment-Like Expression in the Developing Brain. Mech Dev. 70, 49-63.

  20. Ryu, B., Kim, D. S., Deluca, A. M., and Alani, R. M. (2007) Comprehensive Expression Profiling of Tumor Cell Lines Identifies Molecular Signatures of Melanoma Progression. PLoS One. 2, e594.

  21. Wang, Q., Kumar, S., Slevin, M., and Kumar, P. (2006) Functional Analysis of Alternative Isoforms of the Transcription Factor PAX3 in Melanocytes in Vitro. Cancer Res. 66, 8574-8580.

  22. Wang, Q., Kumar, S., Mitsios, N., Slevin, M., and Kumar, P. (2007) Investigation of Downstream Target Genes of PAX3c, PAX3e and PAX3g Isoforms in Melanocytes by Microarray Analysis. Int J Cancer. 120, 1223-1231.

  23. Charytonowicz, E., Matushansky, I., Castillo-Martin, M., Hricik, T., Cordon-Cardo, C., and Ziman, M. (2011) Alternate PAX3 and PAX7 C-Terminal Isoforms in Myogenic Differentiation and Sarcomagenesis. Clin Transl Oncol. 13, 194-203.

  24. Vogan, K. J., and Gros, P. (1997) The C-Terminal Subdomain Makes an Important Contribution to the DNA Binding Activity of the Pax-3 Paired Domain. J Biol Chem. 272, 28289-28295.

  25. Vogan, K. J., Underhill, D. A., and Gros, P. (1996) An Alternative Splicing Event in the Pax-3 Paired Domain Identifies the Linker Region as a Key Determinant of Paired Domain DNA-Binding Activity. Mol Cell Biol. 16, 6677-6686.

  26. Du, S., Lawrence, E. J., Strzelecki, D., Rajput, P., Xia, S. J., Gottesman, D. M., and Barr, F. G. (2005) Co-Expression of Alternatively Spliced Forms of PAX3, PAX7, PAX3-FKHR and PAX7-FKHR with Distinct DNA Binding and Transactivation Properties in Rhabdomyosarcoma. Int J Cancer. 115, 85-92.

  27. Bang, A. G., Papalopulu, N., Kintner, C., and Goulding, M. D. (1997) Expression of Pax-3 is Initiated in the Early Neural Plate by Posteriorizing Signals Produced by the Organizer and by Posterior Non-Axial Mesoderm. Development. 124, 2075-2085.

  28. Meulemans, D., and Bronner-Fraser, M. (2004) Gene-Regulatory Interactions in Neural Crest Evolution and Development. Dev Cell. 7, 291-299.

  29. Buckingham, M., and Rigby, P. W. (2014) Gene Regulatory Networks and Transcriptional Mechanisms that Control Myogenesis. Dev Cell. 28, 225-238.

  30. Blake, J. A., and Ziman, M. R. (2005) Pax3 Transcripts in Melanoblast Development. Dev Growth Differ. 47, 627-635.

  31. Wu, M., Li, J., Engleka, K. A., Zhou, B., Lu, M. M., Plotkin, J. B., and Epstein, J. A. (2008) Persistent Expression of Pax3 in the Neural Crest Causes Cleft Palate and Defective Osteogenesis in Mice. J Clin Invest. 118, 2076-2087.

  32. Kioussi, C., Gross, M. K., and Gruss, P. (1995) Pax3: A Paired Domain Gene as a Regulator in PNS Myelination. Neuron. 15, 553-562.

  33. Tremblay, P., Kessel, M., and Gruss, P. (1995) A Transgenic Neuroanatomical Marker Identifies Cranial Neural Crest Deficiencies Associated with the Pax3 Mutant Splotch. Dev Biol. 171, 317-329.

  34. Conway, S. J., Godt, R. E., Hatcher, C. J., Leatherbury, L., Zolotouchnikov, V. V., Brotto, M. A., Copp, A. J., Kirby, M. L., and Creazzo, T. L. (1997) Neural Crest is Involved in Development of Abnormal Myocardial Function. J Mol Cell Cardiol. 29, 2675-2685.

  35. Franz, T. (1989) Persistent Truncus Arteriosus in the Splotch Mutant Mouse. Anat Embryol (Berl). 180, 457-464.

  36. Daston, G., Lamar, E., Olivier, M., and Goulding, M. (1996) Pax-3 is Necessary for Migration but Not Differentiation of Limb Muscle Precursors in the Mouse. Development. 122, 1017-1027.

  37. Goulding, M., Lumsden, A., and Paquette, A. J. (1994) Regulation of Pax-3 Expression in the Dermomyotome and its Role in Muscle Development. Development. 120, 957-971.

  38. Pourquie, O., Coltey, M., Teillet, M. A., Ordahl, C., and Le Douarin, N. M. (1993) Control of Dorsoventral Patterning of Somitic Derivatives by Notochord and Floor Plate. Proc Natl Acad Sci U S A. 90, 5242-5246.

  39. Ordahl, C. P., and Le Douarin, N. M. (1992) Two Myogenic Lineages within the Developing Somite. Development. 114, 339-353.

  40. Ridgeway, A. G., and Skerjanc, I. S. (2001) Pax3 is Essential for Skeletal Myogenesis and the Expression of Six1 and Eya2. J Biol Chem. 276, 19033-19039.

  41. Watanabe, A., Takeda, K., Ploplis, B., and Tachibana, M. (1998) Epistatic Relationship between Waardenburg Syndrome Genes MITF and PAX3. Nat Genet. 18, 283-286.

  42. Bondurand, N., Pingault, V., Goerich, D. E., Lemort, N., Sock, E., Le Caignec, C., Wegner, M., and Goossens, M. (2000) Interaction among SOX10, PAX3 and MITF, Three Genes Altered in Waardenburg Syndrome. Hum Mol Genet. 9, 1907-1917.

  43. Potterf, S. B., Furumura, M., Dunn, K. J., Arnheiter, H., and Pavan, W. J. (2000) Transcription Factor Hierarchy in Waardenburg Syndrome: Regulation of MITF Expression by SOX10 and PAX3. Hum Genet. 107, 1-6.

  44. Wilkie, A. L., Jordan, S. A., and Jackson, I. J. (2002) Neural Crest Progenitors of the Melanocyte Lineage: Coat Colour Patterns Revisited. Development. 129, 3349-3357.

  45. Yasumoto, K., Takeda, K., Saito, H., Watanabe, K., Takahashi, K., and Shibahara, S. (2002) Microphthalmia-Associated Transcription Factor Interacts with LEF-1, a Mediator of Wnt Signaling. EMBO J. 21, 2703-2714.

  46. Lang, D., Lu, M. M., Huang, L., Engleka, K. A., Zhang, M., Chu, E. Y., Lipner, S., Skoultchi, A., Millar, S. E., and Epstein, J. A. (2005) Pax3 Functions at a Nodal Point in Melanocyte Stem Cell Differentiation. Nature. 433, 884-887.

  47. Jiao, Z., Mollaaghababa, R., Pavan, W. J., Antonellis, A., Green, E. D., and Hornyak, T. J. (2004) Direct Interaction of Sox10 with the Promoter of Murine Dopachrome Tautomerase (Dct) and Synergistic Activation of Dct Expression with Mitf. Pigment Cell Res. 17, 352-362.

  48. Ludwig, A., Rehberg, S., and Wegner, M. (2004) Melanocyte-Specific Expression of Dopachrome Tautomerase is Dependent on Synergistic Gene Activation by the Sox10 and Mitf Transcription Factors. FEBS Lett. 556, 236-244.

  49. Epstein, J. A. (2000) Pax3 and Vertebrate Development. Methods Mol Biol. 137, 459-470.

  50. Epstein, J. A., Shapiro, D. N., Cheng, J., Lam, P. Y., and Maas, R. L. (1996) Pax3 Modulates Expression of the c-Met Receptor during Limb Muscle Development. Proc Natl Acad Sci U S A. 93, 4213-4218.

  51. Lang, D., and Epstein, J. A. (2003) Sox10 and Pax3 Physically Interact to Mediate Activation of a Conserved c-RET Enhancer. Hum Mol Genet. 12, 937-945.

  52. Lang, D., Chen, F., Milewski, R., Li, J., Lu, M. M., and Epstein, J. A. (2000) Pax3 is Required for Enteric Ganglia Formation and Functions with Sox10 to Modulate Expression of c-Ret. J Clin Invest. 106, 963-971.

  53. Mayanil, C. S., George, D., Mania-Farnell, B., Bremer, C. L., McLone, D. G., and Bremer, E. G. (2000) Overexpression of Murine Pax3 Increases NCAM Polysialylation in a Human Medulloblastoma Cell Line. J Biol Chem. 275, 23259-23266.

  54. Liu, Y., Zhu, H., Liu, M., Du, J., Qian, Y., Wang, Y., Ding, F., and Gu, X. (2011) Downregulation of Pax3 Expression Correlates with Acquired GFAP Expression during NSC Differentiation Towards Astrocytes. FEBS Lett. 585, 1014-1020.

  55. Nakazaki, H., Reddy, A. C., Mania-Farnell, B. L., Shen, Y. W., Ichi, S., McCabe, C., George, D., McLone, D. G., Tomita, T., and Mayanil, C. S. (2008) Key Basic Helix-Loop-Helix Transcription Factor Genes Hes1 and Ngn2 are Regulated by Pax3 during Mouse Embryonic Development. Dev Biol. 316, 510-523.

  56. Barber, T. D., Barber, M. C., Tomescu, O., Barr, F. G., Ruben, S., and Friedman, T. B. (2002) Identification of Target Genes Regulated by PAX3 and PAX3-FKHR in Embryogenesis and Alveolar Rhabdomyosarcoma. Genomics. 79, 278-284.

  57. Mayanil, C. S., Pool, A., Nakazaki, H., Reddy, A. C., Mania-Farnell, B., Yun, B., George, D., McLone, D. G., and Bremer, E. G. (2006) Regulation of Murine TGFbeta2 by Pax3 during Early Embryonic Development. J Biol Chem. 281, 24544-24552.

  58. Guo, X. L., Ruan, H. B., Li, Y., Gao, X., and Li, W. (2010) Identification of a Novel Nonsense Mutation on the Pax3 Gene in ENU-Derived White Belly Spotting Mice and its Genetic Interaction with c-Kit. Pigment Cell Melanoma Res. 23, 252-262.

  59. Yavuzer, U., and Goding, C. R. (1994) Melanocyte-Specific Gene Expression: Role of Repression and Identification of a Melanocyte-Specific Factor, MSF. Mol Cell Biol. 14, 3494-3503.

  60. Galibert, M. D., Yavuzer, U., Dexter, T. J., and Goding, C. R. (1999) Pax3 and Regulation of the Melanocyte-Specific Tyrosinase-Related Protein-1 Promoter. J Biol Chem. 274, 26894-26900.

  61. Moriyama, M., Osawa, M., Mak, S. S., Ohtsuka, T., Yamamoto, N., Han, H., Delmas, V., Kageyama, R., Beermann, F., Larue, L., and Nishikawa, S. (2006) Notch Signaling Via Hes1 Transcription Factor Maintains Survival of Melanoblasts and Melanocyte Stem Cells. J Cell Biol. 173, 333-339.

  62. Pani, L., Horal, M., and Loeken, M. R. (2002) Rescue of Neural Tube Defects in Pax-3-Deficient Embryos by p53 Loss of Function: Implications for Pax-3- Dependent Development and Tumorigenesis. Genes Dev. 16, 676-680.

  63. Underwood, T. J., Amin, J., Lillycrop, K. A., and Blaydes, J. P. (2007) Dissection of the Functional Interaction between p53 and the Embryonic Proto-Oncoprotein PAX3. FEBS Lett. 581, 5831-5835.

  64. Li, H. G., Wang, Q., Li, H. M., Kumar, S., Parker, C., Slevin, M., and Kumar, P. (2007) PAX3 and PAX3-FKHR Promote Rhabdomyosarcoma Cell Survival through Downregulation of PTEN. Cancer Lett. 253, 215-223.

  65. Moncaut, N., Rigby, P. W., and Carvajal, J. J. (2013) Dial M(RF) for Myogenesis. FEBS J. 280, 3980-3990.

  66. Sommer, A., Young-Wee, T., and Frye, T. (1983) Previously Undescribed Syndrome of Craniofacial, Hand Anomalies, and Sensorineural Deafness. Am J Med Genet. 15, 71-77.

  67. Asher, J. H.,Jr, Sommer, A., Morell, R., and Friedman, T. B. (1996) Missense Mutation in the Paired Domain of PAX3 Causes Craniofacial-Deafness-Hand Syndrome. Hum Mutat. 7, 30-35.

  68. Barr, F. G., Galili, N., Holick, J., Biegel, J. A., Rovera, G., and Emanuel, B. S. (1993) Rearrangement of the PAX3 Paired Box Gene in the Paediatric Solid Tumour Alveolar Rhabdomyosarcoma. Nat Genet. 3, 113-117.

  69. Baldwin, C. T., Hoth, C. F., Macina, R. A., and Milunsky, A. (1995) Mutations in PAX3 that Cause Waardenburg Syndrome Type I: Ten New Mutations and Review of the Literature. Am J Med Genet. 58, 115-122.

  70. WAARDENBURG, P. J. (1951) A New Syndrome Combining Developmental Anomalies of the Eyelids, Eyebrows and Nose Root with Pigmentary Defects of the Iris and Head Hair and with Congenital Deafness. Am J Hum Genet. 3, 195-253.

  71. Tassabehji, M., Read, A. P., Newton, V. E., Harris, R., Balling, R., Gruss, P., and Strachan, T. (1992) Waardenburg's Syndrome Patients have Mutations in the Human Homologue of the Pax-3 Paired Box Gene. Nature. 355, 635-636.

  72. Robson, E. J., He, S. J., and Eccles, M. R. (2006) A PANorama of PAX Genes in Cancer and Development. Nat Rev Cancer. 6, 52-62.

  73. Epstein, J. A., Song, B., Lakkis, M., and Wang, C. (1998) Tumor-Specific PAX3-FKHR Transcription Factor, but Not PAX3, Activates the Platelet-Derived Growth Factor Alpha Receptor. Mol Cell Biol. 18, 4118-4130.

  74. Baxter, L. L., Hou, L., Loftus, S. K., and Pavan, W. J. (2004) Spotlight on Spotted Mice: A Review of White Spotting Mouse Mutants and Associated Human Pigmentation Disorders 1. Pigment Cell Res. 17, 215-224.

  75. Bennett, D. C., and Lamoreux, M. L. (2003) The Color Loci of Mice--a Genetic Century. Pigment Cell Res. 16, 333-344.

  76. Epstein, D. J., Vogan, K. J., Trasler, D. G., and Gros, P. (1993) A Mutation within Intron 3 of the Pax-3 Gene Produces Aberrantly Spliced mRNA Transcripts in the Splotch (Sp) Mouse Mutant. Proc Natl Acad Sci U S A. 90, 532-536.

  77. Moase, C. E., and Trasler, D. G. (1992) Splotch Locus Mouse Mutants: Models for Neural Tube Defects and Waardenburg Syndrome Type I in Humans. J Med Genet. 29, 145-151.

  78. Vogan, K. J., Epstein, D. J., Trasler, D. G., and Gros, P. (1993) The Splotch-Delayed (Spd) Mouse Mutant Carries a Point Mutation within the Paired Box of the Pax-3 Gene. Genomics. 17, 364-369.

  79. DICKIE, M. M. (1964) New Splotch Alleles in the Mouse. J Hered. 55, 97-101.

  80. Moase, C. E., and Trasler, D. G. (1989) Spinal Ganglia Reduction in the Splotch-Delayed Mouse Neural Tube Defect Mutant. Teratology. 40, 67-75.

  81. Epstein, D. J., Vekemans, M., and Gros, P. (1991) Splotch (Sp2H), a Mutation Affecting Development of the Mouse Neural Tube, shows a Deletion within the Paired Homeodomain of Pax-3. Cell. 67, 767-774.

  82. Goulding, M., Sterrer, S., Fleming, J., Balling, R., Nadeau, J., Moore, K. J., Brown, S. D., Steel, K. P., and Gruss, P. (1993) Analysis of the Pax-3 Gene in the Mouse Mutant Splotch. Genomics. 17, 355-363.

  83. Fleming, J., Pearce, A., Brown, S. D., and Steel, K. P. (1996) The Sp4H Deletion may Contain a New Locus Essential for Postimplantation Development. Genomics. 34, 205-212.

  84. Xiao, Y., Zhang, L., He, K., Gao, X., Yang, L., He, L., Ma, G., and Guo, X. (2011) Characterization of a Novel Missense Mutation on Murine Pax3 through ENU Mutagenesis. J Genet Genomics. 38, 333-339.

  85. Hornyak, T. J., Hayes, D. J., Chiu, L. Y., and Ziff, E. B. (2001) Transcription Factors in Melanocyte Development: Distinct Roles for Pax-3 and Mitf. Mech Dev. 101, 47-59.