Phenotypic Mutation 'Widget' (pdf version)
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Mutation Type critical splice donor site (1 bp from exon)
Coordinate78,122,590 bp (GRCm38)
Base Change C ⇒ T (forward strand)
Gene Pax3
Gene Name paired box 3
Synonym(s) Pax-3
Chromosomal Location 78,101,267-78,197,134 bp (-)
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]
Accession Number

NCBI RefSeq: NM_008781, NM_001159520; MGI:97487

Mapped No 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000004994] [ENSMUSP00000084320]
SMART Domains Protein: ENSMUSP00000004994
Gene: ENSMUSG00000004872

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

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
Phenotypic Category
Phenotypequestion? Literature verified References
Alleles Listed at MGI

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

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01642:Pax3 APN 1 78196663 critical splice donor site probably null
IGL02249:Pax3 APN 1 78195325 missense probably damaging 0.98
IGL02271:Pax3 APN 1 78195332 missense probably damaging 1.00
IGL02376:Pax3 APN 1 78132292 missense probably damaging 1.00
IGL02530:Pax3 APN 1 78121787 missense possibly damaging 0.87
IGL02950:Pax3 APN 1 78103360 missense probably benign 0.06
Nidoqueen UTSW 1 78132232 missense probably damaging 1.00
stemware UTSW 1 78132347 missense
R0049:Pax3 UTSW 1 78103504 missense probably damaging 1.00
R0049:Pax3 UTSW 1 78103504 missense probably damaging 1.00
R0523:Pax3 UTSW 1 78195441 missense possibly damaging 0.83
R1575:Pax3 UTSW 1 78103484 missense probably benign 0.00
R1831:Pax3 UTSW 1 78132340 missense probably damaging 1.00
R1934:Pax3 UTSW 1 78103480 missense possibly damaging 0.90
R2420:Pax3 UTSW 1 78196864 unclassified probably null
R2473:Pax3 UTSW 1 78122590 critical splice donor site probably null
R4430:Pax3 UTSW 1 78195324 missense probably damaging 1.00
R4693:Pax3 UTSW 1 78196746 missense probably benign 0.00
R4818:Pax3 UTSW 1 78132232 missense probably damaging 1.00
R4860:Pax3 UTSW 1 78192456 missense possibly damaging 0.78
R4860:Pax3 UTSW 1 78192456 missense possibly damaging 0.78
R5302:Pax3 UTSW 1 78121612 missense possibly damaging 0.88
R5475:Pax3 UTSW 1 78103418 missense probably benign 0.06
R5855:Pax3 UTSW 1 78121651 missense probably damaging 0.99
R6102:Pax3 UTSW 1 78132347 missense probably damaging 1.00
R6190:Pax3 UTSW 1 78192549 missense possibly damaging 0.63
R6856:Pax3 UTSW 1 78132419 missense probably damaging 1.00
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
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-->


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


Tissue expression

Expression in cancers



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



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


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



Transcript lacks intron 8 and translation proceeds from exon 8 to 9; protein does not have a portion of the TA domain


Transcript contains exons 8, 9, and 10 but lacks introns 8 and 9; protein is similar to PAX3D

Decreases differentiated melanocyte proliferation and migration





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


Predominant in neuroblastoma



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


Predominant in neuroblastoma



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


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.


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).


Table 2. Select Pax3 targets


Brief description of function

PAX3-associated affect



Receptor tyrosine kinase

Promotes cell migration during limb muscle and melanocyte development



Promotes cell migration during enteric ganglia formation



Factors involved in myelination of nerves in the peripheral nervous system

Maintains a nonmyelinated axonal phenotype for Schwann cells



Inhibits neural stem cell differentiation towards astrocytes


Enhances nerve cell survival and differentiation


Enhances L1 expression to support Schwann cell precursor migration to the peripheral nervous system


Pax3 represses expression; cell-cell adhesion


Transcription factor

Initiates neuronal lineage specification


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



Transcription factor

Melanocyte development; Mitf binding to Dct, Tyr, Tyrp1, and c-Kit (see the record for Pretty2) initiates the melanogenic cascade



Pigment enzyme

Maintains the “stemness” of migratory neural cells to inhibit melanocyte differentiation



Pigment enzyme; see the record for chi

Promotes melanogenic cascade



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


Trp53 (alternatively, p53), Pten, Bcl2l1 (alternatively, Bcl-XL)

Antiapoptotic factors (see lentil for information about Trp53bp1)

Promotes survival of melanocytes (and possibly melanoma cells)


Six1, Eya2, Myod1, Myf5, Myog, Sostdc1

Myogenic regulatory factors

Skeletal myogenesis


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/ETBR 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 Gα 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/ETBR signaling pathway induces the phosphorylation of Ser298 on MITF through activation of the phospholipase C/PIP2/DAG/PKC pathway; the ET-3/ETBR merges with the ERK signaling pathway downstream of c-Kit. For a more comprehensive view of ETBR 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 Splotch4H (a large deletion in Pax3) and Splotch2H (an intragenic deletion) (77;81-83). Each of these mutants disply variable white belly spotting in the heterozygote. Two ENU-induced Pax3 mutants (Pax3wbs and Pax3Sp-1Xzg) exhibit variable white belly spotting in heterozygous mice (58;84). The Pax3wbs 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 Pax3wbs 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.
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsKimberly Hawkins and Jamie Russell
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