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Branchio-oculo-facial syndrome (BOFS) is a rare autosomal-dominant cleft palate-craniofacial disorder with variable expressivity. The major features include cutaneous anomalies (cervical, infra- and/or supra-auricular defects, often with dermal thymus), ocular anomalies, characteristic facial appearance (malformed pinnae, oral clefts), and, less commonly, renal and ectodermal (dental and hair) anomalies. The molecular basis for this disorder is heretofore unknown. We detected a 3.2 Mb deletion by 500K SNP microarray in an affected mother and son with BOFS at chromosome 6p24.3. Candidate genes in this region were selected for sequencing on the basis of their expression patterns and involvement in developmental pathways associated with the clinical findings of BOFS. Four additional BOFS patients were found to have de novo missense mutations in the highly conserved exons 4 and 5 (basic region of the DNA binding domain) of the TFAP2A gene in the candidate deleted region. We conclude BOFS is caused by mutations involving TFAP2A. More patients need to be studied to determine possible genetic heterogeneity and to establish whether there are genotype-phenotype correlations.

Neuroblastoma, the most common extracranial solid tumour of childhood, is derived from neural crest progenitor cells. The TFAP2a transcription factor regulates neural crest patterning. We analysed TFAP2a protein expression in 97 primary neuroblastic tumors and report that TFAP2a was strongly expressed in poorly differentiated neuroblastomas. TFAP2a expression in tumor cells of differentiated neuroblastic tumors was below detection. TFAP2a was strongly expressed in 4 of 6 neuroblastoma cell lines tested, and TFAP2a siRNA mediated knock down in SH-EP cells reduced proliferation and induced a more differentiated phenotype associated with an increase in the expression of the differentiation marker neurotensin.

Transcription factor AP2 (Tfap2) genes play essential roles in development of the epidermis and migratory cells of the neural crest (NC) in vertebrate embryos. These transcriptional activators are among the earliest genes expressed in the ectoderm and specify fates within the epidermis/crest through both direct and indirect mechanisms. The Tfap2 family arose from a single ancestral gene in a chordate ancestor that underwent gene duplication to give up to five family members in living vertebrates. This coincided with the acquisition of important roles in NC development by Tfap2 genes suggesting that this gene family was important in ectodermal evolution and possibly in the origin of NC. Here, we show that a zebrafish tfap2c is expressed in the nonneural ectoderm during early development and functions redundantly with tfap2a in NC specification. In zebrafish embryos depleted of both tfap2a and tfap2c, NC cells are virtually eliminated. Cell transplantation experiments indicate that tfap2c functions cell-autonomously in NC specification. Cells of the enveloping layer, which forms a temporary skin layer surrounding the ectoderm, also fail to differentiate or to express appropriate keratins in tfap2c deficient embryos. The role of Tfap2 genes in epidermal and NC development is considered here in the broader context of ectodermal evolution. Distinct, tissue-specific functions for Tfap2 genes in different vertebrates may reflect subfunctionalisation of an ancestral gene that consequently led to the gain of novel roles for different subfamily members in patterning the epidermis and NC.

Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin `purse strings', which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

Cleft lip or palate (or the two in combination) is a common birth defect that results from a mixture of genetic and environmental factors. We searched for a specific genetic factor contributing to this complex trait by examining large numbers of affected patients and families and evaluating a specific candidate gene.We identified the gene that encodes interferon regulatory factor 6 (IRF6) as a candidate gene on the basis of its involvement in an autosomal dominant form of cleft lip and palate, Van der Woude's syndrome. A single-nucleotide polymorphism in this gene results in either a valine or an isoleucine at amino acid position 274 (V274I). We carried out transmission-disequilibrium testing for V274I in 8003 individual subjects in 1968 families derived from 10 populations with ancestry in Asia, Europe, and South America, haplotype and linkage analyses, and case-control analyses, and determined the risk of cleft lip or palate that is associated with genetic variation in IRF6.Strong evidence of overtransmission of the valine (V) allele was found in the entire population data set (P10(-9)); moreover, the results for some individual populations from South America and Asia were highly significant. Variation at IRF6 was responsible for 12 percent of the genetic contribution to cleft lip or palate and tripled the risk of recurrence in families that had already had one affected child.DNA-sequence variants associated with IRF6 are major contributors to cleft lip, with or without cleft palate. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling.

The neural crest is a uniquely vertebrate cell type that gives rise to much of the craniofacial skeleton, pigment cells and peripheral nervous system, yet its specification and diversification during embryogenesis are poorly understood. Zebrafish homozygous for the lockjaw (low)mutation show defects in all of these derivatives and we show that low (allelic with montblanc) encodes a zebrafish tfap2a, one of a small family of transcription factors implicated in epidermal and neural crest development. A point mutation in lowtruncates the DNA binding and dimerization domains of tfap2a, causing a loss of function. Consistent with this, injection of antisense morpholino oligonucleotides directed against splice sites in tfap2a into wild-type embryos produces a phenotype identical to low. Analysis of early ectodermal markers revealed that neural crest specification and migration are disrupted in low mutant embryos. TUNEL labeling of dying cells in mutants revealed a transient period of apoptosis in crest cells prior to and during their migration. In the cranial neural crest, gene expression in the mandibular arch is unaffected in low mutants, in contrast to the hyoid arch, which shows severe reductions in dlx2 and hoxa2 expression. Mosaic analysis, using cell transplantation,demonstrated that neural crest defects in low are cell autonomous and secondarily cause disruptions in surrounding mesoderm. These studies demonstrate that low is required for early steps in neural crest development and suggest that tfap2a is essential for the survival of a subset of neural crest derivatives.

Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Most IRFs regulate the expression of interferon-alpha and -beta after viral infection, but the function of IRF6 is unknown. The gene encoding IRF6 is located in the critical region for the Van der Woude syndrome (VWS; OMIM 119300) locus at chromosome 1q32-q41 (refs 2,3). The disorder is an autosomal dominant form of cleft lip and palate with lip pits, and is the most common syndromic form of cleft lip or palate. Popliteal pterygium syndrome (PPS; OMIM 119500) is a disorder with a similar orofacial phenotype that also includes skin and genital anomalies. Phenotypic overlap and linkage data suggest that these two disorders are allelic. We found a nonsense mutation in IRF6 in the affected twin of a pair of monozygotic twins who were discordant for VWS. Subsequently, we identified mutations in IRF6 in 45 additional unrelated families affected with VWS and distinct mutations in 13 families affected with PPS. Expression analyses showed high levels of Irf6 mRNA along the medial edge of the fusing palate, tooth buds, hair follicles, genitalia and skin. Our observations demonstrate that haploinsufficiency of IRF6 disrupts orofacial development and are consistent with dominant-negative mutations disturbing development of the skin and genitalia.

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.

The AP-2 family of transcription factors consists of five different proteins in humans and mice: AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2e. Frogs and fish have known orthologs of some but not all of these proteins, and homologs of the family are also found in protochordates, insects and nematodes. The proteins have a characteristic helix-span-helix motif at the carboxyl terminus, which, together with a central basic region, mediates dimerization and DNA binding. The amino terminus contains the transactivation domain. AP-2 proteins are first expressed in primitive ectoderm of invertebrates and vertebrates; in vertebrates, they are also expressed in the emerging neural-crest cells, and AP-2α -/- animals have impairments in neural-crest-derived facial structures. AP-2β is indispensable for kidney development and AP-2γ is necessary for the formation of trophectoderm cells shortly after implantation; AP-2α and AP-2γ levels are elevated in human mammary carcinoma and seminoma. The general functions of the family appear to be the cell-type-specific stimulation of proliferation and the suppression of terminal differentiation during embryonic development.

The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.

Knockdown studies suggest transcription factor AP-2 alpha (Tfap2a), is required for neural crest induction in frog embryos. Because tfap2a is expressed in neural crest and in presumptive epidermis, a source of signals that induce neural crest, it was unclear whether this requirement was cell autonomous. Moreover, neural crest induction occurs normally in zebrafish tfap2a and mouse Tcfap2a mutant embryos, so it was unclear if a requirement for Tfap2a in this process was evolutionarily conserved. Here we show that zebrafish tfap2c, encoding AP-2 gamma (Tfap2c), is expressed in non-neural ectoderm including transiently in neural crest. Inhibition of tfap2c with antisense oligonucleotides does not visibly perturb development. However, simultaneous inhibition of tfap2a and tfap2c utterly prevents neural crest induction, and disrupts development of cranial placode derivatives, although gene expression characteristic of the pre-placodal domain is normal. Transplant studies support a cell-autonomous role for Tfap2a and Tfap2c in neural crest induction. Unexpectedly, Rohon-Beard sensory neurons, which previous studies indicate are derived from the same precursor population as neural crest, are reduced by less than half in tfap2a/tfap2c doubly deficient embryos, implying non-neural crest origin for a subset of them. These results reveal a requirement for Tfap2-type activity for early development of all non-neural ectoderm derivatives.

The AP-2alpha transcription factor is required for multiple aspects of vertebrate development and mice lacking the AP-2alpha gene (tcfap2a) die at birth from severe defects affecting the head and trunk. Several of the defects associated with the tcfap2a-null mutation affect neural crest cell (NCC) derivatives including the craniofacial skeleton, cranial ganglia, and heart outflow tract. Consequently, there is considerable interest in the role of AP-2alpha in neural crest cell function in development and evolution. In addition, the expression of the AP-2alpha gene is utilized as a marker for premigratory and migratory neural crest cells in many vertebrate species. Here, we have specifically addressed how the presence of AP-2alpha in neural crest cells affects development by creating a conditional (floxed) version of tcfap2a which has subsequently been intercrossed with mice expressing Cre recombinase under the control of Wnt1 cis-regulatory sequences. Neural crest-specific disruption of tcfap2a results in frequent perinatal lethality associated with neural tube closure defects and cleft secondary palate. A small but significant fraction of mutant mice can survive into adulthood, but have retarded craniofacial growth, abnormal middle ear development, and defects in pigmentation. The phenotypes obtained confirm that AP-2alpha directs important aspects of neural crest cell function. At the same time, we did not observe several neurocristopathies affecting the head and heart that might be expected based on the phenotype of the AP-2alpha-null mouse. These results have important implications for the evolution and function of the AP-2 gene family in both the neural crest and the vertebrate embryo.

Subunit switches in the BAF chromatin remodeler are essential during development. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause neurodevelopmental disorders, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we leveraged ARID1B+/− Coffin-Siris patient-derived iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF). ARID1B-BAF regulates exit from pluripotency and lineage commitment by attenuating thousands of enhancers and genes of the NANOG and SOX2 networks. In iPSCs, these enhancers are maintained active by ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A- to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch, and maintain ARID1A-BAF at the pluripotency enhancers throughout all stages of CNCC formation. This leads to persistent NANOG/SOX2 activity which impairs CNCC formation. Despite showing the typical neural crest signature (TFAP2A/SOX9-positive), ARID1B-haploinsufficient CNCCs are also aberrantly NANOG-positive. These findings suggest a connection between ARID1B mutations, neuroectoderm specification and a pathogenic mechanism for Coffin-Siris syndrome. Mutations in the ARID1B subunit of the BAF chromatin remodeling complex are associated with the neurodevelopmental Coffin-Siris syndrome. Here the authors reveal that there is a transition from ARID1A-containing complexes to ARID1B during cranial neural crest cell differentiation that is impaired in Coffin-Siris patient-derived cells, which is important for exit from pluripotency.