RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

Ttc30a affects tubulin modifications in a model for ciliary chondrodysplasia with polycystic kidney disease.

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci (ift80 and ift172) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies.

Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia.

Primary cilia contain specific proteins to achieve their functions as cellular antennae. Ciliary protein trafficking is mediated by the intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes. Mutations in genes encoding the IFT-A subunits (IFT43, IFT121/WDR35, IFT122, IFT139/TTC21B, IFT140 and IFT144/WDR19) often result in skeletal ciliopathies, including cranioectodermal dysplasia (CED). We here characterized the molecular and cellular defects of CED caused by compound heterozygous mutations in IFT144 [the missense variant IFT144(L710S) and the nonsense variant IFT144(R1103*)]. These two variants were distinct with regard to their interactions with other IFT-A subunits and with the IFT-B complex. When exogenously expressed in IFT144-knockout (KO) cells, IFT144(L710S) as well as IFT144(WT) rescued both moderately compromised ciliogenesis and the abnormal localization of ciliary proteins. As the homozygous IFT144(L710S) mutation was found to cause autosomal recessive retinitis pigmentosa, IFT144(L710S) is likely to be hypomorphic at the cellular level. In striking contrast, the exogenous expression of IFT144(R1103*) in IFT144-KO cells exacerbated the ciliogenesis defects. The expression of IFT144(R1103*) together with IFT144(WT) restored the abnormal phenotypes of IFT144-KO cells. However, the coexpression of IFT144(R1103*) with the hypomorphic IFT144(L710S) variant in IFT144-KO cells, which mimics the genotype of compound heterozygous CED patients, resulted in severe ciliogenesis defects. Taken together, these observations demonstrate that compound heterozygous mutations in IFT144 cause severe ciliary defects via a complicated mechanism, where one allele can cause severe ciliary defects when combined with a hypomorphic allele.

Ras/MAPK dysregulation in development causes a skeletal myopathy in an activating BrafL597V mouse model for cardio-facio-cutaneous syndrome.

BACKGROUND: Cardio-facio-cutaneous (CFC) syndrome is a human multiple congenital anomaly syndrome that is caused by activating heterozygous mutations in either BRAF, MEK1, or MEK2, three protein kinases of the Ras/mitogen-activated protein kinase (MAPK) pathway. CFC belongs to a group of syndromes known as RASopathies. Skeletal muscle hypotonia is a ubiquitous phenotype of RASopathies, especially in CFC syndrome. To better understand the underlying mechanisms for the skeletal myopathy in CFC, a mouse model with an activating BrafL597V allele was utilized. RESULTS: The activating BrafL597V allele resulted in phenotypic alterations in skeletal muscle characterized by a reduction in fiber size which leads to a reduction in muscle size which are functionally weaker. MAPK pathway activation caused inhibition of myofiber differentiation during embryonic myogenesis and global transcriptional dysregulation of developmental pathways. Inhibition in differentiation can be rescued by MEK inhibition. CONCLUSIONS: A skeletal myopathy was identified in the CFC BrafL597V mouse validating the use of models to study the effect of Ras/MAPK dysregulation on skeletal myogenesis. RASopathies present a novel opportunity to identify new paradigms of myogenesis and further our understanding of Ras in development. Rescue of the phenotype by inhibitors may help advance the development of therapeutic options for RASopathy patients.

C-type natriuretic peptide improves growth retardation in a mouse model of cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome, a genetic disorder caused by germline mutations in BRAF, KRAS, MAP2K1 and MAP2K2, is characterized by growth retardation, heart defects, dysmorphic facial appearance and dermatologic abnormalities. We have previously reported that knock-in mice expressing the CFC syndrome-associated mutation, Braf Q241R, showed growth retardation because of gastrointestinal dysfunction. However, other factors associated with growth retardation, including chondrogenesis and endocrinological profile, have not been examined. Here, we show that 3- and 4-week-old BrafQ241R/+ mice have decreased body weight and length, as well as reduced growth plate width in the proximal tibiae. Furthermore, proliferative and hypertrophic chondrocyte zones of the growth plate were reduced in BrafQ241R/+ mice compared with Braf+/+ mice. Immunohistological analysis revealed that extracellular signal-regulated kinase (ERK) activation was enhanced in hypertrophic chondrocytes in BrafQ241R/+ mice. In accordance with growth retardation and reduced growth plate width, decreased serum levels of insulin-like growth factor 1 (IGF-1) and IGF binding protein 3 (IGFBP-3) were observed in BrafQ241R/+ mice at 3 and 4 weeks of age. Treatment with C-type natriuretic peptide (CNP), a stimulator of endochondral bone growth and a potent inhibitor of the FGFR3-RAF1-MEK/ERK signaling, increased body and tail lengths in Braf+/+ and BrafQ241R/+ mice. In conclusion, ERK activation in chondrocytes and low serum IGF-1/IGFBP-3 levels could be associated with the growth retardation observed in BrafQ241R/+ mice. Our data also suggest that CNP is a potential therapeutic target in CFC syndrome.

Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment.

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

Ectodermal dysplasias: Classification and organization by phenotype, genotype and molecular pathway.

An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias (EDs) that would integrate both clinical and molecular information. We propose the following, a working definition of the EDs building on previous classification systems and incorporating current approaches to diagnosis: EDs are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with EDs that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non-syndromic traits of the causative gene (e.g., non-syndromic hypodontia or missing teeth associated with pathogenic variants of EDA "ectodysplasin"). Information for categorization and cataloging includes the phenotypic features, Online Mendelian Inheritance in Man number, mode of inheritance, genetic alteration, major developmental pathways involved (e.g., EDA, WNT "wingless-type," TP63 "tumor protein p63") or the components of complex molecular structures (e.g., connexins, keratins, cadherins).

Incontinentia pigmenti in adults.

Incontinentia Pigmenti (IP; MIM 308300) is an X-linked dominant genodermatosis caused by pathogenic variant in IKBKG. The phenotype in adults is poorly described compared to that in children. Questionnaire survey of 99 affected women showed an age at diagnosis from newborn to 41 years, with 53 diagnosed by 6 months of age and 30 as adults. Stage I, II, and III lesions persisted in 16%, 17%, and 71%, respectively, of those who had ever had them. IP is allelic to two forms of ectodermal dysplasia. Many survey respondents reported hypohidrosis and/or heat intolerance and most had Stage IV findings. This suggests that "Stage IV" may be congenitally dysplastic skin that becomes more noticeable with maturity. Fifty-one had dentures or implants with 26 having more invasive jaw or dental surgery. Half had wiry or uncombable hair. Seventy-three reported abnormal nails with 27 having long-term problems. Cataracts and retinal detachment were the reported causes of vision loss. Four had microphthalmia. Respondents without genetic confirmation of IP volunteered information suggesting more involved phenotype or possibly misassigned diagnosis. Ascertainment bias likely accounts for the low prevalence of neurocognitive problems in the respondents.

Master regulatory role of p63 in epidermal development and disease.

The transcription factor p63 is a master regulator of epidermal development. Mutations in p63 give rise to human developmental diseases that often manifest epidermal defects. In this review, we summarize major p63 isoforms identified so far and p63 mutation-associated human diseases that show epidermal defects. We discuss key roles of p63 in epidermal keratinocyte proliferation and differentiation, emphasizing its master regulatory control of the gene expression pattern and epigenetic landscape that define epidermal fate. We subsequently review the essential function of p63 during epidermal commitment and transdifferentiation towards epithelial lineages, highlighting the notion that p63 is the guardian of the epithelial lineage. Finally, we discuss current therapeutic development strategies for p63 mutation-associated diseases. Our review proposes future directions for dissecting p63-controlled mechanisms in normal and diseased epidermal development and for developing therapeutic options.

Rescue of neurodegeneration in the Fig4 null mouse by a catalytically inactive FIG4 transgene.

The lipid phosphatase FIG4 is a subunit of the protein complex that regulates biosynthesis of the signaling lipid PI(3,5)P2. Mutations of FIG4 result in juvenile lethality and spongiform neurodegeneration in the mouse, and are responsible for the human disorders Charcot-Marie-Tooth disease, Yunis-Varon syndrome and polymicrogyria with seizures. We previously demonstrated that conditional expression of a wild-type FIG4 transgene in neurons is sufficient to rescue most of the abnormalities of Fig4 null mice, including juvenile lethality and extensive neurodegeneration. To evaluate the contribution of the phosphatase activity to the in vivo function of Fig4, we introduced the mutation p.Cys486Ser into the Sac phosphatase active-site motif CX5RT. Transfection of the Fig4(Cys486Ser) cDNA into cultured Fig4(-/-) fibroblasts was effective in preventing vacuolization. The neuronal expression of an NSE-Fig4(Cys486Ser) transgene in vivo prevented the neonatal neurodegeneration and juvenile lethality seen in Fig4 null mice. These observations demonstrate that the catalytically inactive FIG4 protein provides significant function, possibly by stabilization of the PI(3,5)P2 biosynthetic complex and/or localization of the complex to endolysosomal vesicles. Despite this partial rescue, later in life the NSE-Fig4(Cys486Ser) transgenic mice display significant abnormalities that include hydrocephalus, defective myelination and reduced lifespan. The late onset phenotype of the NSE-Fig4(Cys486Ser) transgenic mice demonstrates that the phosphatase activity of FIG4 has an essential role in vivo.

Adams-Oliver syndrome review of the literature: Refining the diagnostic phenotype.

The Adams-Oliver syndrome (AOS) is defined as aplasia cutis congenita (ACC) with transverse terminal limb defects (TTLD). Frequencies of associated anomalies are not well characterized. Six causative genes have been identified: ARHGAP31, DOCK6, EOGT, RBPJ, NOTCH1, and DLL4. We review 385 previously described individuals (139 non-familial and 246 familial probands and family members) and add clinical data on 13 previously unreported individuals with AOS. In addition to ACC and TTLD, the most commonly associated anomalies included a wide variety of central nervous system (CNS) anomalies and congenital heart defects each seen in 23%. CNS anomalies included structural anomalies, microcephaly, vascular defects, and vascular sequelae. CNS migration defects were common. Cutis marmorata telangiectasia congenita (CMTC) was found in 19% of the study population and other vascular anomalies were seen in 14%. Hemorrhage was listed as the cause of death for five of 25 deaths reported. A relatively large number of non-familial probands were reported to have hepatoportal sclerosis with portal hypertension and esophageal varices. Non-familial probands were more likely to have additional anomalies than were familial probands. The data reported herein provide a basis for refining the diagnostic features of AOS and suggest management recommendations for probands newly diagnosed with AOS. © 2017 Wiley Periodicals, Inc.

Impaired Osteogenesis of Disease-Specific Induced Pluripotent Stem Cells Derived from a CFC Syndrome Patient.

Cardiofaciocutaneous (CFC) syndrome is a rare genetic disorder caused by mutations in the extracellular signal-regulated kinase (ERK) signaling. However, little is known about how aberrant ERK signaling is associated with the defective bone development manifested in most CFC syndrome patients. In this study, induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts of a CFC syndrome patient having rapidly accelerated fibrosarcoma kinase B (BRAF) gain-of-function mutation. CFC-iPSCs were differentiated into mesenchymal stem cells (CFC-MSCs) and further induced to osteoblasts in vitro. The osteogenic defects of CFC-MSCs were revealed by alkaline phosphatase activity assay, mineralization assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting. Osteogenesis of CFC-MSCs was attenuated compared to wild-type (WT)-MSCs. In addition to activated ERK signaling, increased p-SMAD2 and decreased p-SMAD1 were observed in CFC-MSCs during osteogenesis. The defective osteogenesis of CFC-MSCs was rescued by inhibition of ERK signaling and SMAD2 signaling or activation of SMAD1 signaling. Importantly, activation of ERK signaling and SMAD2 signaling or inhibition of SMAD1 signaling recapitulated the impaired osteogenesis in WT-MSCs. Our findings indicate that SMAD2 signaling and SMAD1 signaling as well as ERK signaling are responsible for defective early bone development in CFC syndrome, providing a novel insight on the pathological mechanism and therapeutic targets.

New BRAF knockin mice provide a pathogenetic mechanism of developmental defects and a therapeutic approach in cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

Enhanced SMAD1 Signaling Contributes to Impairments of Early Development in CFC-iPSCs.

Cardio-facio-cutaneous (CFC) syndrome is a developmental disorder caused by constitutively active ERK signaling manifesting mainly from BRAF mutations. Little is known about the role of elevated ERK signaling in CFC syndrome during early development. Here, we show that both SMAD1 and ERK signaling pathways may contribute to the developmental defects in CFC syndrome. Induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of a CFC syndrome patient (CFC-iPSCs) revealed early developmental defects in embryoid body (EB) development, ß-catenin localization, and neuronal differentiation. Both SMAD1 and ERK signalings were significantly activated in CFC-iPSCs during EB formation. Most of the ß-catenin was dissociated from the membrane and preferentially localized into the nucleus in CFC-EBs. Furthermore, activation of SMAD1 signaling recapitulated early developmental defects in wild-type iPSCs. Intriguingly, inhibition of SMAD1 signaling in CFC-iPSCs rescued aberrant EB morphology, impaired neuronal differentiation, and altered ß-catenin localization. These results suggest that SMAD1 signaling may be a key pathway contributing the pathogenesis of CFC syndrome during early development.

Objective studies of the face of Noonan, Cardio-facio-cutaneous, and Costello syndromes: A comparison of three disorders of the Ras/MAPK signaling pathway.

Noonan, Cardio-facio-cutaneous, and Costello syndromes are disorders of the Ras/MAPK pathway that share many clinical features. This observational and anthropometric study was conducted to describe the key facial features of each syndrome in order to improve discrimination between the three conditions, particularly in young children where diagnosis is most challenging. Direct measurement of the head and face was used to enhance diagnostic accuracy, and identify the most unusual or specific dimensions. The Noonan syndrome cohort included 123 individuals, aged 6 months to 41 years. There were 20 children and adolescents with Cardio-facio-cutaneous syndrome, and 28 individuals with Costello syndrome, aged 1-32 years. The facial phenotypes of these syndromes, particularly Noonan syndrome, are well-described but objective data have not been published in peer-reviewed literature. In this study, subjective observations, in the main, were validated by anthropometry with one exception. In individuals with Costello syndrome, mouth width was normal, thus the impression of wide mouth is likely due to full lips or the mouth being viewed in relation to a narrow lower face. When the three conditions were compared objectively, syndrome-specific pattern profiles showed high concordance in early life. At older ages, Cardio-facio-cutaneous syndrome was distinguished by increased width of the mid/lower face, and reduced growth of maxillary and mandibular dimensions was noted in both Noonan and Costello syndromes. Despite substantial similarities in face shape in older individuals with these two conditions, bulbous nasal tip, full lips, and an apparently wide mouth in those with Costello Syndrome facilitate discrimination from Noonan syndrome. © 2016 Wiley Periodicals, Inc.

Crystal structure of a vFlip-IKKgamma complex: insights into viral activation of the IKK signalosome.

Key to the pathogenicity of several viruses is activation of the canonical nuclear factor-kappaB (NF-kappaB) transcriptional pathway. Subversion of this tightly regulated mechanism is achieved through the production of host mimetic viral proteins that deregulate the transcription process. One such protein is ks-vFLIP (produced by the Kaposi's sarcoma herpes virus [KSHV]), which associates with IKKgamma, an essential component of the IKK complex or signalosome. This interaction renders the canonical NF-kappaB pathway constitutively active and has been linked to Kaposi's sarcoma and other malignancies. In order to elucidate the molecular basis underpinning ks-vFLIP-induced activation of the IKK signalosome, we have determined the crystal structure of a complex involving a fragment of IKKgamma bound to ks-vFLIP at 3.2 A. In addition to identifying and subsequently probing the ks-vFLIP-IKKgamma interface, we have also investigated the effects of a mutation implicated in the genetic disorder anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID).

APR-246/PRIMA-1(MET) rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations.

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET.

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.