Key role of p63 in BMP-4-Induced Epidermal Commitment of Embryonic Stem Cells

In vivo studies, transgenic and knock-out mice have demonstrated that p63 isoforms play pivotal roles in ectodermal and epidermal development but their respective function remains highly controversial. Since embryonic stem (ES) cells can be differentiated into many cell types, they represent an effective tool to recapitulate in vitro the main steps of embryonic development. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells and clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation through ΔNp63 gene activation. ΔNp63 is not required for ectodermal fate but enhances ES-derived ectodermal cell proliferation and epidermal commitment. This unique cellular model should further provide a powerful tool for identifying the molecular mechanisms controlling normal skin development and in p63-ectodermal dysplasia human congenital pathologies.     

Embryonic stem cells and skin: from cellular model to therapeutic potential

Embryonic stem (ES) cells are pluripotent cells able to differentiate into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported their efficient ability to recapitulate ectodermal and epidermal fates and form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. Thus, ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, characteristic of embryonic skin formation. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation. We further demonstrated that p63 stimulates ectodermal cell proliferation and is necessary for epidermal commitment. We provided further evidence that this unique cellular model provides a powerful tool to identify the molecular mechanisms controlling normal skin development and to investigate human ectodermal dysplasia congenital pathologies linked to p63 (in p63-ectodermal dysplasia human congenital pathologies). Epidermal stem cell activity has been used for years to repair skin injuries, but ex vivo keratinocyte amplification has limitations and grafted skin homeostasis is not totally satisfactory. Human ES cells raise hopes that the understanding of developmental steps leading to the generation of epidermal stem cells will once be translated into therapeutic benefit. We recently demonstrated that human embryonic stem cells can give rise to a stable somatic ectodermal cell population. Its finite population doubling, normal cell cycle kinetics and the absence of teratoma formation strongly suggest that, although derived from human embryonic stem cells, these ectodermal cells represent a clinically safe somatic cell population. They could thus be particularly useful as a source for committed, homogeneous, non-tumorigenic cell populations to be employed in clinical trials for epithelial stem cell loss.     

Embryonic stem cells as a cellular model for neuroectodermal commitment and skin formation

Embryonic stem (ES) cells can be differentiated into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells were able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1(+) neural precursors to undergo specific apoptosis while inducing epidermal differentiation through DeltaNp63 gene activation. We further demonstrated that DeltaNp63 enhances ES-derived ectodermal cell proliferation and is necessary for epidermal commitment. This unique cellular model further provides a powerful tool for identifying the molecular mechanisms controlling normal skin development and for investigating p63-ectodermal dysplasia human congenital pathologies.     

Zahnanomalien bei Genodermatosen

Hair, nails and sweat glands as well as the teeth do all represent adnexal structures of the ectoderm. Hence, it is conceivable why mutations within genes controlling embryonic development may simultaneously involve the skin and the teeth. Autosomal dominant phenotypes showing a combination of cutaneous and dental anomalies include tuberous sclerosis, tricho-dento-osseous syndrome, AEC syndrome, EEC syndrome, Witkop tooth-nail syndrome, amelogenesis imperfecta with terminal onycholysis, and Böök syndrome. Autosomal recessive genodermatoses associated with dental defects are Papillon-Lefèvre syndrome, GAPO syndrome, Steijlen syndrome, and junctional epidermolysis bullosa. X-linked male-lethal disorders involving both skin and teeth include incontinentia pigmenti, Goltz syndrome, and OFD1 syndrome. Within the group of X-linked non-lethal phenotypes, Christ-Siemens-Touraine syndrome is a well-known disease, whereas ectodermal dysplasia of Zonana represents a recently delineated entity that is caused by “hypomorphic” mutations within the NEMO gene. This disorder is likewise associated with pronounced hypohidrosis, which is why the term “X-linked hypohidrotic ectodermal dysplasia” has become ambiguous and should no longer be used as a synonym for Christ-Siemens-Touraine syndrome.     

Special AT-rich binding protein-2 (SATB2) differentially affects disease-causing p63 mutant proteins

p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins.     

ΔNp63 Is Essential for Epidermal Commitment of Embryonic Stem Cells

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the ΔNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. ΔNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that ΔNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that ΔNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.     

Epidermal differentiation governs engineered skin biomechanics

Engineered skin must be mechanically strong to facilitate surgical application and prevent damage during the early stages of engraftment. However, the evolution of structural properties during culture, the relative contributions of the epidermis and dermis, and any correlation with tissue morphogenesis are not well known. These aspects are investigated by assessing the mechanical properties of engineered skin (ES) and engineered dermis (ED) during a 21-day culture period, including correlations with cellular metabolism, cellular organization and epidermal differentiation. During culture, the epidermis differentiates and begins to cornify, as evidenced by immunostaining and surface electrical capacitance. Tensile testing reveals that the ultimate tensile strength and linear stiffness increase linearly with time for ES, but are relatively unchanged for ED. ES strength correlates significantly with epidermal differentiation (p<0.001) and a composite strength model indicates that strength is largely determined by the epidermis. These data suggest that strategies to improve ES biomechanics should target the dermis. Additionally, time-dependant changes in average ES strength and percent elongation can be used to set upper bound limits on mechanical stimulation profiles to avoid tissue damage.     

Epidermal stem cell fate: what can we learn from embryonic stem cells?

Because of its constant renewal and high propensity for repair, the epidermis is, together with the gut and the hematopoietic system, a tissue of choice to explore stem cell biology. Previous research over many years has revealed the complexity of the epidermis: the heterogeneity of the stem cell compartment, with its rare, slowly cycling, multipotent, hair-follicle, “bulge” stem cells and the more restricted interfollicular, follicle-matrix, and sebaceous-gland stem cells, which in turn generate the large pool of transit-amplifying progeny. Stem cell activity has been used for some considerable time to repair skin injuries, but ex-vivo keratinocyte amplification has its limitations, and grafted skin homeostasis is not totally satisfactory. Human embryonic stem cells raise the hope that the understanding of the developmental steps leading to the generation of epidermal stem cells and the characterization of the key signaling pathways involved in skin morphogenesis (such as p63) will be translated into therapeutic benefit. Our recent results suggest the feasibility not only of identifying but also of amplifying human ES cells, early ectodermal progenitors with an intact multipotent potential that might improve the quality and functionality of grafts, provided that preclinical in vivo studies confirm our expectations from in vitro analysis. The work described here was supported by funds from the Sixth EEC Framework Program under the EPISTEM project, l’Agence Nationale pour la Recherche (ANR projets blancs), INSERM, and the Institut National Contre le Cancer (INCa).     

Derivation of keratinocyte progenitor cells and skin formation from embryonic stem cells

Despite numerous elegant transgenic mice experiments, the absence of an appropriate in vitro model system has hampered the study of the early events responsible for epidermal and dermal commitments. Embryonic stem (ES) cells are derived from the pluripotent cells of the early mouse embryo. They can be expanded infinitely in vitro while maintaining their potential to spontaneously differentiate into any cell type of the three germ layers, including epidermal cells. We recently reported that ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, which are characteristic of embryonic skin formation. Derivation of epidermal cells from murine ES cells has been successfully established by exposing the cells to precisely controlled instructive influences normally found in the body, including extracellular matrix and the morphogen BMP-4. These differentiated ES cells are able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment similar to native skin. This bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling skin development and epidermal stem cell properties.     

A novel c.1037C > G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome

Ectrodactyly – ectodermal dysplasia and cleft lip/palate (EEC) syndrome (OMIM 604292) is a rare disorder determined by mutations in the TP63 gene. Most cases of EEC syndrome are associated to mutations in the DNA binding domain (DBD) region of the p63 protein. Here we report on a three-generation Brazilian family with three individuals (mother, son and grandfather) affected by EEC syndrome, determined by a novel mutation c.1037C > G (p.Ala346Gly). The disorder in this family exhibits a broad spectrum of phenotypes: two individuals were personally examined, one presenting the complete constellation of EEC syndrome manifestations and the other presenting an intermediate phenotype; the third affected, a deceased individual not examined personally and referred to by his daughter, exhibited only the split-hand/foot malformation (SHFM). Our findings contribute to elucidate the complex phenotype-genotype correlations in EEC syndrome and other related TP63-mutation syndromes. The possibility of the mutation c.1037C > G being related both to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM is also raised by the findings here reported.     

DeltaNp63 is essential for epidermal commitment of embryonic stem cells

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.     

Analysis of large phenotypic variability of EEC and SHFM4 syndromes caused by K193E mutation of the TP63 gene

EEC (ectrodactyly, ectodermal dysplasia, clefting; OMIM 604292) is an autosomal dominant developmental disorder resulting mainly from pathogenic mutations of the DNA-binding domain (DBD) of the TP63 gene. In this study, we showed that K193E mutation in nine affected individuals of a four-generation kindred with a large degree of phenotypic variability causes four different syndromes or TP63-related disorders: EEC, Ectrodactyly-ectodermal dysplasia (EE), isolated ectodermal dysplasia, and isolated Split Hand/Foot Malformation type 4 (SHFM4). Genotype-phenotype and DBD structural modeling analysis showed that the K193-located loop L2-A is associated with R280 through hydrogen bonding interactions, while R280 mutations also often cause large phenotypic variability of EEC and SHFM4. Thus, we speculate that K193 and several other DBD mutation-associated syndromes may share similar pathogenic mechanisms, particularly in the case of the same mutation with different phenotypes. Our study and others also suggest that the phenotypic variability of EEC is attributed, at least partially, to genetic and/or epigenetic modifiers.     

Ephrin-B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB–Efnb2 signaling-deficient mice.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile α-motif (SAM) domain (residues 505–579), a region crucial to explaining the human genetic disease ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of free-dom of individual residues and discussion of the possible causes for the pathology.