Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels, and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and fibroblast growth factor signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together, these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair.

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers. EpCAM is a well-established marker for carcinomas of epithelial origin and a potential target for immunotherapy. In vitro analyses have implicated EpCAM in a plethora of different cellular processes, such as adhesion, motility, proliferation, differentiation, and signaling. Strikingly, depending on the context, EpCAM displayed rather opposite effects, either promoting or attenuating cell–cell adhesion versus cell migration and tissue invasion, a phenomenon described as the “double-face” of EpCAM. However, the in vivo relevance of its different effects remained largely unclear. Here, we present the first genetic analysis of EpCAM function in vivo, based on loss-of-function mutants in the zebrafish. As it is in mammals, zebrafish EpCAM is expressed in simple epithelia. Mutant embryos display defects both in epithelial morphogenesis and in epithelial integrity. Reduced epithelial morphogenesis is accompanied, and possibly caused, by an extension of apical junctional complexes and compromised basal protrusive activity. Furthermore, mutant epithelia display alterations in the relative abundance of adherence junction versus tight junction components. In addition, EpCAM tightly cooperates with E-cadherin and has a previously unrecognized trans effect on the morphogenesis and integrity of underlying cell layers. Cell differentiation and proliferation in EpCAM mutants are not, or only secondarily, affected. During later development and adulthood, EpCAM is largely dispensable, reinforcing its suitability as a target for anti-carcinoma immunotherapy with minimal side effects.

Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.

Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease. There are a large number of human genetic syndromes with limb and digit deformities. It has been shown that the genes underlying these syndromes are well conserved in evolution, and most perform the same role even in the fins of fish. One such human syndrome is Fraser Syndrome, characterized by a number of defects including fusion of the fingers (syndactyly). Data obtained with corresponding mouse mutants suggest that all of these defects are due to transient basement membrane disruptions and epithelial blistering during development. Whilst some of the Fraser Syndrome genes have been identified, others are unknown. We show that mutation of the known Fraser Syndrome genes in zebrafish generate comparable blistering defects in the fins. Importantly, we have also identified additional genes and mechanisms required for the same processes. Included in this are hemicentin1, a gene whose function had thus far only been studied in nematodes, and furinA, encoding a proprotein convertase, for which we reveal a novel role in ectodomain shedding of Fras/Frem proteins. This work thus expands our understanding, not only of Fraser Syndrome, but also of the common processes of basement membrane formation and function during fin and limb development.

The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient’s primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation’s aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.