Skin development and wound healing

∆Np63, which we had initially isolated as a transcriptional target of Bmp signalling blocking neural specification in the ventral ectoderm, plays an additional later role in the derivatives of the ventral ectoderm, the epidermis. During larval stages, the epidermis of the zebrafish skin is bi-layered, while it becomes multi-layered in juvenile stages, similar to the stratified organization of the skin in mammals. In all stages, ∆Np63 expression is restricted to the basal layer, in which the skin stem cells are located. Antisense-based knock down of ∆Np63 leads to severe skin lesions, most likely due to precocious differentiation of skin stem cells. This phenotype is very similar to that of human patients suffering from p63 mutations.
To identify more and novel regulators of skin development we carry out different mutagenesis screens. First, we have analyzed the collection of insertional zebrafish lines generated in the laboratory of Nancy Hopkins, MIT, Boston, USA. These mutants have the advantage that the affected genes have already been cloned. Currently, we are following up mutants in two different genes, the epithelial cell adhesion molecule Epcam, and the Hepatocyte growth factor activator inhibitor Hai1. Hai1 is a membrane-bound inhibitor of the extracellular protease Matriptase1. Its loss in mutant embryos leads to an up-regulation of Matriptase1 activity, which in turn causes a loss of epithelial integrity in the embryonic epidermis. Live imaging revealed that mutant keratinocytes loose contacts to each other and the underlying basement membrane and become mobile. We are currently investigating whether this leads to carcinogenesis by metastasizing keratinocytes. In addition, we apply chemical compound screens to identify anti-Matriptase and thereby anti-carcinogenic drugs, and genetic and biochemical approaches to identify the relevant Matriptase1 target proteins.
Second, we are carrying out large-scale screens for ENU-induced mutations, which are performed in the frame of the Integrated EU 6^th framework programme "Zebrafish models for development and human disease". Very recently, we succeeded in cloning a set of genes affected in mutants with specific blistering of the embryonic skin. Two of them turned out as homologues of genes that cause Fraser Syndrome in human, a recessive multi-syndrome and lethal disorder affecting different developmental processes as a consequence of embryonic basement membrane defects. A third gene required for this process encodes a novel putative basement membrane protein. We have established collaborations with human geneticists to study whether the corresponding human gene is affected in thus far unresolved cases of Fraser syndrome. In addition, we will analyze the novel protein on the biochemical and histological level, and will study its interaction with known Fraser syndrome proteins and other proteins identified in our screen.
Other mutations causing specific skin defects are currently cloned, applying meiotic mapping and positional cloning approaches.
During the ENU mutagenesis screens, in addition to analyzing normal skin development, we conduct assays to search for mutants with defects during larval or adult wound healing. Healing of skin wounds is both of academic and medical interest. It involves different cellular processes. First, skin cells at the edge of the wound undergo an epithelial-mesenchymal transition and migrate towards the center of the wound to cover the wounded area. Secondly, epithelial cells increase proliferation rates to rebuild the multi-layered organization of the skin. We have designed our assay so that we can distinguish mutants in either of the two processes. This should allow us to systematically identify essential in vivo regulators of the different steps of wound healing. In addition, we are testing proposed regulators of wound healing, such as Bmps, Fgfs, and Hgf (hepatocyte growth factor), using inducible transgenesis, and treatments with specific chemical blockers.