Philip INGHAM / Tom CARNEY   
                       
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Philip INGHAM Research

 

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Tom CARNEY Research
     
  Tom CARNEY Research
   

The mechanisms animals employ to generate their tissues and organise constituent cells are highly conserved across species. Within an individual, they are also re-deployed in multiple organs at different times of development, and so understanding the patterning of simple tissues can be informative for more complex tissues of other species.

We wish to understand how the epidermis evolved, develops and patterns. The epidermis forms an essential barrier to external insults and infection in all organisms and thus can be considered a first line of defence. In addition, during limb development, the epidermis folds to forms an important signalling centre called the Apical Ectodermal Ridge (AER). How the epidermis forms this structure and the signalling systems it uses to converse with other cells of the developing limb are unclear.

We are using the unique properties of the zebrafish as an experimental model to analyse epidermal development and disease. The zebrafish epidermis shows high conservation with the basal layer of mammalian epidermis at the cellular and genetic levels. Crucially, the zebrafish allows both forward and reverse genetic analysis, with rapid and efficient overexpression, transgenesis, antisense and gene knockout technologies well established. Large scale mutagenesis screens also continue to yield a wealth of data on gene function in numerous developmental processes. The accessibility and transparency of zebrafish embryos allow processes to be analysed at cellular resolution and we make use of in vivo timelapse imaging, transplantation and lineage analysis to study cellular functions in different genetic contexts.

   
    Figure : Figure 1 Labelling of the different layers of the epidermis in the zebrafish fin by a novel transgenic line (R. Lee)
  1. Analysis of epidermal development
   

Through forward and reverse genetic approaches, we have identified zebrafish mutants in a conserved protease system. The serine protease inhibitor Hai1 is essential to maintaining the epithelial state of the epidermis, and its loss in mouse or zebrafish leads to abrogation of epidermal integrity as well as apoptosis, inflammation and hyperproliferation (see Figure 3).

Thus the hai1a mutant presents a lucrative resource for analysis of serine protease/inhibitor biology as well as the associated hai1a phenotypes: enhanced cell motility and invasiveness, as well as chronic inflammation. We are currently screening natural products for modulators of these traits.

We have shown that the defects in this mutant are due entirely to the Hai1a mediated inhibition of the conserved membrane bound serine protease St14, although the targets of this protease in human and fish skin are unclear. We are analyzing st14 mutants to better understand its role in epithelial physiology and homeostasis
     
   
    Figure: Purple p63+ve epidermal cell aggregates in the hai1a zebrafish mutant (white arrow, right panel) compared to the wild-type (left panel). Mutants also show elevated epidermal apoptosis (red) and a concomitant inflammatory response in this mutant (green leukocytes).

  2. Epidermal – dermal cross talk
   


The skin consists of both an epithelial epidermis and matrix-rich dermis. We aim to define how the different cells of the two layers signal and interact. We have identified the origin of the fibroblasts in the fins and are now characterising mutants which show defects in dermal-epidermal adhesion due to mutations in both adhesion components and signalling systems.

Movie 1. Timelapse movie of the tail region of a transgenic embryo tracking dermal fibroblasts emerging  from somites into the subcutaneous region of the fin.

Click the "Play" icon to view the video.

Movie 1

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  3. Origin, formation and repair of bone elements
   


We also apply genetic analysis and cell lineage tracing using the zebrafish system to understanding how bone evolved and forms. We have identified that dermal bone of the zebrafish body is mesodermally derived, suggesting osteogenesis may have been an invention of the somites, not the neural crest.

We have further characterised a mutant strain lacking functional Bmp1a metalloprotease. This mutant displays defects in the collagen matrix of both the dermis and bone, compromising integrity of the skin dermis as well as the skeleton. This mutant thus accurately models the human Brittle Bone disease, Osteogenesis Imperfecta, and we are collaborating with clinical geneticists who have identified BMP1 lesions in patients with Osteogenesis Imperfecta. Being fracture prone, the bmp1a fish mutant also complements our investigations into mechanisms of fracture repair.

   
    Figure: Top Panel: A fish scale stained for a mesodermal lineage tracer (purple) and mineralising marker (green). Bottom panel: Micro CT scan of a bmp1a mutant adult zebrafish showing defective skeletogenesis and sponta.