Epithelial bending in mammalian tooth and salivary gland morphogenesis

Genes drive and control the embryonic development of tissues and organs of the body in the way that software drives a computer. We are beginning to understand "gene programmes" written in the genome. However, we know much less about how the software drives the hardware: how do genes drive the physical shapes that we see? This question needs to be answered if we want one day to fix the hardware using biological methods. To answer this question, the extremely complicated and elaborate process of physical construction of the body (known as "morphogenesis") needs to be broken down into more easily analysed sub-processes. One of these is the bending of a sheet of cells to make a groove or pit. This is called "invagination". Invagination is medically important because it occurs in making the brain and spinal cord and its failure is a major class of birth defects (which includes, for example, spina bifida). We propose to look at something simpler but that will give answers about several different kinds of invagination. We will examine the formation of teeth and salivary glands ("ectodermal organs") in mice. Each of these organs begins with an invagination that becomes gradually deeper. We will use some new techniques to detect more precisely than ever before the outlines of cells that make up the invaginating sheet so that we can get good, engineering-style measurements of their structures. Finally, we can grow bits of the tissues in a dish and see which kinds of chemical "inhibitors" (which interfere with certain known proteins in the cell) will block which parts of the morphogenesis. This will link the cell movements and shape changes to particular proteins. Since proteins come from known genes, these studies, together with the mutant studies. will link cell shapes and movements to genes and be able to fit them in with the genetic software written in the DNA. Ultimately this kind of knowledge should make it possible to use chemical signals (software) to drive structural repairs (hardware).

Epithelial invagination is a fundamental and widespread morphogenetic motif during development of embryos and their organs. The ectodermal organs tooth and salivary gland provide good models for studying mammalian invagination because they are amenable to in vivo and ex-vivo (explant culture) analysis as well as being of interest in their own right. This project is first to map cell morphologies in these organs by genetic mosaic cell labelling during invagination. Cell shape analysis - specifically, correlation of the degree of bending with basal and apical areas and aspect ratios, cell height above the basal lamina and epithelial thickness - will be used to define which possible epithelial bending mechanisms occur when, revealing whether apical constriction, basal wedging or basal relaxation - each with its characteristic cell morphologies - predominates at each location and stage of the invaginating primordium. We will also establish the mechanism of "local stratification" (the generation of suprabasal, sometimes called inner, epithelial cells) of tooth germs and salivary glands by live imaging of explants. We have found a way of tracking cells in live explants and will use this to establish cell delamination processes (i.e. extrusion versus oriented cell division) associated with generation of the supraepithelial cells in the tooth and early salivary gland primordia. Finally, we will test the role of cytoskeletal components, cell proliferation and signalling by FGF (a candidate morphogenetic driver) using well-validated specific inhibitors on explants. By controlling the timing of inhibition and distinguishing which cytoskeletal system and which FGF pathway branches are required, we will establish molecular causes of the cell and tissue shape changes. These studies will relate tooth and salivary gland morphogenesis to morphogenesis of other invaginations in development and help establish distinctive and generalisable mechanisms.

This proposal is for fundamental basic research whose impact is hard to predict but which, on the basis of fundamental research of the past, is potentially significant. Broadly put, this project aims to clarify the physical mechanisms of tissue building at an unprecedented (i.e. cellular) level of resolution.

Beneficiaries within the commercial private sector:
As infrastructure-type knowledge building, this may be of benefit to biotechnology companies engaged in cell-based therapy development, especially in the area of tissue engineering and regeneration. Skin regeneration is already a highly developed area where the commercial sector is engaged while tooth repair and pulp restoration are active avenues in dentistry. The research may help open up improvements in these technologies with potential commercial benefit, both for medical and cosmetic purposes. The timescale for this type of output is highly unpredictable, but active contacts with stem cell companies and bioengineers (and the Business Development department of King's College London) by the PI will ensure that such avenues are pursued actively as appropriate.

Policy-makers who would benefit from this research:
As lab-based research on animal models, this project is not likely to have an impact in the governmental/regulatory domain.

Beneficiaries within the public sector, third sector or any others who might use the results to their advantage:
This research is highly visual and easily appreciated by the public. The PI is actively engaging with programmes that have track records of collaboration between the sciences and the arts with a view to establishing a better representation of the aesthetic "fine art" appreciation of biological landscapes. Collaborations between this project and visual artists and fabric designers are envisaged during the course of the project.

Beneficiaries within the wider public:
In the short term, the wider public will be able to enjoy the fascination and beauty of biological subjects and at the same time be introduced to the potential for life-enhancing and wealth-generating new technologies through biomedical research.

Research and professional skills development for staff working on the project:
All staff working on the project will gain and be encouraged in transferable skills, including IT skills (graphics software, data management), writing and communication skills - organising ideas on paper and writing clear persuasive documents, project planning and prioritisation, teaching, managing and mentoring students and others.