Fat-Hippo regulation of osteoblast differentiation

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The overall aim of the proposal is to elucidate the mechanisms by which a newly identified vertebrate signaling pathway, Fat4-Dchs1 controls cell differentiation and morphogenetic processes using osteoblast differentiation as a model system. In Drosophila, the Fat-PCP-Hippo pathway controls tissue growth and planar cell polarity (PCP). How this pathway functions in vertebrates is far less clear and to date only 1 transcriptional target has been identified. Our analysis has shown that there is a significant reduction in ossification of the cranial membrane bones and appendicular endochondral bones in Fat4-/- and Dchs1-/- mouse mutants. Fat4 and Dchs1 mutant osteoblasts also show a deficit in mineralisation in vitro, demonstrating that Fat4/Dchs1 are critical regulators of osteoblast development. This finding provides both an excellent model to dissect Fat4/Dchs1 signalling in vertebrates and identifies a new pathway in bone development. We will dissect how Fat4/Dchs1 regulate osteoblast differentiation using genetic rescue approaches, analysis of osteoblast differentiation and behaviour in vivo and in vitro, micro-CT analysis and microarray analysis to identify transcriptional targets. If we demonstrate a link with either Hippo or PCP, this will be the first demonstration of Fat4-Hippo signalling in mammals and/or PCP regulation of polarised osteoblast behaviour. The microarray analysis will identify transcriptional targets of Fat4 signalling in vertebrates. Therefore, our study will determine Fat4/Dchs1 signalling pathways in osteoblasts, a cell-type that has many critical functions, and will provide a model system for elucidation of Fat4/Dchs1 signalling in vertebrates. Any links we identify may also be relevant to potential Fat-Hippo signalling in other contexts such as cancer, stem cell proliferation and underlie a more generalised facet of Fat-PCP-Hippo signalling in vertebrates both during development and disease.

There are many levels at which the proposed research is likely to have significant impact. It will identify the mechanisms controlling the contribution made by a new pathway to osteoblast differentiation at the signalling level, it will have implications for manipulation of bone formation and perhaps bone regeneration in circumstances where bone mass is compromised by ageing and disease, and provide new routes for promoting osteogenesis in fracture healing as well as many orthopaedic procedures. The proposal addresses fundamental questions about how a newly identified signalling pathway controls osteoblast differentiation. To date just 3 papers have been published on the function of this novel Fat4/Dchs1 pathway in vertebrates. This includes our own, published during a current BBSRC grant, which provided a significant advance of how this pathway achieves its critical roles during many aspects of developmental biology. Based on comparison with the advances that have taken place upon the first identification of other signalling pathways, it is highly likely that these seminal papers will provide the springboard for many further publications, by both us and others; it is envisioned that this will set the foundations for our understanding how this pathway functions, not only in development but post-natally and in disease. We have now created a new team with distinct expertise to analyse a novel and unexpected finding, namely an osteogenic defect in the Dchs1 and Fat4 mutants. We build on and unite existing collaborations of the two groups (PFW and AP), incorporating collaborations with 3 other research groups, that provide us with crucial unpublished reagents and tools to analyse bone development and morphogenesis. The proposal will provide new knowledge which advances the field and enhances research capacity by combining distinct expertise and resources keeping the UK at the forefront of this new and emerging research area.

Defning the mechanism of Fat4/Dchs1 regulation of osteoblast differentiation will be a significant contribution and likely seminal to the field. If we demonstrate a link with the Hippo pathway, this will be the first genetic determination of Fat4-Hippo signalling in vertebrates. If we demonstrate Fat4/Dchs1 regulation of polarised cell behaviour this will be the first link between osteoblast development and PCP. We will also identify new transcriptional targets setting the foundations for analysis of Fat4/Dchs1 signalling during organ development, cell differentiation and disease. Our focus on bone, given the key roles it serves in many aspects of human and animal physiology, will ensure manuscripts gain high prominence amongst developmental, cell biologists, veterinarians and medics.

The proposal will train the post-doctoral research assistant (PDRA) in techniques at the forefront of the field, including microarrays, bioinformatics and CT imaging, expose this scientist to international and national collaborations to enhance success; experience at two renown Institutions and opportunity to publish high impact publications at the forefront of the field. This training will provide the PDRA with opportunities for career progression (e.g. fellowship/independent position). It will allow novel use of the data obtained (e.g. microarray analyses) to facilitate expansion into new directions. These positions are not restricted to the developmental biology field but could be expanded for example, to tissue engineering or cell biology depending on the research interests developed by the PDRA. The skill set would also be applicable to non-academic research.

The proposal has also has an economic and societal impact. It will increase our understanding of bone defects and ultimately, disease processes e.g. osteoporosis, fracture repair mechanisms and stem cell/ tissue engineering approaches that will be used to restore unhealthy bone to normal function and more rapidly repair damaged, otherwise, healthy bone.