Fat-Hippo regulation of osteoblast differentiation

Bone is essential in both humans and animals. Bone enables us to move, protects our internal organs such as our heart and brain and is important for other aspects of our health such as kidney function. Bone is generated by bone-making cells called osteoblasts. Without osteoblasts, bone cannot be made. The aim of this proposal is to understand how two factors called Dchs1 and Fat4 control how osteoblasts develop. This will be a major advance because these factors are vital parts of a brand new type of cell control - this makes our application timely and exciting. We have shown that when Dchs1 and Fat4 are absent, the bones form but they do not grow properly ; they are smaller and softer. The little we know about these new Dchs1 and Fat4 factors indicates that their function is to instruct cell behaviour e.g. move in a certain direction, alter cell shape or they inhibit growth of cells and promote the formation of specialized cell types within the body. We will determine how Fat4 and Dchs1 control the osteoblast which make bone.

Defects in bone development are relatively common and can result in severe deformities such as sciolosis, the curvature of the spine, and osteoporosis, the loss of bone which occurs frequently in post-menopausal women. Any bone loss will increase the chances of bone fracture. The elderly are particularly vulnerable to falls and fractures which typically lead to increased morbidity and mortality. Similar fractures due to weaker bones are also common in many animals particularly production poultry.

The current proposal will discover new ways of controlling osteoblast behavior in order that we will be able to repair bone defects more quickly and increase bone mass by stimulating the bodies own cells to make bone or by generating more new osteoblasts using stem cell techniques. The aim is also to delay bone loss during ageing which diminish fracture incidence in both animals and humans. Both of these aims would have significant impact on a population's health and well being particularly of the aged.

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. The proposal addresses fundamental questions about how a newly identified signalling pathway controls osteoblast differentiation. It will have implications for manipulation of bone formation and bone regeneration in for example, during ageing and disease, and provide new routes for promoting osteogenesis in fracture healing as well as many orthopaedic procedures. To date just 3 papers have been published on this novel Fat4/Dchs1 pathway in vertebrates. This includes our own, published during a current BBSRC grant, which provided a significant advancement on our understanding of how this pathway controls 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 these initial findings will set the foundations in our understanding of 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.
The demonstration of 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 such as microarray analysis, bioinformatic processing of data and CT analysis, expose the young scientist to international and national collaborations which will enhance success and career progression, an experience of research at two renowned Institutions and opportunity to publish high impact publications at the forefront of the research field. This training will provide the PDRA with opportunities for career progression (e.g. a fellowship or independent position). It will allow novel use of the data obtained (e.g. from microarray analyses) to facilitate expansion into new directions in Fat4/Dchs1 signalling in remodelling/ageing. 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.