Helen McNeill, Ph.D.
Department of Developmental Biology Developmental Biology
RESEARCH ACHIEVEMENTS AND FUTURE GOALS
The overall goal of my research is to understand how tissue growth and tissue organization are coordinately regulated. We are focusing on how Fat cadherins function in Hippo pathway-regulated growth control, planar cell polarity (PCP) tissue organization and metabolism. Fat cadherins are enormous cell adhesion molecules that bind via cadherin-cadherin interactions to another large cadherin called Dachsous (Ds). The Hippo pathway is a highly conserved signaling pathway that regulates proliferation and apoptosis via control of the activity of the transcriptional co-activators Yorkie/YAP, which has been implicated in basic growth control and cancer development. We have used Drosophila as a genetically tractable organism to investigate the basic and conserved mechanisms of Fat function and the control of Hippo pathway activity. We have combined this with biochemical analysis of Fat-cadherins in both Drosophila and tissue culture models, and explored the relevance of our finding to mammalian health with mouse models of Ft cadherins. We continue to explore the highly conserved Fat/Ds pathway, and its regulation of the Hippo kinase pathway, to determine its role in normal development and how it controls tissue growth and patterning.
Now we are poised to extend our knowledge of Fat cadherins and the Hippo pathway in flies, and to explore its relevance to human disease by exploring these functions in mouse development and disease models, and by collaborating with clinicians to human development and disease. We collaborated with an international team to demonstrate that mutations in FAT4 and DCHS1 lead to Van Maldergem syndrome. Recently, we have collaborated with the laboratory of Friedhelm Hildebrandt, to show that mutations in FAT1 leads to a nephrotic syndrome. Our data on Fat4 in cystic disease has led to collaborations with scientists in the Netherlands, who are sequencing FAT pathway mutations in kidney disease patients. In addition, collaborating with Dr. Kirk Campbell, we have found that mutations in the Hippo pathway YAP, the transcriptional co-activator that leads to focal and segmental glomerulosclerosis (FSGS). Thus, our studies have shed light on genes involved in human diseases.
However, although it is now clear that disruptions of Fat cadherins and the Hippo pathway have critical roles in human diseases, it is poorly understood how to treat these diseases. For this we need to better understand the biology of Fat cadherin signaling, and how Fat and other upstream pathway components regulate the Hippo pathway. In addition, since our studies have revealed that the Hippo pathway plays critical roles in shaping the kidney and the lower urinary tract, we will explore how Hippo pathway activation regulates kidney morphogenesis.
By studying Fat cadherins and the Hippo pathway in both fly and mouse models, we will capitalize on each system's strengths. We will integrate biochemical studies with genetic analysis to extend our knowledge and test our hypotheses:
1. Use biochemical approaches to dissect Fat cadherin function. We have used classical biochemical analysis to demonstrate that Fat is processed from a 560kDa precursor to generated a cell-surface receptor composed of a 110kDa transmembrane domain and a 450kDa domain composed of 34 cadherin repeats, EGF and LaminG domains that mediates binding to Ds. We showed that binding of Fat to Ds promotes Fat phosphorylation and signaling to the Hippo pathway. We are using proteomic screening to identify Fat cadherin pathway effectors with mass spectrometry (MS) to identify interaction partners in a near physiological context. We used MS to identify proteins that bind the cytoplasmic domain of Fat4, and have excitingly found several binding partners that have the potential to explain some of the effects of loss of Fat4 in PCP and Hippo activity (for example Par3, Vangl2, PTPN14). We will extend this approach to Fat and Ds and as well as the other mammalian Fat cadherins (Fat1-3), and ligands (Dachsous1-2). Co-culture assays of cells expressing Fat4 and Dchs1 will determine how their binding affects the profile of proteins that bind the cytoplasmic domain of each protein. MS analysis of components of the Fat/Ds pathway will identify changes in post-translational modifications upon Fat-Ds binding. For prioritized interactors, we will conduct second-round proteomic studies. Interacting domains will be defined via co-immunoprecipitation experiments and direct interactions assayed with purified proteins.
2. Use Drosophila genetics to clarify the Ft signaling pathway, test the relevance of interactors found in biochemical screens, and identify new Fat pathway components. We will use Drosophila to explore the role of conserved Fat/Fat4 pathway protein interactors identified in our AP-MS experiments, as Fat pathway phenotypes are rapidly and easily analyzed in flies. We will use null alleles or transgenic RNAi to explore the function of Fat4 binding proteins. Proteins that show altered interactions dependent on Fat-Ds binding, or binding regulated posttranslational modifications will also be prioritized for in vivo analysis. In turn, protein interactors that our in vivo analysis in Drosophila indicates are involved in Hippo or PCP will be selected for second-round proteomic interaction studies. We will also conduct genetic screens in Drosophila in a sensitized background (of fat hypomorphic alleles) to identify new Fat pathway components. CRISPR will be used to mutate binding residues in Fat to dissect their contribution in vivo.
3. Understand how Ft cadherins are cleaved to regulate their activity. We showed that Ft cadherins undergo sequential cleavages to generate a receptor for Ds that functions to regulate Hippo pathway activity, and subsequently releases a cytosolic fragment that is imported into mitochondria, where it directly binds Ndufv2, a component of CI. To identify the protease involved, we will conduct a high throughput siRNA screen to identify genes essential for the mitochondrial localization of Fat/Fat4. We will tag endogenous Fat/Fat4 with GFP using CRISPR approaches. This screen should identify both the protease that cleaves the extracellular portion of Fat4, as well as the protease that releases a fragment for mitochondrial import. It has the potential to also identify other regulators of the cleavage, such as kinases or import factors.
4. Investigate how Fat functions in regulation of mitochondrial complex I (CI). We discovered that Fat binds to Ndufv2, a core component of mitochondrial CI. In the absence of Ft, there is a dramatic loss of assembled CI, suggesting that Fat functions either as an assembly or stability factor. We will use CRISPR to generate specific mutations in mitochondrial import and cleavage signals. We will collaborate with the Sicheri lab to determine the structure of Fat-mito in complex with Ndufv2. This will allow us to develop targeted mutations to probe how Fat binding to Ndufv2 promotes CI. Pulse-chase experiments will determine if loss of Fat/Fat4 affects assembly or stability of CI. We will also examine the effects of loss of the protease that cleaves Fat/Fat4 in Drosophila larvae and mammalian cell culture.
5. Determine how Ft cadherins regulate kidney development. We showed that Fat cadherins control PCP in kidney development, and that loss of Fat pathway components leads to cystic disease and defects in nephron progenitor self-renewal and epithelial-mesenchymal transitions. We will use genetic analysis to dissect the contribution of Fat1-4 and Dchs2 binding proteins to these functions in vivo. We will also use high-resolution time-lapse imaging in organ culture to explore the effects of loss of Fat4, and Fat4-pathway components on cell behavior in the developing kidney.
6. Investigate Fat and the Hippo pathway in kidney disease. We have strong collaborations with clinical scientists, who have identified mutations in the Fat/Dachsous pathway in children with congenital kidney disease (Drs Robinson, Hildebrandt and Renkeman). For a few well-validated mutations we will use CRISPR to generate synonomous mutations in mouse models, to critically test the proposal that new mutations are causal in human kidney disease, and to gain insights into which Fat pathway is disrupted in these diseases. We recently identified a novel role for the Hippo pathway in the regulation of branching morphogenesis in the mouse kidney. Using whole animal conditional and organ-culture approaches, we find that loss of NF2 or LATS or overexpression of YAP leads to growth of the collecting ducts in the absence of branching. To understand better how branching is affected, we will conduct high-resolution time-lapse imaging of the induction of branching morphogenesis. We will also assay tissue tension at junctions in the bud, and the trunk of developing branches in the mouse kidney. Cystic kidney disease is often associated with Ciliary defect. We have shown that Fat4 is localized to the primary cilium, and Fat4 mutants have cilia defects in the kidney. We find cilia defects are replicated in cultured cells in Fat4 siRNA knockdown experiments, providing a system for high-resolution analysis of Fat4 function in ciliary establishment and maintenance. Our MS analysis has identified number of Fat4 proteins implicated in cilia function, which we will explore in this system.
Education and Professional Experience
- 1988-1993 Ph.D. in Molecular and Cellular Physiology Stanford University, Stanford, California, USA
- 1981-1985 B.Sc., Biology, Ramapo College of New Jersey, Mahwah, New Jersey, USA
Research and Professional Experience
- Professor, Institute of Medical Science, University of Toronto, January 2011 – present
- Professor, Department of Molecular Genetics, University of Toronto, July 2010 – present
- Senior Investigator, Lunenfeld-Tanenbaum Research Institute (formerly, Samuel Lunenfeld Research Institute), Sinai Health System (formerly, Mount Sinai Hospital), September 2005- present
- Professor of Developmental Biology, Washington University School of Medicine, January 2018- present
Honors and Awards
- 2017- Fellow of the Royal Society of Canada
- 2016-present Canada Research Chair, Tier 1, Lunenfeld-Tanenbaum Research Institute, Toronto, Canada
- 2012-present - Editorial Board Member, eLife, UK
- 2010 - The Lloyd S.D. Folger, Award for Research Excellence
- 2010 - Faculty of 1000 Reviewer, Faculty of 1000 Ltd, UK
- 2009-2012 - Member and the Representative of Canada, North American Drosophila Board of Directors
2007-2013 - Director of Collaborative Program in Developmental Biology,
University of Toronto, Canada
- 2006-2007 - Petro Canada Young Innovator’s Award, Petro Canada, Canada
- 2007-2012 - Editorial Board Member, Developmental Dynamics, USA
- 1993-1996 - Postdoctoral Fellowship, American Cancer Society, USA
- 1991-1992 - Eloise Gerry Predoctoral Fellowship, Stanford University, USA
- 1991 - Katherine McCormick Fellowship, Stanford University, School of Medicine, USA
- 1990 - Woods Hole Summer Student Tuition Scholarship, Marine Biology Laboratories, USA
- 1985 - President’s List, Ramapo College, USA
- 1985 - Women’s Leader Award, Ramapo College, USA
- 1985 - Fellowship in Biomedical Research, New York Medical College, USA
- 1982-1985 - Dean’s List, Ramapo College, USA
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