The Role of Notch-RFX Signaling in Ciliogenesis
Lead Research Organisation:
University College London
Department Name: Institute of Child Health
Abstract
Every organ in the body has unique functions which facilitates the body‘s ability to react to environmental stimuli. An organ is made up of cells with different characteristics which allow that organ to carry out its function. While all cells are initially derived from a single cell type, a programme of genes within a given cell can tell that cell what to become. Chemicals secreted by signal-sending cells can activate a programme of genes in neighbouring cells which receive the signal. As a result, proteins are made within the cell which interact with each other and carry out the special functions of that cell. Sensing changes in the environment is a unique cellular function that is undertaken by structures called cilia which project from the cell surface. Many diseases can result from processes which interfere with the synthesis and function of cilia. We, in the Beales laboratory aim to identify the chemical signals which instruct cells to make cilia. At the same time, we will be investigating genetic defects in a family with a rare cilia-related disease. This work has important implications for the treatment of a wide range of cilia-related diseases that include kidney failure, obesity and diabetes.
Technical Summary
The Role of Notch-RFX Signaling in Ciliogenesis
Background: Ciliopathies encompass a wide range of developmental diseases that include cystic kidney disease, left-right asymmetry, maturity onset obesity and pleiotropic syndromes including Bardet Biedl syndrome (BBS). While genetic mutations encoding components of the primary cilium have been identified in many ciliopathies, little is known about the upstream molecular pathways regulating ciliogenesis or the differentiation of ciliated cells. A conserved ciliogenic role for the family of regulatory transcription factor X (RFX) has been demonstrated in the nematode, fruitfly, zebrafish and mouse. Subsets of ciliogenic genes are regulated by Rfx and encode proteins such as the BBS proteins and those involved in intraflagellar transport (IFT). Recent work proposes that Notch signaling regulates rfx2 expression during ciliated cell fate specification in the developing zebrafish pronephros while genetic and pharmacological inhibition of Notch signalling can rescue the cystic kidney phenotype of certain ciliopathy mutants by upregulating rfx2 expression.
Objectives: To determine whether a conserved role exists for Notch-RFX signaling in specification of mammalian ciliated cell fate during organogenesis and whether certain human ciliopathies may result from defective Notch/RFX signaling.
Design: A spatiotemporal expression analysis of Rfx2 and Rfx3 protein and mRNA will be undertaken during murine nephronogenesis at E13.5 (when nephronogenesis and branching morphogenesis are established), E15.5 and E18.5 (when renal filtration is evident) and postnatal (P) day 21 (when nephronogenesis is complete). A subsequent spatiotemporal expression analysis of Notch ligands, receptors and molecular targets in relation to Rfx expression will be employed at similar developmental time-points. In order to determine the temporal requirement for Notch signalling in determining Rfx expression, I will utilize a novel inducible transgenic mouse model of
RBPJ-kappa deletion (inhibition of canonical Notch signaling) to examine Rfx expression at the same aforementioned time-points of murine nephrogenesis. Genetic sequencing for RFX mutations in a family with a unique syndrome with clinical features similar to Rfx3 null mice will also be undertaken.
Methodology: Techniques such as immunohistochemistry, immunofluorescence microscopy, in situ hybridization, inducible transgenic mouse systems, polymerase chain reaction, generation of zebrafish morphants, zebrafish therapeutics, positional cloning and gene sequencing will be employed. Advanced sequencing techniques such as customized exon arrays of the region (Nimblegen) followed by 454/Solexa sequencing may also be employed.
Scientific opportunities: Determination of a novel conserved pathway during organogenesis and disease. Advancement of knowledge pertaining to the molecular regulation of ciliogenesis
Medical opportunities: Potential therapeutic modulation of molecular pathways for a range of human ciliopathies.
Background: Ciliopathies encompass a wide range of developmental diseases that include cystic kidney disease, left-right asymmetry, maturity onset obesity and pleiotropic syndromes including Bardet Biedl syndrome (BBS). While genetic mutations encoding components of the primary cilium have been identified in many ciliopathies, little is known about the upstream molecular pathways regulating ciliogenesis or the differentiation of ciliated cells. A conserved ciliogenic role for the family of regulatory transcription factor X (RFX) has been demonstrated in the nematode, fruitfly, zebrafish and mouse. Subsets of ciliogenic genes are regulated by Rfx and encode proteins such as the BBS proteins and those involved in intraflagellar transport (IFT). Recent work proposes that Notch signaling regulates rfx2 expression during ciliated cell fate specification in the developing zebrafish pronephros while genetic and pharmacological inhibition of Notch signalling can rescue the cystic kidney phenotype of certain ciliopathy mutants by upregulating rfx2 expression.
Objectives: To determine whether a conserved role exists for Notch-RFX signaling in specification of mammalian ciliated cell fate during organogenesis and whether certain human ciliopathies may result from defective Notch/RFX signaling.
Design: A spatiotemporal expression analysis of Rfx2 and Rfx3 protein and mRNA will be undertaken during murine nephronogenesis at E13.5 (when nephronogenesis and branching morphogenesis are established), E15.5 and E18.5 (when renal filtration is evident) and postnatal (P) day 21 (when nephronogenesis is complete). A subsequent spatiotemporal expression analysis of Notch ligands, receptors and molecular targets in relation to Rfx expression will be employed at similar developmental time-points. In order to determine the temporal requirement for Notch signalling in determining Rfx expression, I will utilize a novel inducible transgenic mouse model of
RBPJ-kappa deletion (inhibition of canonical Notch signaling) to examine Rfx expression at the same aforementioned time-points of murine nephrogenesis. Genetic sequencing for RFX mutations in a family with a unique syndrome with clinical features similar to Rfx3 null mice will also be undertaken.
Methodology: Techniques such as immunohistochemistry, immunofluorescence microscopy, in situ hybridization, inducible transgenic mouse systems, polymerase chain reaction, generation of zebrafish morphants, zebrafish therapeutics, positional cloning and gene sequencing will be employed. Advanced sequencing techniques such as customized exon arrays of the region (Nimblegen) followed by 454/Solexa sequencing may also be employed.
Scientific opportunities: Determination of a novel conserved pathway during organogenesis and disease. Advancement of knowledge pertaining to the molecular regulation of ciliogenesis
Medical opportunities: Potential therapeutic modulation of molecular pathways for a range of human ciliopathies.