Periodic Reporting for period 1 - INTEPOL (Elucidating polarity pathways in the fly and murine intestinal epithelium)
Reporting period: 2018-04-05 to 2020-04-04
Epithelial cells are organized into sheet-like structures and provide a selective and dynamic barrier between body compartments. The ability of epithelial cells to assemble into sheets is based on the coordinated polarization of groups of cells along a common vectorial axis. Epithelial cells polarize along an apical-basal axis, which involves the asymmetric segregation of cytoplasmic factors and plasma membrane domains, polarization of the cytoskeleton, and directional organelle transport, which collectively contribute to epithelial function and integrity. Loss of cell polarity is proposed to account for the malignant transformation of cancer cells, most of which arise from cells of epithelial origin.
Studies on Drosophila epithelia in various tissues have revealed a remarkably. consistent cell polarity programme that is established by evolutionarily conserved factors, including the partitioning defective (PAR), Crumbs (CRB), and Scribble (SCRIB) protein complexes, which mark distinct membrane domains to facilitate the formation of the apical-basal polarity axis. Examples of polarity mechanisms that do not rely on these key regulators have started to emerge, however, and it has become evident that different polarity pathways operate in a context dependent manner.
This proposal was aimed at elucidating polarity mechanisms in a new Drosophila epithelium, the adult gut. Peculiarly, the host laboratory has recently demonstrated that the conserved polarity regulators are entirely dispensable for epithelial polarity in the Drosophila midgut. Unlike any other cell type studied so far in Drosophila, polarity in the gut epithelium requires integrin signalling, which resembles the polarity mechanism commonly observed in mammalian cells. Hence, the gut epithelium may be a better model for mammalian epithelia than other insect epithelia, making it an attractive system in which to investigate polarity.
This proposal described the first large-scale analysis of polarity in the Drosophila adult midgut epithelium. The main aim was to conduct an in vivo forward genetic screen to identify novel epithelial polarity factors in the Drosophila midgut. As polarity in the gut is established in a different manner to all other well-characterized Drosophila epithelia, this project was expected to have a great potential to discover new polarity factors. Results from this study are expected to contribute to a better understanding of epithelial biology and will be of interest to many developmental biologists. Moreover, cell polarity is one among many biological processes that are deregulated in cancer, and polarity loss is proposed to be an event that is intricately connected to their malignant progression. By studying fundamental aspects of cell polarity establishment, this proposal has a potential to contributing to a better understanding of cancer biology.
Studies on Drosophila epithelia in various tissues have revealed a remarkably. consistent cell polarity programme that is established by evolutionarily conserved factors, including the partitioning defective (PAR), Crumbs (CRB), and Scribble (SCRIB) protein complexes, which mark distinct membrane domains to facilitate the formation of the apical-basal polarity axis. Examples of polarity mechanisms that do not rely on these key regulators have started to emerge, however, and it has become evident that different polarity pathways operate in a context dependent manner.
This proposal was aimed at elucidating polarity mechanisms in a new Drosophila epithelium, the adult gut. Peculiarly, the host laboratory has recently demonstrated that the conserved polarity regulators are entirely dispensable for epithelial polarity in the Drosophila midgut. Unlike any other cell type studied so far in Drosophila, polarity in the gut epithelium requires integrin signalling, which resembles the polarity mechanism commonly observed in mammalian cells. Hence, the gut epithelium may be a better model for mammalian epithelia than other insect epithelia, making it an attractive system in which to investigate polarity.
This proposal described the first large-scale analysis of polarity in the Drosophila adult midgut epithelium. The main aim was to conduct an in vivo forward genetic screen to identify novel epithelial polarity factors in the Drosophila midgut. As polarity in the gut is established in a different manner to all other well-characterized Drosophila epithelia, this project was expected to have a great potential to discover new polarity factors. Results from this study are expected to contribute to a better understanding of epithelial biology and will be of interest to many developmental biologists. Moreover, cell polarity is one among many biological processes that are deregulated in cancer, and polarity loss is proposed to be an event that is intricately connected to their malignant progression. By studying fundamental aspects of cell polarity establishment, this proposal has a potential to contributing to a better understanding of cancer biology.
We started the project by conducting a microscopy-based, forward genetic screen to identify novel factors that regulate polarity in the Drosophila midgut. We screened 2100 mutagenized flies, of which 58 (24%) were categorized as “integration defective” phenotype. We first conducted complementation tests of all mutants with previously characterized mutants of the integrin signalling pathway that display polarity defects, i.e. fit1 (kindlin), rhea (talin), and alpha-spectrin. These tests revealed that 6 out of the 58 integration defective mutants were homozygous lethal when crossed with either one of these three polarity mutants, strongly indicating that the gut phenotype that was observed in the screen was caused by a loss of function of these genes.
Next, we carried out a secondary validation screen of all remaining mutants by immunofluorescence microscopy. Based on the penetrance of the polarity phenotypes in the validation screen, we selected 48 mutants for whole genome sequencing to identify the gene of interest in each mutant.
Primary SNP analysis of the sequencing data revealed 0-5 unique SNPs of the mutants. Subsequent additional analysis for large structural variations resulted in the further identification of >200bp deletions in coding regions in two additional mutants. However, we have been unsuccessful in identifying unique mutations in 5 remaining mutants and hence, we are in the process of developing an additional analysis pipeline that would detect InDels that span the sizes within the 20bp-200bp range.
Complementation tests were carried out/are being carried out with deficiency lines and/or previously characterized mutants in order to determine whether the unique SNPs and structural variants identified were causative of the lethality phenotype. In parallel, we are currently validating the gut phenotype with independent mutant alleles whenever available and generating new mutant alleles by means of CRISPR technology in cases where no mutants are available.
Next, we carried out a secondary validation screen of all remaining mutants by immunofluorescence microscopy. Based on the penetrance of the polarity phenotypes in the validation screen, we selected 48 mutants for whole genome sequencing to identify the gene of interest in each mutant.
Primary SNP analysis of the sequencing data revealed 0-5 unique SNPs of the mutants. Subsequent additional analysis for large structural variations resulted in the further identification of >200bp deletions in coding regions in two additional mutants. However, we have been unsuccessful in identifying unique mutations in 5 remaining mutants and hence, we are in the process of developing an additional analysis pipeline that would detect InDels that span the sizes within the 20bp-200bp range.
Complementation tests were carried out/are being carried out with deficiency lines and/or previously characterized mutants in order to determine whether the unique SNPs and structural variants identified were causative of the lethality phenotype. In parallel, we are currently validating the gut phenotype with independent mutant alleles whenever available and generating new mutant alleles by means of CRISPR technology in cases where no mutants are available.
We are expecting to be able to validate a candidate gut polarity gene upon completion of the third validation round involving the use of independent mutant alleles. Genetic and biochemical analyses will be carried out to further characterize the molecular function of the gene in the establishment of polarity in the intestinal epithelial cells.
In addition, we would like to investigate the functional conservation of the novel polarity factor in the murine intestinal model. Although epithelial cells of intestinal organoids are clearly polarized, the mechanisms underlying this process remain elusive. We will apply a conditional, Cre-inducible gene knockout strategy to determine whether the polarity function of the gene identified in the Drosophila gut screen is conserved for the maintenance of epithelial integrity of intestinal organoids by its murine homologue.
Overall, the outcome of this project will be of relevance to the scientific community of developmental and cell biologists and to the fields of cancer and stem cell research. As noted earlier, cell polarity is one among many biological processes that are de-regulated in cancer, and polarity loss is proposed to be an event that is intricately connected to their malignant progression. Understanding the molecular nature of these interactions is important to open new research avenues for the identification of better clinical prognostic factors and for developing new therapeutic intervention strategies.
The research results have been twice disseminated within the scientific community of the University of Cambridge through internal seminars at the Gurdon Institute.
In addition, we would like to investigate the functional conservation of the novel polarity factor in the murine intestinal model. Although epithelial cells of intestinal organoids are clearly polarized, the mechanisms underlying this process remain elusive. We will apply a conditional, Cre-inducible gene knockout strategy to determine whether the polarity function of the gene identified in the Drosophila gut screen is conserved for the maintenance of epithelial integrity of intestinal organoids by its murine homologue.
Overall, the outcome of this project will be of relevance to the scientific community of developmental and cell biologists and to the fields of cancer and stem cell research. As noted earlier, cell polarity is one among many biological processes that are de-regulated in cancer, and polarity loss is proposed to be an event that is intricately connected to their malignant progression. Understanding the molecular nature of these interactions is important to open new research avenues for the identification of better clinical prognostic factors and for developing new therapeutic intervention strategies.
The research results have been twice disseminated within the scientific community of the University of Cambridge through internal seminars at the Gurdon Institute.