Exploring the developmental functions of fat cadherins in Drosophila and mammals

The large cadherin Fat has important functions in morphogenetic processes during development of Drosophila and mammals. Yet, its molecular partners and signaling pathways are poorly understood. Here I studied a physical link between Drosophila Fat and the Hippo pathway regulator Expanded. I found that Expanded interacts with distinct domains of Fat through its FERM domain. This finding offers a possible explanation of how Fat regulates the apical localization of Expanded and its activity in the Hippo pathway. In order to better understand Expanded signaling, I further studied its phosphorylation and determined novel Expanded interactors, including the Yorkie co-factor Mask.
The size and transmembrane nature of the mammalian Fat cadherin Fat4 had previously hindered the use of biochemical approaches to gain insight into its molecular functions. Here I developed several cell culture tools that allowed me to overcome some of these limitations and to study Fat4 localization and function. I performed proximity-dependent biotin identification (BioID) and identified an array of potential novel Fat4 interactors that will serve as a useful resource for future studies. Beside a variety of developmental defects, Fat4 mutant mice exhibit prenatal renal cysts with regions of abnormal primary cilia. Therefore, to understand if Fat4 regulates primary cilia, as has been suggested for several PCP proteins, I tested the effect of Fat4 depletion on cilia formation and maintenance in cell culture. Dramatic effects on cilia maintenance and centrosome positioning and coherence were found by knockdown with several independent siRNAs. However, CRISPR/Cas9-mediated Fat4 knockout did not confirm a requirement of Fat4 in these processes and indicated that cilia and centrosome defects were likely RNAi off-target effects. This highlights the potential pitfalls of RNAi and should be regarded as a cautionary tale.