Analysis Of The Role Of Extracellular Matrix Components In Adipose Tissue Organization And Basement Membrane Assembly In Drosophila

Sheet-like basement membranes(BMs),mainly composed of Type IV Collagen,Laminin,proteoglycan Perlecan and Nidogen(Ndg),are specialized extracellular matrices surrounding tissues and organs in all animals.Adipocytes in both insects and mammals are classified as mesenchymal cells,as they lack polarity and epithelial organization.However,adipocytes associate tightly into compact adipose tissue layers.Neither the mechanisms providing this tissue-level organization nor the specific roles of extracellular matrix components in adipose biology are known.In this thesis,I characterized and performed a functional analysis of two different forms of extracellular matrix in the adipose tissue: canonical planar BMs and a novel form of matrix consisting in discrete Collagen IV Intercellular Concentrations(CIVICs),thicker in section than BMs.We found that in fat body adipocytes,CIVICs serve a structural adhesion role.Absence of Collagen IV in CIVICs,but not other components,caused inter-adipocyte gaps and impaired continuity of the adipose tissue layer.We also found that CIVICs are attached by Integrin and Syndecan receptors,promoting normal adipocyte growth and preventing autophagy through Src/PI3K/Akt signaling.With respect to the traditional planar BMs in larval fat body,I found that Collagen IV affects deposition of all other BM components,whereas Ndg is required for maintaining BM integrity.To confirm this,I isolated null mutations of Ndg using CRISPR/Cas9 and found that Ndg is not essential for organogenesis or viability,but it is required for proper maintenance of certain BMs,such as the ones in the fat body and flight muscles in a tissue-specific way.In addition,systematic structure/function analysis of the different Nidogen protein domains in vivo revealed the Rod domain of Ndg is required but not sufficient for Ndg incorporation into BMs.I also showed that Ndg acts as link bridging Laminin and Collagen IV,with Nidogen G3 domain binding to Laminin,and G1 and G2 redundantly binding Collagen IV.Altogether,these results evidence a surprising non-basement membrane role of Collagen IV in non-epithelial tissue morphogenesis,as well as tissue-specific roles of Nidogen in maintaining BM integrity.