Macromolecular uptake In drosophila pericardial cells requires rudhira function

Abstract

This thesis reports the molecular-genetic and functional characterization of pericardial cells of Drosophila melanogaster. We describe pericardial cells in terms of their number and gene expression in post-embryonic stages. We establish several assays and demonstrate the nephrocyte function of pericardial cells in live animals as well as in live cells. We identify a novel gene rudhira as a specific marker of post-embryonic pericardial cells and show that it is required for pericardial cell function. We further demonstrate that the nephrocyte function of pericardial cells is essential for achieving normal lifespan in Drosophila. This is the first report on the genetic regulation of nephrocyte fiinction of insect pericardial cells. The vertebrate reticuloendothelial system (RES) performs an important function in eliminating physiological and non-physiological wastes too large for glomerular filtration. Different mechanisms are required to eliminate the wide variety of macromolecular waste that the RES encounters. The RES employs two broad categories of cells for thisprofessional phagocytes such as macrophages and professional pinocytes such as scavenger endothelial cells (SECs). Although macrophages are generally considered the primary effector cells of the RES, recent studies have established SECs as an equally important component in maintaining homeostasis. SECs play a fundamental role in eliminating waste by removing an array of soluble and colloidal macromolecules by receptor-mediated endocytosis. In insects RES function is performed by nephrocytes, which include hemocytes, pericardial cells and garland cells. While hemocytes are the primary phagocytes, pericardial The Drosophila heart or dorsal vessel is comprised of contractile cardiac cells flanked by non-contractile accessory pericardial cells. The embryonic origin of pericardial cells from the common dorsal mesodermal precursor pool has been described in detail. However the fate and function of these cells in the post-embryonic stages has not been investigated. Hence we began our study with a comparative analysis of embryonic and post-embryonic pericardial cells. A simple morphological comparison suggested difference in number, size, morphology and arrangement between embryonic and larval pericardial cells. Whereas 50-55 pairs of pericardial cells exist at the end of embryogenesis only 20-22 pairs are observed at the end of the larval stage. Thus pericardial cell number decreases post-embryogenesis. Embryonic pericardial cells can be classified into different subsets based on the expression of transcription factors like Odd-skipped (Odd), Even-skipped (Eve), Tinman (Tin), Seven-up (Svp). We analyzed expression of these embryonic markers in larval stages and find that Odd and Eve were co-expressed in all larval pericardial cells, whereas Tin and Svp showed no pericardial cell expression. This suggests a complex lineage relationship in larval pericardial cells. We also analyzed other markers expressed in larval stages - Pericardin (Pre), a collagen-like extracellular matrix protein is expressed in the dorsal vessel at all stages. Serpent (Srp), a GATA factor is expressed in larval but not embryonic pericardial cells. We had earlier identified a novel cytoplasmic WD40 domain protein, Rudhira (Rudh) by sequence homology with a mouse protein expressed during primitive erythropoiesis and neovascularization. We report Rudh as a specific pericardial cell marker from mid-second larval instar to adult. Rudh is the only cytoplasmic marker reported for pericardial cells. To test the nephrocyte function of Drosophila pericardial cells we tracked movement of hemolymph macromolecules into pericardial cells. Using an in vivo secreted protein (animals expressing secreted GFP) or a foreign macromolecule (ingested Silver nitrate) we demonstrate in live animals that Drosophila pericardial cells function as classical nephrocytes. For further investigation of this function at the cellular level, we developed livecell assays. Using fluorescently labeled bacteria we show that Drosophila pericardial cells are incapable of phagocytosis and hence are microphagocytes. However they take up both soluble (FITC-Dextran and Cy3-m-BSA) and colloidal (Coomasie Brilliant Blue) macromolecules. Maleylated- Bovine Serum Albumin (m-BSA), a ligand for Anion Ligand Binding Receptor (ALBR) is taken up by pericardial cells suggesting that an endogenous receptor similar to ALBR is expressed in these cells. The GTPase, Dynamin is known to regulate macromolecular uptake in many contexts. We show that while uptake of soluble macromolecules is Dynamin-dependent that of colloids occurs independent of Dynamin fimction.