The Slit proteins have emerged as pivotal guidance molecules during embtyogenesis, mediating the migration of axonal growth cones and neuronal precursors through the complex terrain of the developing central nervous system (CNS). The Slits are large, modular secreted ligands that transduce their chemotactic stimuli through interactions with the Roundabout (Robo) family of transmembrane receptors. The Slit/Robo interaction has been postulated to control axonal and neuronal repulsion from a variety of different CNS territories in species as divergent as Drosophila melanogaster and Rattus norvegicus, thus ensuring correct migratory decisions are made to facilitate correct target innervation and neural connectivity within the CNS. The Slit and Robo gene families are also expressed in a broad array of tissues outside the CNS, both in the developing embryo and in the adult. This thesis documents the expression of the Slit and Robo genes during development of the murine metanephros and investigates the role of Slitz during kidney organogenesis.
The three vertebrate Slit genes are expressed in distinct, temporally dynamic patterns during metanephrogenesis, as are their receptors, Robo1 and Robo2. Importantly, the Slit and Robo expression domains are often complementary, suggesting that Slit/Robo signaling may play an essential role during kidney development. The expression of the Slit genes in spatially isolated areas of the growing embryonic kidney also hints at differential roles for these proteins during proliferation and differentiation of the metanephric primordia. By employing a wide array of techniques designed to specifically disrupt Slitz expression in kidney explant assays in vitro, the functional role of this gene was analysed during kidney development.
During the course of this investigation, Slit3, whose expression overlaps somewhat with Slitz in the developing metanephros, was also found to possess a unique subcellular localisation in cultured epithelial cells. In addition to secretion and localisation to the cell membrane, the Slit3 protein was demonstrated to undergo mitochondrial import in cell culture. This dual trafficking event was a completely novel discovery, and implies that the mechanisms of Slit3 biology may extend beyond its role as a secreted ligand.
Finally, the role of the Slit2 protein in development of the nervous system was investigated in vitro using cultured peripheral neurons. Surprisingly, Slit2 was found to promote both survival and neurite extension from three different populations of embryonic chick peripheral neurons. This discovery was the first described for a member of the Slit family, and further extends the broad repertoire of events these molecules are capable of mediating throughout development.