The long-term objective of this research is to understand the factors controlling neurogenesis and cell differentiation in the olfactory (OS) and vomeronasal (VNS) systems. These two major nasal chemosensory systems, found in most terrestrial vertebrates, are the only sensory systems that are known to send information directly to the telencephalon, and they do so via completely separate pathways. The olfactory (OE) and vomeronasal (VNE) epithelia of both mammals and reptiles are similar in that both contain bipolar neurons that turnover during adulthood and show target-specific regeneration following section of their respective nerves. Thus, the OE and VNE of mammals and reptiles have been used successfully in studies of postnatal neurogenesis and comparisons between regeneration and development. Snakes are a valuable research resource for investigating developmental processes in the OS and VNS. Embryonic snakes can be grown in culture, facilitating experimental manipulations. OS and VNS structures are hypertrophied through the telencephalon and mediate a wide variety of snake behaviors. Ultimately, I hope to examine the consequences of cell differentiation, the OS and VNS, on neonatal and embryonic behavior.
The specific aims of this research proposal are: 1) to identify stem cells in the OS and VNS of snakes, using markers for cell cycle events, 2) to determine the fate of neuronal precursors during normal development and 3) to identify the spatial and temporal distributions of epidermal (EGF) and neuronal (NGF) growth factor receptors and their relationship to fos expression in OS and VNS structures of snakes. Using immunocytochemistry (ICC) and 3H-thymidine autoradiography (ARG), the purpose of the first aim is to establish a method for identifying reliably stem cells within OS and VNS structures. I will determine how and if the number of neuronal precursors changes during development of the receptor epithelia and telencephalic structures of the OS and VNS. By double labelling cells with cell cycle markers and 3H-thymidine ARG, correlations between cell cycle marker expression and DNA synthesis will examine the fate of cells incorporating 3H-thymidine during the course of normal development by increasing survival times after 3H-thymidine administration into embryos grown in vitro and into neonates. The state of differentiation will be assessed using ICC with antibodies and in situ hybridization (ISH) with cDNA probes to markers for neuronal development and maturation. These studies will establish the time course of neuronal differentiation in OS and VNS structures.
The third aim will determine if OS and VNS structures express EGF and NGF receptors and determine if their temporal expression correlates to periods of differentiation. The expression of the immediate-early gene, fos, will be correlated to the appearance of growth factor receptors. The results of this research plan should shed light on how cell cycle control and growth factors influence the differentiation and maturation of neuronal precursors.
