371A Poster - 04. Stem cells, regeneration and tissue injury
Thursday April 07, 2:00 PM - 4:00 PM

A Screen for Amino Acid Transporters Involved in Nutrient-Dependent Reactivation of Quiescent Neuroblasts


Authors:
Erik Miao; Jonathan Day; Susan Doyle; Bharath Sunchu; Sarah Siegrist

Affiliation: University of Virginia

Keywords:
c. neural stem cells; o. stem cells

In the developing Drosophila nervous system, neural stem cells, called neuroblasts, undergo a period of quiescence at the end of embryogenesis. Neuroblasts exit from quiescence after larvae hatch and begin feeding. Dietary amino acids are required for neuroblast reactivation, however little is known regarding the exact nature of amino acids required and the cell and tissue types involved in sensing them. Insulin signaling is a key regulator of nutrient-dependent growth and metabolism in neuroblasts, and Drosophila insulin-like peptides (Dilps), produced by the insulin-producing cells (IPCs) of the brain, activate PI3-kinase signaling in neuroblasts. Recently, two L-type amino acid transporters expressed in the IPC’s have been shown to be required for release of Dilp2 in response to the amino acid leucine. Therefore, we hypothesized that amino acids directly sensed by the IPCs may play an important role in nutrient-dependent reactivation of neuroblasts from developmental quiescence.

To test this hypothesis, we used the Gal4/UAS binary expression system to disrupt transmembrane amino acid transporters in the IPCs. UAS-RNAi lines for the 26 genes in the solute carrier family that encode AA transporters were expressed under the control of an IPC-specific GAL4 driver (dilp2-GAL4), in combination with a pcnaGFP transgene to assess cell proliferation. Freshly hatched larvae were raised on a standard Bloomington diet, and after 24 hours of feeding, live larvae were removed from food, rinsed and mounted on a glass slide with a coverslip to immobilize them. Brains were imaged in whole mount through the cuticle using epifluorescence, and reactivation from quiescence scored based upon GFP expression.

After screening through 21 of the proposed RNAi lines, no significant reduction of reactivation was found with comparison to the control line (OregonR). This suggests that redundancy exists in nutrient uptake pathways, such that knocking out one AA transporter is not enough to show a significant reduction in reactivation. In the future, we plan to expand our amino acid transporter screen by using Gal4 drivers to express AA transporter RNAi lines in the fat body, glia, and neuroblasts themselves. The results of these experiments may give us valuable information on the tissues and nutrients required to reactivate neural stem cells from quiescence, and in addition may lend insight into improving human stem cell regeneration.