| dc.description.abstract | Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-Hydroxy Kinases, generating a lipid signaling in eukaryotic cells, but the biological outcomes of these signaling pathways remain unclear. Herein, we exploit genetic techniques, shRNA, and an in utero electroporation approach to investigate the role of PITP-dependent inositol lipid signaling in the embryonic neural stem cell (NSC) pool. The chief discoveries of this work are 1) that two type-1 START-like PITPs, PITPNA and PITPNB are specifically required for maintaining the NSC pool (meaning that the functional role of PITPNA/PITPNB in NSCs self-renewing cannot be accounted for by a simple PtdIns-gradient model) and 2) PITPNA and PITPNB are specifically required to cooperate in maintaining neural stem cell (NSCs) self-renewal via a PtdIns-4-P and GOLPH3-dependent mechanism, a critical process for proper neuronal development.
This discovery is the culmination of a number of smaller discoveries. First, we found that the combination of a Pitpna null mouse line and Pitpnb silencing evokes a dramatic depletion of NSC reserves via accelerating asymmetric differentiation cell division in embryonic brain (see 2.4.1, 2.4.2). While a plasmid that is isogenic to the wild-type PITPNA or PITPNB rescued this NSC depletion, PITPNA mutant clones that prevent phosphatidylinositol or phosphatidylcholine binding failed to rescue the NSC depletion during in utero electroporation experiments (see 2.4.3). Moreover, neither Sec14p, (a structurally unrelated yeast PITP with similar lipid-binding/transfer activities and cellular localization) nor PITPNC1 (a mouse homolog of PITPNA/PITPNB that also shows similar PtdIns-transfer activity but with PtdOH as the secondary ligand), restored the NSC pool in Pitpna-/- embryos expressing Pitpnb shRNA (see 2.4.4). We observed that GOLPH3-dependent Golgi positioning is required for controlling NSC self-renewing and maintaining the NSC pool, and that shRNA for GOLPH3 yielded a similar NSC pool reduction phenotype to PITPNA/PITPNB-/- (see 2.4.5, 2.4.6). Furthermore, we confirmed that PITP deficiencies cause a significant reduction of the Notch intracellular domain (NICD) in embryonic NSCs (see 2.4.7). We propose a mechanism where PITPNA/PITPNB drive PtdIns-4-P-dependent recruitment of GOLPH3 to Golgi membranes so as to promote an asymmetric Golgi network where Notch receptor is maturated to control the NSC self-renewing. | en |
