title: Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm.
abstract: PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 91895, USA. Electronic address: marycwallingford@gmail.com.
authors.forename: Mary C
authors.initials: MC
authors.lastname: Wallingford
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 91895, USA. Electronic address: ceci@uw.edu.
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Giachelli
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics / Q000502:physiology; D004705:Endocytosis / Q000235:genetics / Q000502:physiology; D004707:Endoderm / Q000196:embryology / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007074:Immunoglobulin G / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution; D015017:Yolk Sac / Q000196:embryology* / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D008840:Microfilament Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; C493587:Slc20a1 protein, mouse; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; C058992:Tagln protein, mouse
keywords: Endocytosis; Microautophagy; Phosphate transport; PiT-1; Slc20a1; Yolk sac development