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. 2016 Sep;204(1):153-62.
doi: 10.1534/genetics.116.188532. Epub 2016 Jul 22.

Linking Gene Expression in the Intestine to Production of Gametes Through the Phosphate Transporter PITR-1 in Caenorhabditis Elegans

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Free PMC article

Linking Gene Expression in the Intestine to Production of Gametes Through the Phosphate Transporter PITR-1 in Caenorhabditis Elegans

Zita Balklava et al. Genetics. .
Free PMC article

Abstract

Inorganic phosphate is an essential mineral for both prokaryotic and eukaryotic cell metabolism and structure. Its uptake into the cell is mediated by membrane-bound transporters and coupled to Na(+) transport. Mammalian sodium-dependent Pi cotransporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III. Despite being discovered more than two decades ago, very little is known about requirements for NaPi-III transporters in vivo, in the context of intact animal models. Here we find that impaired function of the Caenorhabditis elegans NaPi-III transporter, pitr-1, results in decreased brood size and dramatically increased expression of vitellogenin by the worm intestine. Unexpectedly, we found that the effects of pitr-1 mutation on vitellogenin expression in the intestine could only be rescued by expression of pitr-1 in the germline, and not by expression of pitr-1 in the intestine itself. Our results indicate the existence of a signal from the germline that regulates gene expression in the intestine, perhaps linking nutrient export from the intestine to production of gametes by the germline.

Keywords: Caenorhabditis elegans; PiT; germline signaling; phosphate sensing; sodium-dependent phosphate transporter; yolk.

Figures

Figure 1
Figure 1
Overaccumulation of yolk in b1028 mutants. (A–F) YP170::GFP trafficking in adult hermaphrodites. In WT worms, YP170::GFP is efficiently endocytosed by oocytes (A). In the b1028 mutant worms, YP170::GFP accumulation in the body cavity is greatly increased (B). (C and D) Both WT and b1028 mutant worms show comparable levels of YP170::GFP in oocytes. WT worms show detectable levels of YP170::GFP in the intestine, while b1028 worms show increased accumulation of enlarged YP170::GFP positive vesicles in the intestine (E–F). (G and H) The localization of yolk receptor RME-2::GFP is unaltered in b1028 mutant oocytes. Images were taken through the middle focal plane of oocytes. Insets show enlarged top focal plane images of oocytes, demonstrating RME-2::GFP positive cortical endosomes. Bars (A and B), 100 μm; (C–F), 20 μm. (I) YP170::GFP fluorescence quantification in one- and two-cell embryos, results are expressed as arbitrary units of fluorescence intensity ± SD, asterisks indicate statistical differences (t-test, P ≤ 0.001). (J) Size measurement of WT and various rme mutant worms (n ≥ 12), results are expressed in micrometers ± SD, asterisks indicate statistical differences (t-test, P ≤ 0.001). (K) Real-time PCR analysis of vit-2 expression in WT and b1028 mutant worms. eif-3.C was used as a reference mRNA. Results are expressed as a fold change ± SD; asterisks indicate statistical differences (t-test, P ≤ 0.05).
Figure 2
Figure 2
Predicted membrane topology of C48A7.2. (A) Diagram of the C48A7.2 locus. Exons are in black; b1028 mutation is indicated with an arrowhead; ok2116 deletion is indicated with a gray box. (B) Putative topology of PITR-1 protein, based on TM domain prediction; insertion site of GFP into rescuing transgene is indicated by an arrowhead.
Figure 3
Figure 3
Sequence similarity of worm and human type III sodium-dependent phosphate transporters. Comparison of C48A7.2 with ortholog from human (GenBank NP 005406) reveals 42% identity.
Figure 4
Figure 4
pitr-1 is strongly expressed in C. elegans germline. Expression of endogenous PITR-1::GFP is indicated in (A and B) germline of young adults; (C) germline of larvae; (D) quickly degrading in embryos; (E) body-wall muscles of larvae; and (F) pharynx, head muscles, and muscle arms. Bar, 20 μm.
Figure 5
Figure 5
pitr-1 rescue. (A–C) The defect in YP170 expression was rescued by expressing the pie-1 promoter-driven PITR-1::GFP in the germline. (A) WT worms expressing pwIs88 (vit-2::rfp), (B) pitr-1(b1028) worms expressing pwIs88, (C) pitr-1(b1028) worms expressing pwIs88 and rescuing transgene pwIs676 (pie-1p::pitr-1::gfp). (D–F) Subcellular localization of PITR-1 and YP170 in pitr-1(b1028) rescued animal. Note that cell surface localization of PITR-1::GFP in these animals may have been quenched due to oxidation of the GFP(S65C) variant used in these experiments. (D) Localization pattern of PITR-1::GFP in oocytes, (E) localization of YP170::RFP in the oocytes, and (F) merged image showing that PITR-1::GFP and YP170::RFP positive structures do not colocalize. Bars (A–C), 100 μm; (D–F), 20 μm. (G) YP170::RFP fluorescence quantification in one- and two-cell embryos; results are expressed as arbitrary units of fluorescence intensity ± SD; asterisks indicate statistical difference (t-test, P ≤ 0.001). (H) Brood size count of WT, pitr-1(b1028), and transgenic worms; for each bar n = 20 ± SD; asterisks indicate statistical difference (t-test, P ≤ 0.001). Decreased brood size of pitr-1(b1028) is rescued by expressing PITR-1::GFP in the germline. (I) SDS/PAGE and WB analysis of YP170 levels in WT and the indicated mutant genotypes. Increased accumulation of yolk in pitr-1(b1028) worms is rescued by expressing PITR-1::GFP in the germline. Actin was used as a loading control. (J) SDS/PAGE and WB analysis of YP170 levels in F1 N2 worms fed either with control (L4440) or two different RNAi clones (RNAi no. 1 and RNAi no. 2) targeting pitr-1. Actin was used as a loading control.

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