Tran U , Pickney LM , Ozpolat BD , Wessely O .

5R21DK070671-02 NIDDK NIH HHS , R21 DK070671-01 NIDDK NIH HHS , R21 DK070671-02 NIDDK NIH HHS , R21 DK070671 NIDDK NIH HHS

	bicc1 (bicaudal C homolog 1) gene expression in Xenopus laevis embryos, NF stage 38, as assayed by in situ hybridization, ateral view, anterior left, dorsal up.
	ift88 (intraflagellar transport 88 homolog ) gene expression in Xenopus laevis embryos, NF stage 36, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	foxj1 (forkhead box J1) expression in Xenopus laevis, NF stage 35 & 36 embryo, via in situ hybridization, lateral view, anterior left, dorsal up.
	Fig. 1. Pronephros expression of xBic-C. (A–C) Whole mount in situ hybridization analysis of xBic-C at (A) stage 30 and (B) stage 38 showing expression in the pronephric tubules and duct. (C) Close up of B; arrows indicate the expression of Bicaudal-C in the three nephrostomes. (D) Schematic diagram of the amphibian pronephros. (E, F) Anterior and posterior transverse section of (B) showing xBic-C expression in the tubules and duct, respectively. (G) Double in situ hybridization with WT-1 (purple) and xBic-C (turquoise). Note that xBic-C is expressed only in the epithelial components of the pronephros, whereas WT-1 is expressed specifically in the glomus. Inserts in E–G are close-ups of the pronephric region highlighting the epithelium-specific expression of Bicaudal-C.
	Fig. 2. Inhibition of xBic-C leads to edema formation. (A) Sequence of the two pseudoalleles of Xenopus Bicaudal-C. The positions of the two antisense morpholino oligomers are indicated. (B) In vitro transcription/translation of xBic-C in the presence or absence of xBic-C-MO1. Lane 1, pCSII; lane 2 and 3, xBic-C-HA-pCSII; lane 4 and 5, xBic-C*-HA-pCSII. (C, D) Morphology of uninjected and xBic-C-MO1 + 2-injected embryo displaying large edema. (E) Quantification of edema formation at stage 43 of uninjected control embryos and Xenopus embryos injected radially at the 2–4 cell stage with xBic-C-MO2-scrambled (1 pM), xBic-C-MO1 (1 pM and 2 pM), xBic-C-MO2 (1 pM and 2 pM), and xBic-C-MO1 + 2 (1 pM each morpholino oligomer).
	Fig. 3. Bicaudal-C is not required for pronephros formation. (A–D) Histological analysis of transverse sections from uninjected and xBic-C-MO1 + 2-injected embryos at stage 45 (A, B) and stage 38 (C, D). Note the edema and the enlarged pronephric tubules in embryos lacking xBic-C. (E, F) Whole mount in situ hybridization of β1-Na/K ATPase of uninjected and xBic-C-MO1 + 2-injected embryos. (G) Fluorescence-labeled Dextran injected into the heart of xBic-C-MO1 + 2-injected Xenopus embryos is excreted via the pronephros. Arrow indicates the cloaca. en, endoderm; no, notochord; nt, neural tube; pn, pronephros; so, somite.
	Fig. 4. Expression analysis of pronephric markers. (A–H′) Whole mount in situ hybridization analysis of uninjected control and xBic-C-MO1 + 2-injected embryos at stage 38. (A, A′) Nephrin; (B, B′) xSGLT-1K; (C, C′) NKCC2; (D, D′) xNBC1; (E, E′) CAII; (F, F′) ClC-K; (G, G′) NCC; (H, H′) ROMK. (I) Schematic diagram of the pronephric marker genes used. Arrows point to the late distal pronephric tubule, arrowheads indicate the pronephric duct.
	Fig. 5. Expression analysis of transcription factors. (A–F′) Whole mount in situ hybridization analysis of uninjected control and xBic-C-MO1 + 2-injected embryos at stage 35. (A, A′) GATA-3; (B, B′) HNF1β; (C, C′) Lim-1; (D, D′) Pax-2; (E, E′) Pax-8; (F, F′) WT-1. Note that the expression of Lim-1 in the late distal tubule is lost upon microinjection of xBic-C-MO1 + 2 (indicated by arrowhead in C, C′). (G, H) Rescue of NBC1 expression in the late distal tubule in xBic-C-MO1 + 2-injected embryos with a single injection (2 ng) of xBic-C*, xBic-C-δKH and xBic-C-δSAM mRNA. (G) Transverse section showing expression of NBC1 in the early proximal tubule (ept) and the late distal tubule (ldt). Note that the expression of NBC1 in the late distal tubule could only be detected on the side injected with xBic-C* mRNA. (H) Quantification of the expression of NBC1 in the late distal tubule; black—bilateral expression; white—no expression; gray—unilateral expression rescued by co-injected mRNA. Each of the rescue experiments was performed at least three independent times and the graph depicts the cumulative numbers of all experiments.
	Fig. 6. Bicaudal-C and cilia formation. (A–D) Whole mount in situ hybridization of uninjected (A–C) or xBic-C-MO1 + 2-injected Xenopus embryos (D) at stage 36 using (A) FoxJ1, (B) Axonemal Dynein Heavy Chain 9 (ADHC9) and (C, D) polaris. (E–J′) Immunocytochemistry using anti-acetylated α-tubulin antibody (red) on uninjected control, xBic-C-MO1 + 2 and xPol-MO-injected embryos at stage 42. (E, E′, H, H′) Ciliated cells of the epidermis; (F, F′, I, I′) cilia of the nephrostomes; (G, G′, J, J′) individual primary cilia present on the cells of the pronephric duct. (K, K′) Immunocytochemistry using anti-acetylated α-tubulin (red) and anti-NBC1 (green) antibodies on uninjected control and xBic-C-MO1 + 2-injected embryos at stage 42. Images were obtained by confocal microscopy; nuclei were counterstained with DAPI (blue).

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