Xenopus Bicaudal-C is required for the differentiation of the amphibian pronephros

Abstract

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which--when disrupted--results in PKD.

Figure 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. Insert in E-G are close-ups of the pronephric region highlighting the epithelium-specific expression of Bicaudal-C.

Figure 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 pMol), xBic-C-MO1 (1 pMol and 2 pMol), xBic-C-MO2 (1 pMol and 2 pMol), and xBic-C-MO1+2 (1 pMol each morpholino oligomer).

Figure 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.

Figure 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.

Figure 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.

Figure 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).