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???displayArticle.abstract??? Podocytes are highly specialized cells in the vertebrate kidney. They participate in the formation of the size-exclusion barrier of the glomerulus/glomus and recruit mesangial and endothelial cells to form a mature glomerulus. At least six transcription factors (wt1, foxc2, hey1, tcf21, lmx1b and mafb) are known to be involved in podocyte specification, but how they interact to drive the differentiation program is unknown. The Xenopus pronephros was used as a paradigm to address this question. All six podocyte transcription factors were systematically eliminated by antisense morpholino oligomers. Changes in the expression of the podocyte transcription factors and of four selected markers of terminal differentiation (nphs1, kirrel, ptpru and nphs2) were analyzed by in situ hybridization. The data were assembled into a transcriptional regulatory network for podocyte development. Although eliminating the six transcription factors individually interfered with aspects of podocyte development, no single gene regulated the entire differentiation program. Only the combined knockdown of wt1 and foxc2 resulted in a loss of all podocyte marker gene expression. Gain-of-function studies showed that wt1 and foxc2 were sufficient to increase podocyte gene expression within the glomus proper. However, the combination of wt1, foxc2 and Notch signaling was required for ectopic expression in ventral marginal zone explants. Together, this approach demonstrates how complex interactions are required for the correct spatiotemporal execution of the podocyte gene expression program.
Fig. 1. Spatiotemporal expression of podocyte genes. (A-A′) Schematics depicting the glomus (black) in whole Xenopus embryos (A) and in transverse section at low (A′) and high (A′) magnification at stage 35. (B-J′) In situ hybridization of podocyte transcription factors wt1 (B-B′), foxc2 (C-C′), tcf21 (D-D′), mafb (E-E′) and lmx1b (F-F′) and of markers of podocyte differentiation kirrel (G-G′), nphs1 (H-H′), ptpru (I-I′) and nphs2 (J-J′) at stage 35 in whole-mounts (B-J) and transverse sections (B′-J′). (K) Temporal profile of podocyte gene expression in the glomus. en, endoderm; g, pronephric glomus; lpm, lateral plate mesoderm; no, notochord; nt, neural tube; so, somites; t, pronephric tubules.
Fig. 2. Knockdown of wt1 results in a glomus phenotype. (A) Sequences of the two pseudo-alleles of Xenopus wt1 and the location of wt1-MO1 and wt1-MO2. Mismatches between wt1-MO and the second allele of wt1 are indicated in red. The translational start site is boxed. (B) Schematic of the wt1-GFP reporter constructs. (C-E) Fluorescence of uninjected control embryos (C,D), embryos injected with the wt1-GFP reporter mRNA (C′), with wt1-MO1+2 and the wt1-GFP reporter (C′), wt1(mut)-GFP (D′) or with wt1-MO1+2 and the wt1(mut)-GFP (D′) at stage 10. The results of multiple experiments were quantified (E); the number of embryos analyzed is indicated above the bars. (F-K) Phenotype, histology and β1-Integrin immunofluorescence of wt1-MO1+2-injected embryos (G,I,K) and sibling controls (F,H,J) at stage 40 (F,G) and stage 42 (H-K). Red arrowheads indicate glomus in H.
Fig. 3. wt1 regulates podocyte gene expression. (A-I′) High magnification of representative transverse sections of whole-mount in situ hybridizations comparing the expression of podocyte genes wt1 (A,A′), tcf21 (B,B′), lmx1b (C,C′), mafb (D,D′), foxc2 (E,E′), ptpru (F,F′), nphs1 (G,G′), kirrel (H,H′) and nphs2 (I,I′) between wt1 morphants and sibling control Xenopus embryos at stage 35. (J) Wire diagram summarizing the in situ hybridization data. Arrows, positive regulation; T-bars, negative regulation; thick lines, strong effects; thin lines, weak effects. Note that none of the interactions is necessarily direct and might involve intermediary players. (K-L) In situ hybridization of nphs1 in uninjected controls (K), embryos injected with wt1-MO1 (K′) and with wt1* mRNA and wt1-MO1 (K′). The results of multiple experiments were quantified (L). Black, strong bilateral expression; white, loss of expression; gray, unilateral expression rescued by wt1* mRNA.
Fig. 4. wt1 and foxc2 are key regulators of the podocyte gene regulatory network. (A) Wire diagram combining the data for the individual knockdowns of the six podocyte transcription factors, showing all the connections. Note that none of the interactions is necessarily direct and might involve intermediary players. (B-G) Whole-mount in situ hybridization for kirrel mRNA expression in uninjected control embryos (B) and Xenopus embryos injected radially with wt1-MO1+2 alone (C) and in the presence of tcf21-MO (D), mafb-MO (E), foxc2-MO (F) or lmx1b-MO (G). (H,H′) kirrel expression in a Xenopus embryo injected unilaterally with wt1-MO1+2 and hey1-MO showing the uninjected (H) or injected (H′) side.
Fig. 5. wt1 and foxc2 are required together for expression of podocyte genes. (A-I′) High magnification of transverse sections of whole-mount in situ hybridizations comparing the expression of podocyte genes wt1 (A,A′), tcf21 (B,B′), lmx1b (C,C′), mafb (D,D′), foxc2 (E,E′), ptpru (F,F′), nphs1 (G,G′), kirrel (H,H′) and nphs2 (I,I′) between wt1-MO1 plus foxc2-MO co-injected Xenopus embryos (A′-I′) and sibling controls (A-I) at stage 35.
Fig. 7. wt1, foxc2 and NICD are sufficient to activate podocyte gene expression. (A-B′) Transverse sections of whole-mount in situ hybridizations comparing kirrel (A,A′) and nphs1 (B,B′) expression in wt1* plus foxc2* mRNA-injected Xenopus embryos (A′,B′) and uninjected sibling controls (A,B) at stage 35. Injected and uninjected sides are indicated. (C) RT-PCR analysis comparing the expression of podocyte terminal differentiation genes nphs1, kirrel, ptpru and nphs2 and the transcription factors wt1 and foxc2 in uninjected whole embryos (WE control, lane 1), uninjected ventral marginal zone explants (VMZ control, lane 2) or ventral marginal zones injected with wt1* plus foxc2* mRNA (VMZ wt1*/foxc2*, lane 3), with NICD mRNA (VMZ NICD, lane 4), or co-injected with NICD, wt1* and foxc2* mRNA (VMZ NICD/wt1*/foxc2*, lane 5). (D,D′) Whole-mount in situ hybridization of control embryos (D) and embryos co-injected with NICD, wt1* and foxc2* mRNA (D′) for nphs1 expression at stage 35. Arrow indicates an ectopic patch of nphs1 expression. (E) Quantification of ectopic nphs1 patches in embryos injected with wt1*/foxc2*, NICD, wt1*/NICD, foxc2*/NICD and wt1*/foxc2*/NICD mRNA. (F) Schematic of Xenopus glomus development. The three transcription factors, wt1, foxc2 and hey1, activate the PGRN (podocyte gene regulatory network). Subsequently, differentiated podocytes secrete Vegf to recruit endothelial cells and mesangial cells to form the differentiated glomus (see Discussion for further details).
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