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Fig. 1. Expression pattern of XOsr genes. Panels A, E are vegetal and panels B–D, F–P are lateral views. Insets in panels A, C, D, E, G, H, L, M and N are transverse vibratome sections through the dashed lines in the main panels. (A) Early gastrula stage (stg). XOsr1 is expressed in the involuting mesoderm and endoderm (arrowhead and arrow in inset, respectively). (B, C) During neurulation, XOsr1 mRNA is detected in the pronephric territory. (D) At tailbud, XOsr1 is expressed in the ducts (arrowhead in inset) and in the rectal diverticulum (arrow). (E–G) Expression of XOsr2 is similar, but stronger. In the prospective kidney territory, XOsr2 is detected earlier than XOsr1 (stage 11.5–12, inset in panel F; arrowhead marks the prospective kidney domain), and earlier than other pronephric markers (see inset in panel I for the expression of Xlim1 at this stage; arrowhead marks the prospective kidney domain). (H) At tailbud, XOsr2 is expressed in the tubules (arrow) and in a broad domain adjacent to the ducts (arrowhead in inset). (I, J, M, N) During neurula, expressions of XOsr2 and Xlim1 (I, M) largely overlap in the pronephric region (double in situ hybridization, J, N). (K, O) During neurula, XPax8 is also detected in the pronephric territory. (L, P) At tailbud, Xlim1 and XPax8 are expressed in the tubules and ducts. Inset in panel L show Xlim1 expression in the duct (arrowhead).
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Fig. 3. Xenopus Osr morphant embryos have severely impaired kidneys. (A–H) Lateral views of stage 25 Xenopus tropicalis embryos injected with 20 ng of MOXOsr1 (A–D) or 20 ng of MOXOsr2 (E–H) and 300 pg of LacZ mRNA to determine the injected side. Purple staining shows the expression of Xlim1 (A, B, E, F) or XPax8 (C, D, G, H), and brown staining the somitic muscles, labeled with the monoclonal antibody 12/101. The MO injected embryos show a reduced expression of the kidney markers on the injected sides (arrows in panels B, D, F and H; compare with the control sides shown in panels A, C, E and G). (I, M) Transverse section of stage 25 MOXOsr1 (I) or MOXOsr2 (M) injected embryos triple labeled for Xlim1 (pronephros, purple), muscles (brown) and Sox2 (neural tissue, cyan). Note the strong reduction of the pronephric tissue in the injected sides (arrows). (J–L and N–P) Stage 37 Xenopus tropicalis embryos injected with MOXOsr1 (J–L) or MOXOsr2 (N–P) and stained with the monoclonal antibody 3G8. Note the strong reduction of the kidney tissue in the injected sides (arrows in panels K, L, O, P). Insets are closer views. This reduction is clearly visible in transverse sections (arrow in panels L and P). (Q–T) Lateral views of stage 35 Xenopus tropicalis embryos co-injected with MOXOsr1 (Q, R) or MOXOsr2 (S–T) and Dextran-Fluorescein in the V2.2 blastomere at the 8–16 cell stage. The expression of XSGLT1K (Q, S; purple) and XNKCC2 (R, T; purple) is impaired in the injected side (Fluorescein distribution is visible in cyan). Brown staining in panels Q and T shows the somitic muscles labeled with the monoclonal antibody 12/101. Insets show the control un-injected side. (U) Target sequences for Xenopus Osr Morpholinos (MOs). In all sequences, the first methionine of the corresponding gene is underlined. Identical bases are in blue and mismatches in red. Note that the MOs for each Xenopus Osr gene have one mismatch with the corresponding Xenopus laevis and Xenopus tropicalis target sequences. In contrast, the MO against one of the paralogues has five or more mismatches with the sequence of the other gene. MOs with only one mismatch can efficiently block translation while five or more mismatches make an MO inactive.
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Fig. 5. Overexpression of Osr genes promotes ectopic kidney development. (A–L) Lateral views of stage 25 (A–F), stage 30 (G, H) or stage 37 (I–L) Xenopus embryos, or 48 hpf zebrafish embryo (T). (M–P) Transverse sections of stage 25 (M, N) or stage 37 (O, P) Xenopus embryos. (Q–T) Dorsal views of four somites (Q) or 24 hpf (R, S) zebrafish embryos. Embryos were injected with 50–100 pg of Xenopus or zebrafish Osr mRNAs. Xenopus embryos were co-injected with 300 pg of LacZ mRNA as a lineage tracer. (A–D) Embryos injected with XOsr1 mRNA showed ectopic patches of Xlim1 (A, B) or XPax8 (C, D) expression in the injected sides (arrowheads in panels B, D). In addition, many embryos have enlarged pronephros (arrows in panels B and D; compare with control sides in panels A and C). (E, F) Stage 25 Xenopus embryos injected with XOsr2 (E) or zOsr1 (F) mRNAs and doubly hybridized for Xlim1 and XPax8. The first chromogenic reaction, to detect Xlim1 expression, is shown in the main panels (cyan), and the second chromogenic reaction, to detect XPax8, in the insets (purple). Note that the same cells express ectopically both markers (arrowheads). (G, H) Embryos injected with 100 pg of Xenopus Osr1 mRNA showed ectopic patches of XSGLT1K (G, arrowheads) and XNKCC2 (H, arrowhead). Note that these embryos have gastrulated properly. (I, J) Enlarged (arrow) and ectopic (arrowhead) kidney tissue, as determined by 3G8 staining, in stage 37 Xenopus embryos injected with XOsr1 (I) or XOsr2 (J) mRNAs. Insets show magnification of ectopic renal tissue in other injected embryos. (K, L) Stage 37 Xenopus embryos co-injected with MOXOsr1 and MTXOsr1 mRNA (K) or MOXOsr2 and MTXOsr2 mRNA (L) and stained for 3G8 monoclonal antibody. Note that these MO insensitive mRNAs rescue the MO-induced kidney marker reduction (arrow) (see panels K and O in Fig. 3 for comparison) and promote ectopic renal tissue (arrowhead). (M–P) Transverse sections on stage 25 (M, N) or stage 37 (O, P) Xenopus embryos injected with XOsr1 (M, O) or XOsr2 (N, P) mRNAs. The embryos in panels M and N show a triple staining for Xlim1 (pronephros, purple), monoclonal antibody 12/101(somitic muscles, brown) and Sox2 (neural tube, magenta). The embryos in panels O, P show differentiated kidneys labeled with the monoclonal antibody 3G8. Note that the ectopic renal tissue is always found close to the neural tube (arrowhead). In addition, these embryos show a clear enlargement of the neural tube and the endogenous pronephros (arrows). (Q–T) Zebrafish embryos injected with zOsr1 (Q, R) or zOsr2 (S, T) mRNAs showing zlim1 expression at 4-somite stage (Q), zPax2.1 expression at 24 hpf (R, S) and differentiated renal structures, as determined by 3G8 monoclonal antibody staining (T). Note the enlarged pronephros (arrowheads) and the ectopic renal tissue (T, arrow). Insets in panels Q, R and T show control non-injected embryos.
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Supplementary Fig. 2. Specificity of Osr morpholinos. To test the specificity of the Xenopus and zebrafish Osr1 and Osr2 morpholinos (MOs), we generated Xenopus laevis or zebrafish Osr constructs bearing an Myc epitope either at the amino-(MT-XOsr1, MT-zOsr1, MT-XOsr2 and MT-zOsr2) or at the carboxy-terminus (XOsr1-MT, zOsr1-MT, XOsr2-MT and zOsr2-MT). MOs are antisense oligonucleotides that block translation efficiently only if their target sequence is within 25 bases from the translation start site. This is the case for Osr-MT mRNA but not for the MT-Osr transcripts. As controls, we examined if, for each species, the MO against one of the paralogues affects the translation of the other. Assays were performed in Xenopus laevis embryos. (A, D G and J) Myc staining of stage 12 embryos injected with 0.5 ng of MT-Osr mRNAs and 10 ng of the corresponding MO. The MOs are unable to block MT-Osr translation. (B, E, H and K) Myc staining in stage 12 embryos injected with 0.5 ng of Osr-MT mRNAs and 10 ng of the corresponding MO. Each MO effectively blocks the translation of its corresponding Osr-MT mRNA. Note that Xenopus MOs, which have one mismatch with the Xenopus laevis targeted sequence, are perfectly efficient in blocking translation. However, the MO against one Osr-MT mRNA, which differs in five or more bases with the target sequence, cannot impair the translation of the other gene (C, F, I and L).
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