DEV023697 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Irx4 and Irx5 expression pattern in Xenopus. Lateral views of Xenopus embryos at different developmental stages, as indicated. (A-C) Irx4 expression is detected in the pronephros at stage 30. Inset in C show a detail of this expression. (D-F) No expression of Irx5 is observed in the pronephros at these stages.

• Supplemental Figure S2 -

  Fig. S2. Specificity of Irx morpholinos. To test the specificity of the morpholinos (MOs), we generated Xenopus laevis Irx constructs bearing a Myc epitope either at the N (MT-Irx) or at the C terminus (Irx-MT). MOs are antisense oligonucleotides that block translation efficiently only if their target sequence is within 25 bases of the translation start site. This is the case for Irx-MT mRNA but not for the MT-Irx transcripts. (A,D,G,J,M) Myc staining of stage 12 embryos injected with 0.5 ng of MT-Irx mRNAs and 10 ng of the corresponding MO. The MOs are unable to block MT-Irx translation. (B,E,H,K,N) Myc staining in stage 12 embryos injected with 0.5 ng of Irx-MT mRNAs. (C,F,I,L,O) Myc staining in stage 12 embryos injected with 0.5 ng of Irx-MT mRNAs and 10 ng of the corresponding MO. Each MO effectively blocks the translation of its corresponding Irx-MT mRNA. The different MOs can only block the translation of their corresponding genes (not shown).

• Supplemental Figure S3 -

  Fig. S3. Irx2, Irx4 and Irx5 are not required for kidney formation. All embryos were injected with specific Irx MOs and GFP mRNA in V2.2 blastomere of 8- to 16-cell stage embryos. Stage 22 embryos injected and triple labelled for Sox2 and Lim1 in purple and 12/101 in brown. (A,D,G) Control uninjected sides. (B,E,H) Morpholino-injected sides. Insets show the GFP distribution in the injected embryos. Impairment of Irx2, Irx4 or Irx5 does not affect Lim1 expression. Transverse sections of injected embryos are shown in C,F,I.

• Supplemental Figure S4 -

  Fig. S4. Transplanted Irx morphant tissue showed impaired pronephros, but not muscle or neural tissue, development. The same embryo, stained for markers for pronephros (Lim1, purple), neural tissue (Sox2, blue) and somatic muscles (12/101, brown) is shown in all panels. (A) Control side showed normal Lim1 expression (arrowhead). (B) The transplanted Irx1 and Irx3 morphant tissue, outlined in red, showed reduced Lim1 expression (arrowhead). No effect was observed in muscles or neuroectoderm. (C) GFP localization on the side containing the transplanted tissue. (D) Overlay of B and C showing the extent of the transplanted area. Inset in D shows a transversal section of this embryo. Lim1 (arrowhead), but not Sox2 or 12/101, levels are reduced in the transplanted side.

• Supplemental Figure S5 -

  Fig. S5. Irx genes are required for maintaining the expression of kidney genes. Embryos injected with a mix of Irx1 and Irx3 MOs were assayed for the expression of different kidney genes at early late neurula-early tailbud stages. (A,C,E,G) Control uninjected sides. (B,D,F,H) Depletion of Irx activity downregulates the expression of Osr2 (B), Nhf1β (D), Wnt4 (F) and Wt1 (H)