Alarcón P et al. (2008), A dual requirement for Iroquois genes during Xe...

XB-ART-38298

Development 2008 Oct 01;13519:3197-207. doi: 10.1242/dev.023697.

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A dual requirement for Iroquois genes during Xenopus kidney development.

Alarcón P , Rodríguez-Seguel E , Fernández-González A , Rubio R , Gómez-Skarmeta JL .

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The Iroquois (Irx) genes encode evolutionary conserved homeoproteins. We report that Xenopus genes Irx1 and Irx3 are expressed and required during different stages of Xenopus pronephros development. They are initially expressed during mid-neurulation in domains extending over most of the prospective pronephric territory. Expression onset takes place after kidney anlage specification, but before pronephric organogenesis occurs. Later, during nephron segmentation, expression becomes restricted to the intermediate tubule region of the proximal-distal axis. Loss- and gain-of-function analyses, performed with specific morpholinos and inducible wild-type and dominant-negative constructs, reveal a dual requirement for Irx1 and Irx3 during pronephros development. During neurula stages, these genes maintain the specification of the pronephric territory and define its size. This seems to occur, at least in part, through positive regulation of Bmp signalling. Subsequently, Irx genes are required for proper formation of the intermediate tubule. Finally, we find that retinoic acid signalling activates both Irx1 and Irx3 genes in the pronephros.

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Species referenced: Xenopus laevis
Genes referenced: bmp7.1 gata3 irx1 irx2 irx3 irx4 irx5 lhx1 mst1 myc osr2 pax8 ptch1 slc12a1 slc4a4 slc5a1.2 slc5a9 smad1 sox2 wnt4 wt1
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	Fig. 1. Expression patterns of Xenopus laevis Irx genes during pronephros development. Embryos are shown in lateral views (except when indicated); red arrowheads indicate the kidney territory. (A-D) Expression pattern of Irx1 at indicated developmental stages. At mid-(A) or late (B) neurula, Irx1 is detected in the dorsal pronephric territory. Insets show pronephric territory of an embryo double-stained for Lim1 or Pax8 (blue) and Irx1 (purple). Irx1 expression is restricted dorsally. During tailbud (C) or tadpole (D) stages, Irx1 expression shifts to a more ventral region that will form the intermediate tubule. Inset in C indicates an embryo double-stained for Pax8 (blue) and Irx1 (purple). Note the ventral position of Irx1 in the future intermediate tubule. Inset in D indicates a higher magnification of the pronephric Irx1 territory. Note the expression of Irx1 in the migrating ventral mesoderm (blue arrowheads). (E-H) Irx2 shows an expression pattern similar to that of Irx1, although it is not expressed in ventral migrating mesoderm. (I-L) Spatial distribution of Irx3 mRNA. (I) At mid-neurula, Irx3 mRNA is detected in a broad domain that contains most of the pronephric territory. Inset indicates pronephric territory of an embryo double-stained for Lim1 (blue) and Irx3 (purple). (J) At late neurula/early tailbud stages, Irx3 becomes restricted to the ventral pronephric territory. Inset indicates Irx3 ventral restriction in an embryo co-stained for Pax8 (blue). (K,L) From tailbud stages, Irx3 expression is detected in the intermediate tubule. Inset in K indicates embryo double-stained for Pax8 (blue) and Irx3 (purple). Inset in L indicates high magnification of the pronephric Irx3 territory. (M,N) Lateral view (M) and transverse section (N) of late neurula embryos showing Lim1 (blue) and Irx1 (purple) expression. Irx1 expression is restricted to the dorsal pronephric anlage. (O,P) Double staining for Sglt1k (green) and Irx1 (red) in tailbud (O) or tadpole (P) embryos. Insets indicate single Irx1 red channel. The Irx1 expression domain is located just distal to that of Sglt1k. (Q,R) Lateral view (Q) and transverse section (R) of late neurula embryos showing Lim1 (blue) and Irx3 (purple) expression. There is initial broad expression of Irx3 in most of the pronephric anlage (Q) and a later restriction to the ventral pronephros (R). (S,T) Double staining for Nkcc2 (green) and Irx3 (red) in tailbud (S) or tadpole (T) embryos. Insets show single Irx3 red channel. The expression domains of both genes largely overlap, but the Irx3 domain extends proximally into the proximal tubule, whereas Nkcc2 extends distally into the distal tubule.
	Fig. 2. Irx1 and Irx3 are necessary for kidney formation in Xenopus. Embryos are shown in lateral views; red arrowheads indicate the kidney territory. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. Control and injected sides of the same embryo are shown, respectively, of the same specimen. The gene examined in each condition is indicated in the right upper corner of the panels in all figures. (A-H) Embryos injected with MOIrx1 showed reduced Lim1 expression at late neurula (A,B) and downregulation of Sglt1k (C,D), Nkcc2 (E,F) and Nbcc1 (G,H) expression at tadpole stages. Inset in (A) indicates a transverse section of the embryo shown in the major panel. (I-P) Similar results were found upon MOIrx3 injection.
	Fig. 3. Irx genes are not required for the initial activation of the early kidney genes. Embryos are shown in lateral views (except A and B, which are dorsal views); red arrowheads indicate the kidney territory. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage. Xenopus embryos injected with a mix of Irx1 and Irx3 MOs and lacZ mRNA were assayed for the expression of Pax8 and Lim1 genes at early (A,B) or late (C-F) neurula stages. (A,B) Impairment of Irx gene function does not affect early expression of Pax8 (A) or Lim1 (B). (C-F) By contrast, depletion of Irx activity downregulates the expression of these genes at late neurula stage. (G,H) Tadpole embryos injected with Irx1 and Irx3 MOs and triple labelled for muscle (12/101, brown), pronephric tubules (3G8, blue) and duct (4A6, purple). The injected side (H) shows strong impairment of kidney tissue (red arrowheads) and reduced number of ventral muscle fibres (blue arrowheads) when compared with the control side (G). (I) Transverse section of the embryo shown in H. (J) The same section after treatment with propidium iodide. An increased number of fibroblast-like cells in the injected right side compared with the control left side (arrowheads).
	Fig. 4. Overexpression of Irx genes in Xenopus expands the pronephric territory. Embryos are shown in lateral views (except when indicated). Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. Neurula (A-P) or tadpole (Q-R) embryos injected with different mRNAs. Transverse sections of tadpole embryos are shown in S,T. (A-H) Overexpression of 300 pg of MT-Irx1-GR (A-D) or MT-Irx3-GR (E-H) mRNAs expands (arrowheads) ventrally the expression of Lim1 (A,B,E,F) and Pax8 (C,D,G,H) upon addition of dexamethasone (Dex) at stage 14, whereas no effect was observed in the absence of Dex (not shown). (I-P) Embryos co-injected with a mix of Irx1 and Irx3 MOs and MT-Irx3-GR mRNAs show strong downregulation of Lim1 (I,J; arrowhead) and Pax8 (K,L; arrowhead) in the absence of Dex. (M-P) This phenotype is rescued upon addition of hormone at stage 14. (Q-T) Tadpole embryos injected with MT-Irx1-GR mRNAs and Dex treated at stage 14 show enlarged kidneys (Q,R; arrowhead), as determined by staining with the 3G8 antibody. (S) Transverse section of a similarly injected embryo. (T) The same section treated with propidium iodide for nuclear staining. The control and the enlarged pronephros show the same cellular morphology.
	Fig. 5. Early Irx gene requirement for pronephros development occurs at neurula stages. Embryos are shown in lateral views. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. Late neurula-early tailbud Xenopus embryos co-injected with 500 pg of HD-E1A-GR (A-H), HD-GR (I-P) or HD-EnR-GR (I,M, inset) mRNAs and assayed for expression of Lim1. (A-H) Embryos injected with a hormone-inducible activating form of Irx (HD-E1A-GR) show expanded Lim1 only when Dex was added during mid neurula stages (arrowheads). (I-P) Embryos injected with a hormone-inducible dominant negative (HD-GR) form of Irx show reduced Lim1 expression only when Dex was added during mid neurula stages (arrowheads). The same results were found with a hormone-inducible repressing form of Irx (HD-EnR-GR) (I and M, inset and not shown).
	Fig. 6. Irx gene loss of function kidney defects are partially rescued by increased Smad1 activity in Xenopus. Embryos are shown in lateral views and red arrowheads indicate the kidney territory. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. (A,B) Injection of 500 pg of Smad1GR mRNA, upon addition of dexamethasone (Dex) at stage 14, expanded Pax8 expression. No effect was observed in the absence of hormone (not shown). (C-F) In embryos co-injected with 500 pg of Smad1GR mRNA and 4 ng of each Irx1 and Irx3 MOs Pax8 expression was downregulated (C,D) or rescued (E,F) in the absence or presence of Dex, respectively. (G,H) Depletion of Irx1 and Irx3 impaired Bmp7 expression.
	Fig. 7. Injection of different doses of Irx1 or Irx3 MOs reveal an early and a late requirement of this gene during pronephric development in Xenopus. Embryos are shown in lateral views and red arrowheads indicate the kidney territory. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. (A-D) Injection of low doses (4 ng) of Irx1 MO had little effect on Sglt1k expression (A,B) but downregulated the proximal domain of Nkcc2 (C,D). (E-L) Injection of low doses (4 ng) of two different Irx3 MOs downregulated the distal expression of Sglt1k (E,F,I,J) and the proximal domain of Nkcc2 (G,H,K,L). No effect was observed in the duct, as determined by Gata3 expression (E,F,I,J). (M-P) Injection of high doses (8 ng) of MOIrx3.2 strongly downregulated Sglt1k (M,N) and Nkcc2 (O,P). Most injected embryos were malformed, as shown in O,P. Nevertheless, a few displayed normal morphology, like that shown in M,N.
	Fig. 8. Temporal requirement for Irx function during proximal-distal pronephric patterning. Embryos are shown in lateral views and red arrowheads indicate the kidney territory. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. (A-H) Xenopus embryos injected with HD-GR mRNA. (A-D) Impairment of Irx activity during neurula stages downregulated Sglt1k, Gata3 (A,B) and Nkcc2 (C,D) expression. (E-H) Impairment of Irx activity during tailbud stages did not affect Sglt1k or Gata3 (E,F) but reduced Nkcc2 (G,H) expression. (I-P) Embryos injected with MT-Irx1-GR mRNA. (I-L) Increasing Irx1 function during neurula caused ectopic, patched Sglt1k (I,J) and enlarged Nkcc2 expression domains (K,L). No effect was observed on the duct marker Gata3 (I,J). (M-P) Increasing Irx1 function during tailbud did not affect Sglt1k or Gata3 (M,N) but enlarged Nkcc2 (O,P) expression. (Q-X) Similar results were found in embryos injected with MT-Irx3-GR mRNA.
	Fig. 9. Irx1 and Irx3 are positively regulated by retinoic acid signalling. Embryos are shown in lateral views and red arrowheads indicate the kidney territory. Embryos were injected in a single blastomere (V2.2) at the 8- to 16-cell stage and lacZ mRNA was used as linear tracer. All panels show Xenopus tadpole embryos. (A,B,E,F) Embryos injected with a dominant negative RA receptor mRNA (RAR-DN) showed impaired Irx1 (A,B) and Irx3 (E,F) expression. (C,D,G,H). Embryos injected with a constitutive RA receptor mRNA (RAR-VP16) showed a strong expansion of Irx1 (C,D) and Irx3 (G,H) expression. (I-Y) Embryos treated at different developmental stages (as indicated) with DMSO (I,M,Q,U), with the inhibitor of RA signalling pathway DEAB (J-L,R-T) or with RA (N-P,V-Y), and analyzed for Irx1 (I-P) or Irx3 (Q-Y) expression. Both genes negatively or positively responded to DEAB (J,R) or RA (N,V), respectively, only when the drugs were added at stage 12.5.
	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.
	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).
	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.
	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.
	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), Nhf1Î² (D), Wnt4 (F) and Wt1 (H)