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Experiment details for lhx1

Alarcón P et al. (2009) Assay

A dual requirement for Iroquois genes during Xenopus kidney development.

Gene Clone Species Stages Anatomy
lhx1 xenopus NF stage 13 pronephric mesenchyme , lateral plate mesoderm
lhx1 xenopus NF stage 22 pronephric mesenchyme

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  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).

Gene Clone Species Stages Anatomy
lhx1 xenopus NF stage 15 pronephric mesenchyme , neural tube
lhx1 xenopus NF stage 17 pronephric mesenchyme , neural tube
lhx1 xenopus NF stage 20 pronephric mesenchyme , early proximal tubule
lhx1 xenopus NF stage 22 pronephric mesenchyme , early proximal tubule , neural tube

  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).

Gene Clone Species Stages Anatomy
lhx1.L laevis NF stage 19 to NF stage 20 pronephric mesenchyme
lhx1.L laevis NF stage 22 to NF stage 25 pronephric mesenchyme

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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.

Gene Clone Species Stages Anatomy
lhx1.L laevis NF stage 21 pronephric mesenchyme

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  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.

Gene Clone Species Stages Anatomy
lhx1.L laevis NF stage 22 pronephric mesenchyme

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  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.