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Gene/Clone	Species	Stage	Anatomy Item	Experimenter
lhx1	xenopus		dorsal lateral plate mesoderm

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Experiment details for lhx1

Buisson I et al. (2014) Assay

Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development.

Gene	Clone	Species	Stages	Anatomy
lhx1.L		laevis	NF stage 18	dorsal lateral plate mesoderm

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Supplementary Material. Fig. S4. Hnf1b, but notosr2 and lhx1 expression, is inhibited in the neurula KF in response to the co-injection of MoPax8 and MoPax8.B. In situ hybridization for lhx1, osr2 and hnf1b genes. Embryos were injected with MoPax8.B alone or in co-injection with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. At neurula stage 18, they were fixed, transversely bisected at the level of the pronephric area and analyzed by in situ hybridization with the indicated probes. For each embryo, the dotted line indicates the frontier between the injected side (on the left) and the uninjected side (on the right). Hnf1b expression is inhibited in the pronephric area (arrow) on the injected side.

Gene	Clone	Species	Stages	Anatomy
lhx1.L		laevis	NF stage 25	dorsal lateral plate mesoderm

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Fig. 3. Pax8 but not Pax2 loss of function inhibits tubule marker genes expression and pronephric tubule anlage formation at the early tailbud stage. (A) Whole mount in situ hybridization for tubule marker genes. Embryos were injected with MoPax8 (ae), MoPax2 (fj) or MoC (ko) at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until tailbud stage 25, fixed and analyzed by in situ hybridization with the indicated probes. For each embryo, injected (ao) and uninjected (a, o) sides are shown. Pax8 but not Pax2 depletion strongly reduces expression of lhx1, hnf1b, pax2, hrt1, and evi1 in the pronephric area. (B) Immunofluorescence with an anti-laminin-1 antibody on transverse sections of stage 25 embryos injected with MoPax8 (p, q) or MoPax2 (r, s) on one side. For each embryo, injected (ps) and uninjected (ps) sides are shown. DAPI staining is shown in blue (q, q, s, s). On uninjected sides and MoPax2 injected side, the pronephric tubule anlage is visualized as a group of somatic cells radially organized, surrounded by laminin-1 immunostaining (p, q, r, s, r, s). No such cell arrangement is observed on the MoPax8 inejcted side. (pq). Laminin-1 is only detected in the extracellular matrix between the epidermis and the somatic mesoderm, as well as between the splanchnic mesoderm and the endoderm. Scale bar: 140 m.

Gene	Clone	Species	Stages	Anatomy
lhx1.L		laevis	NF stage 25	dorsal lateral plate mesoderm

Fig. 4. Inhibition of lhx1 expression in response to MoPax8 is rescued by expressing Pax8. Embryos were first injected at the 4-cell stage in both left blastomeres with MoPax8. They were split into two groups. Embryos from one group were injected again with RNA Pax8-GR. Dexamethasone was added to the culture medium at the end of gastrulation (stage 13). Phenotype was assessed by studying lhx1 expression by in situ hybridization at tailbud stage 25. Phenotypes were classified into three categories: strong inhibition (a), weak inhibition (b) and normal expression (c). Inhibition of pronephric lhx1 expression caused by MoPax8 was partially rescued by inducing Pax8 function just after completion of gastrulation. Histogram shows quantitative results from three independent experiments (d). n=number of analyzed embryos in total.

Gene	Clone	Species	Stages	Anatomy
lhx1.L		laevis	NF stage 25	dorsal lateral plate mesoderm

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Supplementary Material. Fig. S3 Pronephric phenotype induced by co-injection of MoPax8 and MoPax8.B. Whole mount in situ hybridization for tubule and glomus marker genes. Embryos were injected with MoPax8.B alone or in co-injection with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until the tailbud stages 3335 (A), early tailbud stage 2325 (B) and analyzed by in situ hybridization with specific probes for the indicated genes. For each embryo, injected (av) and uninjected (av) sides are shown. A strong reduction of the tubule marker genes (pax2, odf3, smp30, lhx1, hnf1b, pax2, hrt1, and evi1) expression is observed in response to the injection while the glomus markers wt1 and nephrin are still expressed. Insets show higher magnification of the anterior pronephric territory.