Cirio MC , Hui Z , Haldin CE , Cosentino CC , Stuckenholz C , Chen X , Hong SK , Dawid IB , Hukriede NA .

R01DK069403 NIDDK NIH HHS , R01 DK069403 NIDDK NIH HHS , R01 HD053287 NICHD NIH HHS , Intramural NIH HHS

	Figure 1. Over-expression of lhx1 induces expansion of the pronephric kidney. Embryos were injected (1xV2) with 200 pg of LL-VP16 mRNA at the 8-cell stage. (A) In situ hybridization of embryos at stage 20 for the early pronephric marker pax8. (A) Uninjected embryo. (B) Control and injected sides of the same embryo are shown. (C) Expansion of pax8 expression was observed in 83% of the embryos (n = 33). Kidney fields are highlighted with black brackets. (D) 3G8 and 4A6 whole-mount immunostaining were carried out at stages 32 and 37/38, respectively. (D, G) Uninjected embryos. (E, F, H, I) Control and injected sides of the same embryo. (F, I) Larger tubule epithelium with 3G8 was observed in 81% of the embryos (n = 32) and expanded intermediate and distal tubules with 4A6 were observed in 80% of the embryos (n = 30). (D) Insets show enlargements of 3G8 staining of the proximal tubule. (J) 3G8, 4A6 double whole-mount immunostaining of stage 42 embryos. (K) Magnification of the pronephric kidney of uninjected embryo. (M) Magnification of the pronephric kidney of injected embryo. (N) Bar graph showing the percentage of embryos injected at the different doses of LL-VP16 that showed expansion of pax8 expression. LL-VP16 mRNA 50 pg: expansion of pax8 expression was observed in 35% of the embryos (n = 26). LL-VP16 mRNA 100 pg: expansion of pax8 expression was observed in 53% of the embryos (n = 30). LL-VP16 mRNA 200 pg: expansion of pax8 expression was observed in 70% of the embryos (n = 30). LL-VP16 mRNA 300 pg: expansion of pax8 expression was observed in 93% of the embryos (n = 27).
	Figure 2. Inducible activation of Lhx1, defines a temporal window of kidney field expansion.Embryos were injected (1xV2) with 200 pg of LL-VP16-GR mRNA at the 8-cell stage. (A–L) In situ hybridization for pax8 of embryos at stage 31, followed by MZ15 whole-mount immunostaining. (A, C, E, G, I, K) Uninjected embryos. (B, D, F, H, J, L) Injected embryos. Activation of LL-VP16-GR was controlled by addition of dexamethasone (Dex) at specified stages. Dex was added to uninjected and injected embryos at: (A, B) stage 10; (C, D) stage 12.5; (E, F) stage 15; (G, H) stage 18; (I, J) stage 21; (K, L) stage 24. (M) Bar graph with the percentage of injected embryos that showed expansion after Dex treatment at different stages. Expansion of pax8 expression was observed in 53% of the embryos for stage 10 (n = 36), 56% for stage 12.5 (n = 32), 84% for stage 15 (n = 32), 79% for stage 18 (n = 34), 40% for stage 21 (n = 45) and 24% for stage 24 (n = 21).
	Figure 3. Ectopic lhx1 expression causes a fate transformation event.Embryos were injected (1xV2) with 200 pg of LL-VP16 mRNA at the 8-cell stage. Embryos were treated with hydroxyurea and aphidicolin (HUA) at stage 10.5/11. (A–D) In situ hybridization of embryos at stage 20 for pax8. (A) Uninjected embryo. (B) Uninjected embryo treated with HUA. (C) Injected embryo. Expansion of pax8 expression was observed in 91% of the embryos (n = 35). (D) Injected embryo treated with HUA. Expansion of pax8 expression was observed in 82% of the embryos (n = 33). (E–H) Whole-mount immunostaining analysis of proliferation with anti-phospho-Histone H3 antibody, αPH3. (E) Uninjected embryo. (F) Uninjected embryo treated with HUA. (G) Injected embryo. (H) Injected embryo treated with HUA. (I–L) In situ hybridization of embryos for the paraxial mesoderm marker myoD. (I, J) Uninjected and injected embryos at stage 20. Reduced myoD expression was observed in 63% of the embryos (n = 51). (K, L) Control and injected sides of the same embryo at stage 26. Reduced myoD expression was observed in 87% of the embryos (n = 30). Anterior somites are highlighted with black brackets. (M–P) In situ hybridization of embryos for the lateral plate mesoderm marker scl. (M, N) Uninjected and injected embryos at stage 20 (n = 45). Midline of the embryos is marked with a dotted line with anterior to the left. (O, P) Control and injected sides of the same embryo at stage 26 (n = 43). The injected side of the embryos is marked with an asterisk.
	Figure 4. Depletion of lhx1 results in a loss of the pronephric kidney.Embryos were injected (1xV2) with 300 pg of ctrl-AS or lhx1-AS at the 8-cell stage. (A, D, G, J) Uninjected embryos. (B, E, H, K) Embryos injected with ctrl-AS. (C, F, I, L) Embryos injected with lhx1-AS. (A–C) In situ hybridization of embryos at stage 20 for the pronephric marker pax8. (C) Reduced or absent pax8 expression was observed in 47% and 53% of the embryos, respectively (n = 34). (D–F) 3G8 whole-mount immunostaining was carried out at stage 32. (E) Reduced 3G8 staining was observed in 5% of the embryos (n = 41). (F) Reduced 3G8 staining was observed in 76% of the embryos (n = 34). Insets show enlargement of 3G8 staining of the proximal tubule. (G–I) 4A6 whole-mount immunostaining was carried out at stage 37/38. (H) Reduced 4A6 staining was observed in 9% of the embryos (n = 23). (I) Reduced 4A6 staining was observed in 91% of the embryos (n = 34). (J–L) In situ hybridization of embryos at stage 39 for β1-NaK-ATPase. (L) Reduced β1-NaK-ATPase expression was observed in 72% of the embryos (n = 29). (M) Bar graph with the percentage embryos injected with the different doses of lhx1-AS that showed presence, reduction or absence of pax8 expression at stage 20. Lhx1-AS 100 pg: reduced pax8 expression was observed in 89%, absence in 4% and presence in 7% of the embryos (n = 28). Lhx1-AS 200 pg: reduced pax8 expression was observed in 93% and presence in 7% of the embryos (n = 27). Lhx1-AS 300 pg: reduced of pax8 expression was observed in 47% and absent in 53% of the embryos (n = 34). Lhx1-AS 400 pg: reduced pax8 expression was observed in 50% and absent in 50% of the embryos (n = 42). Lhx1-AS 300 pg+zflhx1 mRNA 25 pg: presence of pax8 expression was observed in 51% of the embryos (n = 41).
	Figure 6. Absence of lhx1 affects all the domains of the pronephric kidney.Embryos were injected (1xV2) with 300 pg of lhx1-AS at the 8-cell stage. (A–O) In situ hybridization of embryos at stage 32. (A–C) In situ hybridization for follistatin (fst). (A) Uninjected embryo. (B, C) Control and injected sides of the same embryo are shown. (C) Reduced fst expression was observed in 57% of the embryos (n = 44). (D–F) In situ hybridization for pax2. (D) Uninjected embryo. (E, F) Control and injected sides of the same embryo are shown. (F) Reduced pax2 expression was observed in 86% of the embryos (n = 32). (G–I) In situ hybridization for hoxb7. (G) Uninjected embryo. (H, I) Control and injected sides of the same embryo are shown. (I) Reduced hoxb7 expression was observed in 92% of the embryos (n = 38). (J–L) In situ hybridization for laminin-β1. (J) Uninjected embryo. (K, L) Control and injected sides of the same embryo are shown. (L) Reduced laminin-β1 expression was observed in 94% of the embryos (n = 32). (M–O) In situ hybridization for neuropilin1 (nrp1). (M) Uninjected embryo. (N, O) Control and injected sides of the same embryo are shown. (O) Reduced nrp1 expression was observed in 90% of the embryos (n = 40). (P–U) In situ hybridization of embryos at stage 24 for wt1. (P, Q) Uninjected embryo. (R–U) Control and injected sides of the same embryo are shown. (T, U) Reduced wt1 expression was observed in 89% of the embryos (n = 37). (Q, S, U) Transverse sections of embryos in P and R/T, respectively. Arrows indicate plane of section in appropriate panels.
	Fig S1. Lhx1 and pax8 expression in Xenopus embryos. Expression of lhx1 (A) and pax8 (F) was visualized by in situ hybridization. The intermediate mesoderm (S12.5, S15) (A, B, F, G) and subsequent kidney field (S19.5, S21, S32) (C, H) are highlighted with black brackets.
	Fig S2. Dose-dependent expansion of the kidney field induced by LL-CA. Embryos were injected (1xV2) with different doses of LL-CA mRNA at the 8-cell stage. (A, E, F, H, I, K, L) In situ hybridization of embryos at stage 20 for the early pronephric marker pax8. (D, G, J, M) Visualization of the injected side (asterisk) by the presence of fluorescein dextran. (A) Uninjected embryo. (B) Control and injected (200 pg) sides of the same embryo are shown. (D) Expansion of pax8 expression was observed in 39% of the embryos (n = 31). (E) Control and injected (400 pg) sides of the same embryo are shown. (F) Expansion of pax8 expression was observed in 42% of the embryos (n = 33). (H) Control and injected (800 pg) sides of the same embryo are shown. (I) Expansion of pax8 expression was observed in 62% of the embryos (n = 34). (K) Control and injected (1200 pg) sides of the same embryo are shown. (L) Expansion of pax8 expression was observed in 82% of the embryos (n = 33). (N) Bar graph with the percentage embryos injected with the different doses of LL-CA that showed expansion of pax8 expression.
	Fig S3. Dose-dependent expansion of the kidney field induced by LL-VP16. Embryos were injected (1xV2) with different doses of LL-VP16 mRNA at the 8-cell stage. (A, E, F, H, I, K, L) In situ hybridization of embryos at stage 20 for the early pronephric marker pax8. (D, G, J, M) Visualization of the injected side (asterisk) by the presence of fluorescein dextran. (A) Uninjected embryo. (B) Control and injected (50 pg) sides of the same embryo are shown. (E) Control and injected (100 pg) sides of the same embryo are shown. (H) Control and injected (200 pg) sides of the same embryo are shown. (K) Control and injected (300 pg) sides of the same embryo are shown. (L) Expansion of pax8 expression was observed in 93% of the embryos (n = 27). Arrow indicates the misshapen kidney field.
	Fig S4. Analysis of secondary axis formation. Embryos were injected (1xV2) with 200 pg of LL-VP16-GR mRNA at the 8-cell stage. (A) In situ hybridization for pax8 of embryos at stage 31, followed by 12/101 whole-mount immunostaining. (A, C, E, G, I, K) Uninjected embryos. (B, D, F, H, J, L) Injected embryos. Activation of LL-VP16-GR was controlled by addition of dexamethasone (Dex) at specified stages. Dex was added to uninjected and injected embryos at: (A, B) stage 10; (C, D) stage 12.5; (E, F) stage 15; (G, H) stage 18; (I, J) stage 21; (K, L) stage 24.
	Fig S5. Dose-dependent reduction of the kidney field induced by lhx1 depletion. Embryos were injected (1xV2) with different doses of lhx1-AS at the 8-cell stage. (A, E, F, H, I, K, L) In situ hybridization of embryos at stage 20 for the early pronephric marker pax8. (D, G, J, M) Visualization of the injected side (asterisk) by the presence of fluorescein dextran. (A) Uninjected embryo. (B) Control and injected (100 pg) sides of the same embryo are shown. (E) Control and injected (200 pg) sides of the same embryo are shown. (H) Control and injected (300 pg) sides of the same embryo are shown. (I) Reduction of pax8 expression was observed in 47% of the embryos and absence in 53% of the embryos (n = 34). (K) Control and injected (400 pg) sides of the same embryo are shown.
	Fig S8. Expression of kidney genes identified from the microarray analysis. Whole-mount in situ hybridization of stage 32 embryos was performed. Expression was found in different domains of the pronephric kidney: proximal tubule (PT), early distal tubule (EDT), distal tubule (DT), and connecting tubule (CT). (A, B, H) Genes with expression in the PT. (C) Genes with expression in the EDT. (E) Genes with expression in the DT. (M) Gene with expression in the CT.
	Figure 5. Microarray analysis of lhx1-depletion in organ culture.Venn diagram outlining the distribution of probe sets in the three organ culture treatments. Overlap between probe sets that showed a 4-fold or greater increase in expression in AcRA treated (AcRA) vs untreated (C) explants (pink), probe sets that presented a 2-fold or greater decrease in expression on lhx1-AS/AcRA vs AcRA explants (blue) and probe sets which expression showed less than a 0.5-fold increase/decrease in injected (lhx1-AS) vs C explants were considered unaffected (green).
	Figure 1. Over-expression of lhx1 induces expansion of the pronephric kidney.Embryos were injected (1xV2) with 200 pg of LL-VP16 mRNA at the 8-cell stage. (A–C) In situ hybridization of embryos at stage 20 for the early pronephric marker pax8. (A) Uninjected embryo. (B–C) Control and injected sides of the same embryo are shown. (C) Expansion of pax8 expression was observed in 83% of the embryos (n = 33). Kidney fields are highlighted with black brackets. (D–I) 3G8 and 4A6 whole-mount immunostaining were carried out at stages 32 and 37/38, respectively. (D, G) Uninjected embryos. (E, F, H, I) Control and injected sides of the same embryo. (F, I) Larger tubule epithelium with 3G8 was observed in 81% of the embryos (n = 32) and expanded intermediate and distal tubules with 4A6 were observed in 80% of the embryos (n = 30). (D–F) Insets show enlargements of 3G8 staining of the proximal tubule. (J–M) 3G8, 4A6 double whole-mount immunostaining of stage 42 embryos. (K) Magnification of the pronephric kidney of uninjected embryo. (M) Magnification of the pronephric kidney of injected embryo. (N) Bar graph showing the percentage of embryos injected at the different doses of LL-VP16 that showed expansion of pax8 expression. LL-VP16 mRNA 50 pg: expansion of pax8 expression was observed in 35% of the embryos (n = 26). LL-VP16 mRNA 100 pg: expansion of pax8 expression was observed in 53% of the embryos (n = 30). LL-VP16 mRNA 200 pg: expansion of pax8 expression was observed in 70% of the embryos (n = 30). LL-VP16 mRNA 300 pg: expansion of pax8 expression was observed in 93% of the embryos (n = 27).

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