Figure 4

Analysis of CNS1 activity in transgenesis assay in Xenopus laevis. (a) Reporter constructs used in transgenesis. Wild type CNS1 or CNS1 mutated in the Pax8 binding site (CNS1 mut) was cloned upstream of the β-globin basal promoter and the eGFP reporter gene. (b) Percentage of F0 CNS1-eGFP transgenic embryos generated by the I-SceI meganuclease method showing GFP expression detected by in situ hybridization in the developing pronephros at stages 20, 22, 25 and 28. Results corresponding to three independent experiments. (c) GFP expression detected by in situ hybridization in representative F0 transgenic embryos at stages 28 and 35 generated with reporter constructs as indicated. Transverse cryostat section (16 μm) of CNS1-eGFP transgenic embryos showing GFP reporter gene expression at the level of the proximal part of the pronephros are shown on the right. At stage 28, the dotted line delineates the mesoderm-endoderm frontier. sp splanchnic mesoderm. (d) Percentage of F0 transgenic embryos generated with the indicated reporter constructs expressing GFP in pronephros at stages 28 and 35. Results from three independents experiments. Statistical significance was determined using Fisher’s exact test. P-values are annotated on the figure. (e) Representative stage 40 F0 transgenic tadpole obtained by the REMI method with the CNS1-eGFP construct. Arrowhead indicates the fluorescent pronephros. In (b,d,e), n indicates the total number of analysed embryos.