Cartry J et al. (2006), Retinoic acid signalling is required for specif...

XB-ART-34427

Dev Biol 2006 Nov 01;2991:35-51. doi: 10.1016/j.ydbio.2006.06.047.

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Retinoic acid signalling is required for specification of pronephric cell fate.

Cartry J , Nichane M , Ribes V , Colas A , Riou JF , Pieler T , Dollé P , Bellefroid EJ , Umbhauer M .

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The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 atr cyp26a1 gal.2 lhx1 odc1 pax2 pax8 rab40b rara rarg rgn wt1

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	Fig. 1. Effects of citral and BMS-453 on pronephros marker genes. Embryos were incubated during gastrulation with citral (B, E) or BMS-453 (C, F) or cultured in control medium (A, D). At the late tailbud stage 33, XSMP-30 (A–C) and XPax-2 (D–F) expression was analysed by whole mount in situ hybridization. Panels D′, E′, F′ are high magnification of the pronephric tubules area of panels D, E, F, respectively. XSMP-30 expression is severely reduced in embryos treated with the chemical inhibitors (B, C). XPax-2 expression in connective tubules and nephrostomes (arrowheads in panel D′) is lost in treated embryos (E′, F′) while expression in the duct is not affected.
	Fig. 2. Ectopic expression of XCyp26 or dnRAR inhibits pronephros formation and expression of pronephros marker genes. Four-cell stage embryos were injected with XCyp26 (D–F) or dnRAR (G–I) encoding mRNA together with LacZ mRNA into the four blastomeres (A–I) or the two blastomeres fated to give rise to the left side (J–L). In the later case, the right side of the embryo is used as an internal control. A–I: In situ hybridization (purple staining) for XPax-2 (A, D, G) XSMP-30 (B, E, H) and XWT-1 (C, F, I). X-gal staining is revealed in red (D–I). In control embryos, XPax-2 is expressed in the tubules and the duct while XSMP-30 is restricted to the tubules and XWT-1 expressed in the glomus. No signal for these genes in either of theses structures was detected. J–L: Transverse histological section of tadpole stage 38. Several pronephric tubules (red circle) are clearly visible on the control side (K, and high magnification in panel J) whereas none is observed on the injected side (K, and high magnification in panel L).
	Fig. 4. Increased RA signalling expands the size of the pronephros. Four-cell stage embryos were co-injected with β-galactosidase encoding mRNA and XRaldh2 (A–D) or VP16-xRARα1 (E, F) mRNAs into the two blastomeres fated to give rise to the left side (right column). Embryos in panels A–D were treated 30 min with ATR. XPax-8 (A, B, E, F) and XSMP-30 expression (C, D) was analysed by in situ hybridization at the early tailbud and tadpole stages respectively (purple staining). X-gal staining is revealed in red (B, D) or light blue (F). Panels A′, B′, C′, D′ are pronephros area enlargements of panels A, B, C, D, respectively. XPax-8 and XSMP-30 expression in the pronephros primordium is enlarged on the injected side compare to the uninjected one.
	Fig. 6. RA signalling is required in pronephric precursors. A–C: XSMP-30 expression in embryos injected in the two dorsal blastomeres at the 4-cell stage with XCyp26 mRNA and β-galactosidase encoding mRNA as a lineage tracer. X-gal staining is revealed in blue and XSMP-30 expression in purple. When XCyp26 overexpression is restricted to the dorsal part of the embryos, XSMP-30 expression is not affected (B) whereas it is severely reduced when XCyp26 expression encompasses the pronephric primordium (arrowhead in panel C). An uninjected embryo is shown in panel A. D: Experimental design for the transplantation assay. E–G: Grafted embryo cultured to the tailbud stage 28. Lateral view of the anterior region showing localization of RLDx (red, F) and GFP/dnRAR (green, G) expressing cells. H–M: Grafted embryo cultured to the tadpole stage 38. Lateral view of the anterior region showing localization of RLDx (red, I) and GFP/dnRAR (green, J). Panels K, L and M present the same transverse histological section in the pronephric tubules area showing dapi staining (K), RLDx (L) and GFP (M) localization. Tubules contain RLDx but not GFP/dnRAR cells. In panels E–J, anterior is on the right and dorsal up.
	Fig. 7. RA signalling is required during gastrulation for pronephros formation. Embryos were treated (B–F) or not (A) with BMS-453 during the time windows indicated in the cartoon, cultured until the late tailbud stage analysed for XPax-2 expression by whole mount in situ hybridization. Panels A′, B′, C′, D′, E′, F′ are tubules area enlargements of panels A, B, C, D, E, F respectively. The effect of BMS-453 on XPax-2 expression in the pronephric tubules is much more severe if embryos are treated during gastrulation (B, C, D) rather than after gastrulation is completed (E, F).
	Fig. 8. RAR-α is expressed in pronephric mesoderm. A: RT-PCR analysis of RAR-β expression during Xenopus embryogenesis. Embryonic stages are indicated according to Nieuwkoop and Faber, 1967. ODC is used as an RNA loading control. B–L: In situ hybridization on gastrula stage 11 (B–G) or stage 12 (H–L) for XRaldh2 (B, E, J), RAR-γ (C, F, K), RAR-α (D, G, L), XPax-8 (H) and Xlim-1 (I). In situ hybridization was performed on hemisectioned embryos except in panels B–D. In panels H–L, embryos were cut transversally at the level of the pronephric territory, dorsal is up. RAR-α and XRaldh2 are expressed in the mesodermal layer during gastrulation and their expression domains overlap with Xlim-1 and XPax-8 expression in the pronephric territory at the late gastrula stage.
	Fig. 9. Ectopic XCyp26 mRNA affects early expression of XPax-8 and Xlim-1. Four-cell stage embryos were injected with XCyp26 and β-galactosidase encoding mRNAs into the two blastomeres fated to give rise to the left side. At stage 13, Xlim-1 and XPax-8 expression was analysed by combined in situ hybridization (purple staining) and β-galactosidase activity (red staining). Xlim-1 (A, B) and XPax-8 (C, D) expression in the area fated to give pronephros is severely reduced on the injected side. Note that expression of XPax-8 in the otic placode is also reduced. Dorsal is up, anterior is to the right for the control sides and to the left for the injected sides.
	Fig. 10. Xlim-1 and XPax-8 expression at the late gastrula stage depends on RA signalling and RA induces Xlim-1 expression in absence of protein synthesis. A–H: In situ hybridization for Xlim-1 (A–D) and XPax-8 (E–H) in control embryos (A, B, E, F) or embryos incubated with BMS-453 (C, D, G, H) for 6 h since the gastrula stage 10.5 (C, G) or the neurula stage 17 (D, H). Xlim-1 and XPax-8 staining in the pronephric lineage is reduced when the inhibitor treatment is applied at gastrula stage but not at neurula stage. I–P: Embryos incubated with RA (1 μM) at the late gastrula stage 12.5 and analysed for Xlim-1 (I–N) or XPax-8 (O, P) expression by in situ hybridization 20, 40, 60 or 120 min after the onset of the treatment. Q–T: Xlim-1 expression in control embryos (Q), in embryos treated at the late gastrula stage 12.5 with CHX (R), RA (S) or RA and CHX (T). CHX was applied 30 min before the onset of the 2 h RA treatment.