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Fig. 3. Within alar p2, depletion of Irx1 and Irx2 activities, or an increase in Irx3 activity, promotes ZLI specification. Double ISH or ISH on embryos (A–D) either depleted for both Irx1 and Irx2 activities or (E–H) overexpressing irx3, or (I) overexpressing irx3-GR using barhl2 (A, E), otx2 (B, F), irx3 (C), irx1 (G), shh (D, H, I) probes as indicated. The scale bar stands for 0.5 mm. RNA encoding for irx3, or irx3-GR were co-injected with a MO control used as tracer, coupled to fluorescein detected by immunohistochemistry (red). (A–D) Co-depletion of Irx1/2 shifts the ZLI caudal border. In st. 32 embryos depleted for irx1/2 (A) barhl2 (n=24) expression is not significantly affected, and (B) otx2 (n=20) expression is slightly decreased. We observed (C) a weak increase of irx3 in the thalamic domain (n=36). (D) The ZLI territory, characterized by shh expression, is enlarged (n=36); DISH using barhl2 together with shh probes shows that the barhl2 anterior border and the ZLI anterior border coincided, indicating that the ZLI posterior boundary is caudally shifted. (E–I)irx3 or irx3-GR overexpression promotes ZLI specification. At st. 32 in irx3 overexpressing embryos (E) barhl2 (n=40) expression is modified compared with the CT sides whereas (F) otx2 (n=40) and (G) irx1 (n=40) expression are weakly decreased in the caudal p2 domain. (H) In irx3 overexpressing embryos the surface of the ZLI territory marked by shh is enlarged (n=22). DISH using barhl2 together with shh as probes demonstrates that the ZLI anterior border is not affected. Note that in embryos overexpressing Irx3 the expansion of the ZLI territory is associated to a decrease in shh staining intensity. (I) In embryos overexpressing a hormone-inducible form of irx3 (irx3-GR) and exposed to dexamethasone at st. 20, the surface of the ZLI territory marked by shh is enlarged (n=10). (J) The average width of p2 is not modified in embryos depleted of Irx1/2 or overexpressing Irx3. Using barhl2 as a p2 marker, the width of p2 was measured (Image J) on both the non-injected and the injected sides of embryos injected with GFP (CT, n=11), depleted for Irx1/2 (MOirx1/2, n=12) or overexpressing Irx3 (Irx3, n=14). The ratio of the p2 width of the injected side relative to the control side is shown. The error bars indicate the standard deviation. (K and L) The ZLI surface is significantly increased in Irx3 overexpressing embryos. (K) We delimited the alar/basal plate boundary of the diencephalon by drawing a line at the base of the ZLI area. We measure the ZLI area as the alar p2 area expressing Xshh. (L) Using Image J the surface of the ZLI territory was measured on both the non-injected and the injected sides of embryos injected with GFP (CT, n=9), overexpressing Irx3 (Irx3, n=9), or overexpressing a hormone-inducible form of Irx3 (Irx3-GR, n=9). The average ratio of the ZLI surface of the injected size relative to the control size is shown. The error bars indicate the standard deviation. We observed a significant increase of the ZLI surface in both Irx3 overexpressing embryos (R=1.5±0.19, t-test p≤0.004) and Irx3-GR overexpressing embryos (R=1.2±0.17, t-test p≤0.03). |
