Fig. 2. Pkd2 loss of function affects pax8 expression in the kidney field. (A) In situ hybridization analysis of pax8 expression in pkd2 morphant embryos. Pkd2-MO was injected into the two left blastomeres at the four-cell stage and pax8 expression was analyzed at early neurula stage (NF stage 14–15). Comparison of the left injected side with the right control side shows a strong inhibition of pax8 expression in the kidney field (arrowheads). Extinction, or a strong decrease of pax8 expression, is observed in more than 91% of the embryos analyzed. Pax8 expression in CMO-injected embryos is unaffected. When a pkd2 mRNA lacking the Pkd2-MO target sequence is co-injected with Pkd2-MO, this proportion is reduced to 62%, showing that it results to the specific loss of function of pkd2. OV, ectodermal expression of pax8 in the presumptive otic vesicle; KF, mesodermal expression of pax8 in the kidney field. Arrows indicate orientation of the antero-posterior (AP) axis. (B) RT-QPCR analysis of kidney field gene expression in dissected explants. CMO or Pkd2-MO was injected at the two-cell stage and embryos were cultured until late gastrula stage (NF stage 13) for kidney field explant dissection. Explants were further cultured until early neurula (NF stage 14) or late neurula (NF stage 18) stages for RT-QPCR analysis of pax8, lhx1, osr1 and osr2 expression. Mean6s.e.m. results from three independent experiments are displayed. As expected from the above results performed on whole embryos, there is a dramatic downregulation of pax8 at the early neurula stage. In contrast, lhx1, osr1 and osr2 expression is not affected. At the late neurula stage, pax8 expression is severely inhibited. In contrast, lhx1, osr1 and osr2 expression is not affected, indicating that Ca2+ signaling is acting upstream of pax8, but does not act in a general mechanism of kidney field induction. **P,0.01, ***P,0.001 (paired Student’s t-test).
Image published in: Futel M et al. (2015)
Copyright © 2015. Image reproduced with permission of the Publisher, The Company of Biologists Ltd.
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