XB-IMG-175729
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Figure 3. Prdx1 functions downstream of the RA signaling pathway during proximal tubule formation in the pronephros. (A) prdx1 MOs (40 ng) were injected into both blastomeres of two-cell stage embryos. Animal caps were removed from the injected embryos at the stage 8.5 and incubated with 10 ng/mL activin A, followed by 10−4 M all-trans RA for 3 h. Animal caps were collected from embryos at the stage 33 and used for RT-PCR. RT-PCR results showed the significant reduced expression of lim1, pax2, smp30, and pax8 in prdx1 MO-injected as compared with RA/activin induced. The decreased expression of pronephros markers was rescued by prdx1* RNA. Ornithine decarboxylase (odc) was used as the loading control. A no-RT template in the absence of reverse transcriptase was used as the control. WE, whole embryo; CTL, control animal caps; CTL MO, control MO-injected animal caps. (B) RT-PCR examination of lim1, pax2, smp30 and pax8 expression was confirmed by real time PCR. Significant lower expression level was observed for pronephros markers in the prdx1 MO-injected embryos that was rescued by co-injection with prdx1* RNA except for pax8. WE, whole embryo; CTL, control animal caps; CTL MO, control MO-injected animal caps. (C) prdx1 MOs (40 ng) were co-injected with wild-type prdx1, RARα-vp16 (active rar), pax8, or lim1 (400 pg) into both blastomeres of two-cell stage embryos. Embryos at the stage 33 were used for whole-mount in situ hybridization, and proximal tubules in the pronephros were visualized with a smp30 probe. The pronephros abnormalities of prdx1 morphants were rescued by lim1 mRNA co-injection but not by the activation of active RA receptors or pax8. (D) Graphical representation of pronephros development in prdx1 MO-injected embryos and embryos co-injected with lim1, active RA receptors and pax8 compared to the control embryos (injected with control MO). Pronephros defects were significantly rescued by lim1 mRNA co-injection. (E) Embryos at two-cell stage were injected with dominant-negative retinoic acid receptor (DN-RAR) with or without prdx1. DN-RAR-inhibited pronephros development was rescued by prdx1 overexpression. (F) Expression of pronephros marker lim1 was observed at stage 12.5 in embryos injected with CTL MO and prdx1 MO. lim1 expression was dramatically reduced in Prdx1 morphants.
Image published in: Chae S et al. (2017) © The Author(s) 2017. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
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