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Fig. 5. Knockdown of Dmrt5 reduces Ngnr1 and Ebf2 expression. (A) In vivo translation of a Dmrt5-eGFP reporter construct is specifically inhibited by the Dmrt5-MO. Embryos were injected with 500 pg of Dmrt5-eGFP mRNA or, as a control, 500 pg of eGFP mRNA, alone or in combination with 20 ng of the Dmrt5-MO, as indicated. (B)I) Whole-mount in situ hybridization of early tadpole embryos injected with 20 ng of Dmrt5-MO or a control standard MO and analysed with the indicated probes. (B) and (D)I) Anterior views with dorsal to the top. (C) Horizontal section through the head, anterior to the top. The injected side marked by Xgal staining in light blue is on the right. Note that Dmrt5 morphants show reduced Ngnr1, Ebf2, Aml and Ebf3 expression in the olfactory epithelium while Ep.Keratin and Sox2 expression is unaffected. Injection of a control MO has no effect on Ebf2. (J) and (K) Anterior views of early tadpole stage embryos injected with Ath5 mRNA, with or without 20 ng of DMRT5-MO and hybridised with Ebf2. Note that Ebf2 is expanded in Ath5 injected embryos and reduced in Ath5 overexpressing Dmrt5 depleted embryos. (L) and (M) RT-qPCR analysis of Sox2, Ngnr1 and Ebf2 expression in animal caps derived from embryos injected with Noggin (100 pg), Xenopus MT-Dmrt5 (50 pg), mouse MT-Dmrt5 (mMT-Dmrt5, 50 pg), Xenopus MT-Dmrt4 (xDmrt4, 50 pg), Dmrt4-MO (20 ng) and Dmrt5-MO (20 ng), as indicated. Expression levels were normalized to GAPDH and compared to the level observed in Noggin injected caps, which was set to 1. Note that Noggin-mediated activation of Ngnr1 and Ebf2, but not that of Sox2, is blocked in caps derived from Dmrt5 or Dmrt4 depleted embryos and that their absence can be indifferently rescued by Dmrt5 or Dmrt4 overexpression.
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