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Fig. 4. Fzd4/Fzd4s is required for pancreas development in vitro and in vivo
(A) Fzd4-morpholino (mo) or the corresponding mismatch-morpholino (mmo) were coinjected
along with Vegt and Noggin encoding RNAs. At the equivalent of stage 28, total
RNA was isolated from the programmed explants and subjected to RT-PCR as indicated. (B)
Fzd4-gRNA was co-injected along with RNAs encoding Cas9, Vegt and Noggin into one-cell
stage embryos. Explants were cultivated until the equivalent of stage 35. RT-PCR was for
the genes indicated. Mutation rate is given for Cas9 only or for Cas9 in combination with
Fzd4-gRNA. For both loss of function approaches, approximately 30 explants per condition
from two independent biological replicates were used. (C) Downregulation of Fzd4/Fzd4s by
Fzd4-morpholino injection. 4-cell-stage embryos were injected with RNA coding for β-
galactosidase (glb1) and either Fzd4/Fzd4s-morpholino or the corresponding mismatchmorpholino.
At stage 35/39, embryos from two independent biological replicates were used
for WMISH against indicated pancreatic markers and a real-time PCR analysis for Pdx1,
Ptf1a and Insulin. The graph indicates the fold change of tested markers in relation to Odc
(ornithine decarboxylase). Average values are given as mean and error bars as s.e.m.. ctr,
uninjected embryos. |
