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Fig. 4. Loss of FGFA function disrupts skeletogenesis and gut morphogenesis in
sea urchin. (A) Controls of the efficiency of the
fgfA-splice and fgfA-ATG morpholinos. (a) Scheme of
the intron-exon organization of the fgfA pre-mRNA, indicating the
positions of the target sequence and of the primers used to characterize the
mRNA products generated in the presence of this morpholino. (b) RT-PCR
analysis of control uninjected and embryos injected with the
fgfA-splice morpholino at 0.5, 1 or 1.5 mM. The PCR product in
control embryos had the expected size (101 codons, 303 bp) and predicted
sequence. The PCR product amplified from the fgfA-splice morphants
had the expected size (66 codons, 198 bp) and sequence for a transcript
deleted from exon 2. (c) fgfA-ATG-Mo but not
fgfA-splice-Mo, specifically blocks in vitro translation of a
synthetic fgfA transcript. PCS2-fgfA was in vitro translated
in the presence of fgf9A-ATG-Mo or fgfA-splice at the
indicated concentrations (1.6, 8, 40 µM) and the products were analysed by
PAGE and autoradiography. (Ba-o) Phenotypes caused by microinjection of
the fgfA-splice-Mo (f-j) or fgfA-ATG-Mo (k-o) in the egg.
(Ca-l) Rescue experiment. Eggs were first injected with
fgfA-splice-Mo together with RLDX then subsequently re-injected at
the one- or two-cell stage with a synthetic mRNA encoding FGFA together with
an FLDX.
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