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.