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Fig. 5 | BMC Evolutionary Biology

Fig. 5

From: Tinkering signaling pathways by gain and loss of protein isoforms: the case of the EDA pathway regulator EDARADD

Fig. 5

Human EDARADD B but not EDARADD A rescues skeletal defects observed in MO-injected zebrafish larvae. a Schematic representation of the exon-intron structure of the zebrafish edaradd gene. The location of target sequences for translation-blocking (MO-tsl) and splice-blocking (MO-spl) edaradd morpholinos is indicated by a red and a green line, respectively. Blue arrows indicate the location of the primers used for edaradd mRNA expression analysis during development [see Additional file 4: Figure S4] and for splice-blocking edaradd MO validation by RT-PCR analysis [see Additional file 4: Figure S4]. b Knockdown of Edaradd expression leads to skeletal defects. WT embryos were injected at one-cell stage and skeletal morphogenesis was analyzed through alcian blue and alizarin red staining at 5.5 dpf. The larvae phenotypes observed were classified as severe, intermediate or weak/normal phenotype. The severe phenotype presented no pharyngeal skeleton, a rudimentary neurocranial cartilage and no tooth could be detected. This defect was also accompanied by pericardial and yolk sac edema. Intermediate phenotype showed rudimentary pharyngeal skeleton without second arch and gill arch derivatives and sometimes one rudimentary tooth on each side of the larvae. Weak/normal phenotype demonstrated small gill arch derivatives or no obvious defects and at least one tooth on each side of the larvae could be clearly observed through alizarin red staining. Ventral and lateral views of the head are shown at low magnification (scale bar: 500 μm) and a zoom on the teeth is shown on the right panel (scale bar: 100 μm). c Percentages of the various phenotypic groups obtained following MO injection [see Additional file 4: Figure S4] or MS (Mismatch control) injection. These results are representative of at least 3 independent experiments following injection of at least 70 embryos per condition. d Human EDARADD A and B mRNA effects in rescuing experiments. One-cell stage embryos were co-injected with MO-tsl and/or capped human EDARADD A or B, or capped zebrafish edaradd mRNA with at least 70 embryos injected per condition. The barplot shows the percentage of each phenotypic group in 4 separate experiments and the statistical analyses have been performed on the severe group. e A representative group of 10 larvae, classified according to their phenotype, are shown for each condition

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