Fig 2. Morphological defects during Xenopus laevis development upon Fbl, Ncl, or Ubtf depletion. A, Embryos injected unilaterally at the two-cell stage with the indicated morpholino (MO) were inspected under the microscope at stage 42. As a control, a non-targeting morpholino (Ctrl) was used. Two embryos are presented (#1 and #2), with the left side injected. With the Ctrl morpholino and those targeting ubtf, and ncl expression, two independent experiments were performed, the total number of embryos tested being 100, 94 and 77, respectively. With the morpholino targeting fbl expression, a total of 154 embryos were analyzed in three independent experiments. B, Quantification of the data presented in panels A and E. Percentage of embryos displaying reduced eye size, or craniofacial skeleton defects (head asymmetry). C, Cartilage staining with alcian blue revealed a defect in all the branchial cartilages, namely: ir, infrarostral; m, meckel; p, palatoquadrate; c, ceratohyal; b, other branchial cartilages. Non-dissected and dissected embryos are presented for comparison. Arrowheads highlight striking reduction, or near-complete loss of the branchial arches. D, Western blot analysis of Fbl-depleted and rescued morphants. Total protein was extracted from individual neurula embryos; one embryo equivalent was loaded per lane. The blots were probed with an anti-Flag antibody (top panel) or an anti-Fbl antibody (bottom panel). The anti-Flag antibody detects only the rescue constructs (Flag-Fbl-wt or Flag-Fbl-D238A). The asterisk (*) denotes detection of an aspecific band. The anti-Fbl antibody detects both the endogenous protein and the rescue constructs. The percentage of Fbl expressed with respect to the uninjected control is indicated. The western blots displayed are representative of four independent experiments. As loading control, the blots were probed for alpha-tubulin. E, Both the wild-type Flag-Fbl and the catalytically deficient Flag-Fbl-D238A restored formation of branchial arches to about half the normal size. Representative example of an embryo co-injected with fbl MO and either a MO-resistant wild-type rescue construct (top) or the same construct harboring the D238A mutation (bottom). A total of 77 and 65 embryos were analyzed in two independent experiments with the Flag-Fbl-wt and Flag-Fbl-D238A construct, respectively.
Image published in: Delhermite J et al. (2022)
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