Fig. S15

dpm1 knockdown in zebrafish recapitulates the human phenotypes caused by DPM1 mutations.

Multiple controls to confirm the effectiveness and specificity of the dpm1 morpholinos (MOs) were performed. All error bars represent S.E.M. *=p<0.05, **=p<0.01, ***=p<0.001, unpaired student’s t-test, N’s are indicated for each experiment. (a) Knockdown efficiency was measured in affected embryos using 10ng of dpm1 splice MO at 1dpf. No difference is observed in expression of the housekeeping gene ef1a. (N=3) (b) Splice and start dpm1 MOs caused identical phenotypes as shown in Fig. 4 and in this figure in d and e at different concentrations. As start MOs are usually more effective 4ng were sufficient to generate 100% affected embryos at 1dpf, while the splice MO needed to be injected at higher concentrations. (N=3) (c) Injection of a scrambled control morpholino in parallel to maximally effective concentrations of the start MO had no effect on the embryos, showing that these phenotypes are not due to injection and general MO toxicity. (N=4) (d) Vascular defects reminiscent of those observed in the patients can also be occasionally observed in the dpm1 morphant. The caudal artery (CA) and caudal vein (CV) supply blood flow to the tail in the direction indicated by the arrows in the WT panel. In the morphants blood vessels appear fused and enlarged (outlined by the dotted line) leading to accumulation of blood in the distal portion of the tail. Scale bar: 200µm. (e) Microcephaly, a reduction in head size, and a reduction in eye size are observed in most morphants. Dotted lines in the morphants (MO) panel shows the outline of the wild-type (WT) head in black and of the WT eye in white for reference. Scale bar: 200µm. Quantification of the reduced eye diameter measured on the anterior/posterior (A/P) axis and of head area is shown (N=3). (f) Rescue experiments were performed using 2ng dpm1 start MO injections as described in the Methods and 200pg of DPM1 mRNA, then the number of normal vs. affected embryos were tested. Despite embryos and larvae in the rescue condition appeared less affected than MO injected clutchmates, this difference was not clearly quantifiable due to the multiple phenotypes observed, so the total number of normal vs. affected was used (N=6). (g) To rule out possible gain of function effects of human missense mutations, normal and mutated DPM1 mRNAs were injected in isolation and none caused substantial defects (N=3).