RNA rescue of neural crest defects in mdkb morphants. 100 pg mdkb RNA and 9.4 ng mdkb splice MOs (4.7 ng each) were injected individually or together into early zebrafish embryos. For co-injections, embryos were first injected with Morpholino at the one-cell stage, followed by RNA injection approximately 20 minutes later. 30 embryos each were collected for cDNA synthesis, the remaining embryos were used for in-situ analysis. (A) RT-PCR analysis of mdkb transcripts in non-injected embryos (lane 2), mdkb RNA (lane 3), mdkb splice MO (lane 4) and double injected embryos (lane 5). Top panel shows PCR products using exon specific primers, third panel from top shows products using primers covering the MO targeted intron. Two independent RT-PCRs were run for exon and intron primers. Analysis of actin expression was used as loading control in each experiment. The right panel represents RT controls (cDNA synthesis in the absence of reverse transcriptase). Non-spliced mdkb transcripts were detected in mdkb splice MO and double injected embryos (see lane with intron primer). Note that non-spliced products could not be detected using the exon primers due to excess of competing transcripts of injected RNA. (B-G) Analysis of foxd3 expression in ncc and foxa2 in axial mesoderm. Numbers indicate embryos with respective phenotype. mdkb MO injection results in ncc reduction in vast majority of analyzed embryos (D), while co-injection with RNA leads to a significant number of embryos with normal (E) or expanded ncc (F) indicating partial rescue of ncc defects.
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