RNA interference of Bmp-4 and midface development in postimplantation mouse embryos

Introduction: Clefts of the lip and palate result when the midfacial processes fail to fuse during embryonic development. Many of the cellular and molecular events underlying the fusion of these midfacial processes are unknown. There is evidence from our laboratory and others that Bmp-4 is a signal that regulates the process of fusion in the midface. RNA interference (RNAi) is a relatively new technique used to knock down select RNA levels-and ultimately protein levels-by using short RNA strands complementary to target mRNA in the cytoplasm. This was the first investigation to use RNAi to study the developing wild-type murine midface in vivo. This study used RNAi technology to decrease levels of Bmp-4 RNA during critical stages of midfacial fusion with the hypothesis that correct temporal and spatial expression of Bmp-4 is critical for normal fusion and formation of the midface. Methods: The sample included 19 timed pregnant wild-type (C57B1/6J strain) mice and their litters. Pregnant mice received tail-vein injections of plasmids coding for an epifluorescent label (DsRed) with or without the Bmp-4 RNAi sequence 3 days before typical embryonic midface fusion. Embryos were harvested before, during, and after the typical date of midface fusion, and delivery of the plasmids was confirmed via epifluoroscopy and reverse transcriptase polymerase chain reaction (RT-PCR) for the DsRed label. Results: Alterations in morphology were observed by gross examination and by sectioning the embryos. Levels of Bmp-4 mRNA and protein in the embryonic midface were examined with RT-PCR and immunohistochemistry, respectively. Injections of RNA plasmids via the maternal tail-vein to pregnant mothers reliably delivered plasmids to the developing embryonic midface with an average 46% (± 12.2) knockdown in Bmp-4 RNA levels. Conclusions: Reduced BMP-4 levels caused prefusion delays in the outgrowth of the lateral and medial nasal processes. These processes later recovered, producing no detectable long-term morphological differences when compared with the control embryos. Ectopic Bmp-4 protein expression was observed in the lateral and medial nasal processes mesenchyme in Bmp-4 knockdown embryos superior to the region of primary palatal fusion. These findings implicated Bmp-4 signaling in the outgrowth and development of the midfacial processes. This study introduced the use of RNAi for the study of protein signaling in the developing wild-type murine midface in vivo.