An in vitro mouse model of cleft palate: Defining a critical intershelf distance necessary for palatal clefting

It is unclear whether cleft palate formation is attributable to intrinsic b iomolecular defects in the embryonic elevating palatal shelves or to an ina bility of the shelves to overcome a mechanical obstruction (such as the ton gue in Pierre Robin sequence) to normal fusion. Regardless of the specific mechanism, presumably embryonic palatal shelves are ultimately unable to br idge a critical distance and remain unapproximated, resulting in a clefting defect at birth. We propose to use a palate organ culture system to determ ine the critical distance beyond which embryonic palatal shelves fail to fu se (i.e., the minimal critical intershelf distance). In doing so, we hope t o establish an in vitro cleft palate model that could then be used to inves tigate the contributions of various signaling pathways to cleft formation a nd to study novel in utero treatment strategies. Palatal shelves from CD-1 mouse embryos were micro-dissected on day 13.5 of gestation (EI3.5; term = 19.5 days), before fusion. Using a standardized m icroscope ocular grid, paired palatal shelves were placed on a filter inser t at precisely graded distances ranging from 0 (in contact) to 1.9 mm (0, 0 .095, 0.19, 0.26, 0.38, 0.48, 0.57, 0.76, 0.95, and 1.9 mm). A total of 68 paired palatal shelves were placed in serum-free organ culture for 96 hours (n = 68). Sample sizes of 10 were used for each intershelf distance up to and including 0.48 min (n = 60). For intershelf distances of 0.57 mm and gr eater, two-paired palatal shelves were cultured (n = 8). All specimens were assessed grossly and histologically for palatal fusion. Palatal fusion occurred in our model only when intershelf distances were 0. 38 mm or less. At 0.38 mm, eight of 10 palates appeared grossly adherent, w hereas six of 10 demonstrated clear fusion histologically with resolution o f the medial epithelial seam and continuity of the palatal mesenchyme. None of the 18 palates fused when placed at intershelf distances of 0.48 min or greater. Using our selected intershelf distances as a guideline, we have established an approximate minimal critical intershelf distance (0.48 mm) at which we can reliably expect no palatal fusion. Culturing palatal shelves at intersh elf distances of 0.48 mm or greater results in nonfusion or clefting in vit ro. This model will allow us to study biomolecular characteristics of unfus ed or cleft palatal shelves in comparison with fused shelves. Furthermore, we plan to study the efficacy of grafting with exogenous embryonic mesenchy me or candidate factors to overcome clefting in vitro as a first step towar d future in utero treatment strategies.