Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos.

Embryonic palate development involves bilateral vertical growth of palatal shelves - extensions from the maxillary processes - next to the tongue at embryonic day (E) 13.5. This vertical growth is followed by elevation and fusion above the tongue by E14.5. Recent models indicate that this process of elevation involves a complex vertical to horizontal remodeling of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To further understand the dynamics of palatal shelf elevation, we employed time-restricted pregnancies with a one-hour resolution and magnetic resonance (MR) imaging of intermediate stages. Our data showed that in C57BL/6J mice, palatal shelves are unelevated in 100% of embryos at E14.0 and completely elevated in 80% of embryos at E14.25, which is only a 6-hour timeframe. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a shift towards delayed elevation in female embryos. Even at E14.125, 25% of embryos had completely elevated palatal shelves suggesting that once initiated, the palatal shelves elevated rapidly with elevation completing in less than 3 hours in individual embryos. In contrast to C57BL/6J, in FVB/NJ mice, the elevation window was shifted earlier and was broader to E13.875-E14.25 without any noticeable sex differences. In a proportion of embryos in both strains, we also captured an intermediate stage with unilateral palatal shelf elevation. Both right and left shelves were identified to be unilaterally elevated indicating that it was a random process. It remains to be determined whether palatal shelves elevate in a sequential manner, i.e., one-at-a-time, with an obligate unilateral elevation stage. MR imaging of various stages showed a highly dynamic antero-posterior remodeling of the shelves during elevation, beginning with the formation of bilateral bulges in the posterior palate. These bulges tapered anteriorly, however, palatal shelf fusion still began in the middle part of the palate. The bulge regions did not show changes in cell proliferation, consistent with the notion that rapid elevation was primarily due to cytoskeletal and extracellular matrix changes. We are using finite element analysis to identify the mechanical forces underlying these morphological changes. Thus, our data reveal critical novel insights into the rapid dynamic changes as well as strain and sex differences in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.