Persistent palatal shelf deflection by tongue causes Pierre Robin-cleft in mice

Cleft palate can be caused by failure of elevation of the palatal shelves over the tongue during development. The physical mechanism of elevation is poorly understood. It is thought that elevation is obstructed by the tongue in a condition known as Pierre Robin sequence (PRS) in which the mandible is smaller (micrognathia) and/or further back than normal (retrognathia). Some PRS cases are associated with mutations in the regulatory and coding regions of SOX9. Here, we first examined the timing of palatal shelf elevation in mouse embryonic explants. While elevation in vivo occurs between embryonic days E13.5 and E14.5, we found that palate elevation occurred in both E12.5 and E13.5 explants immediately upon dissection, indicating release of elastic pre-stress by removal of the tongue. These findings suggest that palatal shelves are programmed ab initio to grow horizontally (as they do in birds and most reptiles) but are deflected in mammals by the tongue. Next, to examine this process in utero, we generated a new mtHand2Cre mouse line to delete Sox9 or to express diphtheria toxin specifically in the mandibular mesenchyme. Sox9 deletion resulted in a hypoplastic, dysmorphic, retrognathic mandible with fully penetrant, complete cleft of the secondary palate. In contrast, the majority of R26RDTA;mtHand2Cre embryos showed a reduced mandible (micrognathia) but also displayed a dramatically smaller tongue (microglossia). With a non-obstructing tongue, these mutants exhibited horizontal palatal shelf growth and formed a normal palate. These results are consistent with a mechanism in which physical obstruction by the tongue is the primary cause of downward development of the palatal shelves, far earlier than previously appreciated, and that normal elevation occurs by the release of elastic pre-stress. We experimentally demonstrate that in PRS micrognathia causes cleft palate through tongue malpositioning that prevents release of this palatal shelf pre-stress.