A Highly ConservedShhEnhancer Coordinates Hypothalamic and Craniofacial Development

AbstractEnhancers that are conserved deep in evolutionary time regulate characteristics held in common across taxonomic classes. Here, deletion of the highly conservedShhenhancer SBE2 (Shhbrain enhancer 2) markedly reducedShhexpression within the embryonic brain specifically in the rostral diencephalon; however, no abnormal anatomical phenotype was observed due to compensatory low levels of expression mediated by a second enhancer. In contrast, a further reduction ofShhlevels, achieved by crossing the SBE2 deletion with theShhnull allele, disrupted brain and craniofacial tissue; thus, linking SBE2 regulatedShhexpression to multiple defects and further enabling the study of the effects of differing levels ofShhon embryogenesis. Development of the hypothalamus, derived from the rostral diencephalon, was disrupted along both the anterior-posterior (AP) and the dorsal-ventral (DV) axes. Expression of DV patterning genes and subsequent neuronal population induction were particularly sensitive toShhexpression levels, demonstrating a novel morphogenic context forShh. The role of SBE2, which is highlighted by DV gene expression, is to step-up expression ofShhabove the minimal activity of the second enhancer, ensuring the necessary levels of Shh in a regional-specific manner. We also show that lowShhlevels in the diencephalon disrupted neighbouring craniofacial development, including mediolateral patterning of the bones along the cranial floor and viscerocranium. Thus, SBE2 contributes to hypothalamic morphogenesis and ensures there is coordination with the formation of the adjacent midline cranial bones that subsequently protects the neural tissue.