Abstract
Enhancers that are conserved deep in evolutionary time regulate characteristics held in common across taxonomic classes. Here, deletion of the highly conserved Shh enhancer SBE2 (Shh brain enhancer 2) in mouse markedly reduced Shh expression within the embryonic brain specifically in the rostral diencephalon; however, no abnormal anatomical phenotype was observed. Secondary enhancer activity was subsequently identified which likely mediates low levels of expression. In contrast, when crossing the SBE2 deletion with the Shh null allele, brain and craniofacial development were disrupted; thus, linking SBE2 regulated Shh expression to multiple defects and further enabling the study of the effects of differing levels of Shh on 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 to Shh expression levels, demonstrating a novel morphogenic context for Shh. The role of SBE2, which is highlighted by DV gene expression, is to step-up expression of Shh above the minimal activity of the second enhancer, ensuring the necessary levels of Shh in a regional-specific manner. We also show that low Shh levels 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 protect the neural tissue.
Highlights
Long-distance enhancers that are conserved across multiple classes of vertebrates are implicated in regulating early embryonic development (Nord et al, 2013)
No live Shh null (Shhnull)/ SBE2 offspring were recorded by postnatal (P) 14 days (Figure 1B), these compound heterozygous embryos died perinatally failing to survive beyond P4, with the cause of postnatal lethality currently undetermined
Shh mRNA expression analysis at E10.5 in Shh SBE2/ SBE2 and Shhnull/ SBE2 embryos revealed levels of Shh expression undetected by wholemount in situ hybridisation in the ventral portion of the rostral diencephalon in both crosses (Figures 1C–E and Supplementary Figure S2J)
Summary
Long-distance enhancers that are conserved across multiple classes of vertebrates are implicated in regulating early embryonic development (Nord et al, 2013). The Shh regulatory domain, which extends 1 Mb upstream of the gene contains a number of sequence elements identified by high sequence conservation. These elements appear to be largely responsible for the typical pattern of Shh expression observed at initial stages of organogenesis (Jeong et al, 2008; Anderson et al, 2014). HPE is associated with heterozygous mutations that inactivate the Shh gene (Chiang et al, 1996; Ming and Muenke, 2002) These data suggest that disruption of SBE2 driven expression in the early developing forebrain may contribute to this disorder (Jeong et al, 2008)
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