A Highly Conserved Shh Enhancer Coordinates Hypothalamic and Craniofacial Development

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.

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium

“…1 C). These defects are consistent with SHH deficiency at early neurulation, as seen in Shh mutant mouse embryos ( Carreno et al, 2017 ; Corman et al, 2018 ; Crane-Smith et al, 2021 ; Szabo et al, 2009 ). However, contrary to prior analysis that focused on developmental stages after E9.5, our data uncovered that GAS1 is not required for initial establishment of the SHH domain in the RDVM at E8.5, but is necessary to sustain its activity at later stages of neurulation.…”

“…S2 " type="url"/> ). These changes recapitulated phenotypes seen in Shh mutant mice ( Carreno et al, 2017 ; Corman et al, 2018 ; Crane-Smith et al, 2021 ; Szabo et al, 2009 ) and further substantiated a role for GAS1 in control of SHH activity in the developing forebrain neuroepithelium.

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Impact of Sonic Hedgehog‐dependent sphenoid bone defect on craniofacial growth

“…Phenotype associated with Shh inactivation varied based on the mouse strain, with C57BL/6J background showing a more pronounced effect (Lo et al, 2021). development (Carreno et al, 2017;Crane-Smith et al, 2021;Hamdi-Rozé et al, 2020;Zhao et al, 2012). Recent research also found that mice heterozygous for Six3 displayed a similar pituitary-restricted phenotype due to Six3's control of Shh in the ventral forebrain (Bando et al, 2023).…”

“…This developmental anomaly arises from the abnormal persistence of the tissue connecting Rathke's pouch to the oral ectoderm during development. Studies on conditional knockout mice revealed the crucial role of Shh in the ventral diencephalon for Rathke's pouch development (Carreno et al, 2017 ; Crane‐Smith et al, 2021 ; Hamdi‐Rozé et al, 2020 ; Zhao et al, 2012 ). Recent research also found that mice heterozygous for Six3 displayed a similar pituitary‐restricted phenotype due to Six3's control of Shh in the ventral forebrain (Bando et al, 2023 ).…”

“…Most cases of HPE are caused by a defect in the Sonic Hedgehog (SHH) signaling pathway (Dubourg et al, 2018 ; Gregory et al, 2015 ; Kim et al, 2019 ). The SHH pathway's functions in early brain development have been extensively described (Crane‐Smith et al, 2021 ; Mercier et al, 2013 ). Shh ‐deficient mouse models show the main clinical signs of HPE, including craniofacial morphological defects (Chiang et al, 1996 ; Jeong et al, 2004 ).…”