Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

Abstract

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15th to 24th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.

Gli2 gene-environment interactions contribute to the etiological complexity of holoprosencephaly: evidence from a mouse model

“…This teratogenic exposure regimen has recently been reported to result in severe HPE phenotypes (Heyne et al, 2015a). A remarkable degree of overlap in facial dysmorphology was observed among Gli2 −/− fetuses and those acutely exposed to vismodegib (Fig.…”

“…Vismodegib was purchased from LC Laboratories (Woburn, MA) and suspended at 3 mg ml −1 in 0.5% methyl cellulose with 0.2% Tween 80 as previously described (Heyne et al, 2015a). Individual suspensions were prepared within 30 min of administration.…”

“…For comparison of face and brain morphology, images of Bouin's-fixed tissue were captured and converted to grayscale. The severity of HPE phenotypes in GD17 fetuses was assessed by a single rater that had been blinded to treatment based upon a semi-quantitative scale as previously described (Heyne et al, 2015a; Kietzman et al, 2014). …”

“…In humans and animal models, HPE has been linked to chemical and genetic disruption of the Hedgehog (Hh) signaling pathway (Roessler et al, 1996; Chiang et al, 1996; Heyne et al, 2015a). Initiated by the Sonic Hedgehog (SHH) ligand, Hh signaling is required for ventral patterning and expansion of the medial forebrain, as well as outgrowth of the processes that form the midface.…”

Cichlid fishes as a model to understand normal and clinical craniofacial variation

“…For example, the Wnt signaling network plays successive critical roles during NCC induction (reviewed in (Sauka-Spengler and Bronner-Fraser 2008)), proliferation and outgrowth of facial mesenchyme (Brugmann, et al 2007; Brugmann, et al 2010), fusion of facial prominences (Lan, et al 2006; Song, et al 2009), and osteogenesis (reviewed in (Hartmann 2006; Long 2012)). Likewise, the Hedgehog developmental pathway is necessary for NCC survival (Ahlgren and Bronner-Fraser 1999; Billmyre and Klingensmith 2015), facial patterning and outgrowth at multiple stages (Cordero, et al 2004; Heyne, et al 2015; Hu, et al 2003; Jeong, et al 2004), and chondrogenesis and osteogenesis (Mo, et al 1997; Wada, et al 2005; Yang, et al 2015). This developmental pleiotropy, the iterative usage of a common pathway in multiple tissues or stages, can limit the assessment of later developmental events.…”