Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2. Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2. These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip.
Holoprosencephaly (HPE) is a common and severe human developmental abnormality marked by malformations of the forebrain and face. Although several genetic mutations have been linked to HPE, phenotypic outcomes range dramatically, and most cases cannot be attributed to a specific cause. Gene-environment interaction has been invoked as a premise to explain the etiological complexity of HPE, but identification of interacting factors has been extremely limited. Here, we demonstrate that mutations in Gli2, which encodes a Hedgehog pathway transcription factor, can cause or predispose to HPE depending upon gene dosage. On the C57BL/6J background, homozygous GLI2 loss of function results in the characteristic brain and facial features seen in severe human HPE, including midfacial hypoplasia, hypotelorism and medial forebrain deficiency with loss of ventral neurospecification. Although normally indistinguishable from wild-type littermates, we demonstrate that mice with single-allele Gli2 mutations exhibit increased penetrance and severity of HPE in response to low-dose teratogen exposure. This genetic predisposition is associated with a Gli2 dosage-dependent attenuation of Hedgehog ligand responsiveness at the cellular level. In addition to revealing a causative role for GLI2 in HPE genesis, these studies demonstrate a mechanism by which normally silent genetic and environmental factors can interact to produce severe outcomes. Taken together, these findings provide a framework for the understanding of the extreme phenotypic variability observed in humans carrying GLI2 mutations and a paradigm for reducing the incidence of this morbid birth defect.
Background:Piperonyl butoxide (PBO) is a pesticide synergist used in residential, commercial, and agricultural settings. PBO was recently found to inhibit Sonic hedgehog (Shh) signaling, a key developmental regulatory pathway. Disruption of Shh signaling is linked to birth defects, including holoprosencephaly (HPE), a malformation of the forebrain and face thought to result from complex gene–environment interactions.Objectives:The impact of PBO on Shh signaling in vitro and forebrain and face development in vivo was examined.Methods:The influence of PBO on Shh pathway transduction was assayed in mouse and human cell lines. To examine its teratogenic potential, a single dose of PBO (22–1,800mg/kg) was administered by oral gavage to C57BL/6J mice at gestational day 7.75, targeting the critical period for HPE. Gene–environment interactions were investigated using Shh+/− mice, which model human HPE-associated genetic mutations.Results:PBO attenuated Shh signaling in vitro through a mechanism similar to that of the known teratogen cyclopamine. In utero PBO exposure caused characteristic HPE facial dysmorphology including dose-dependent midface hypoplasia and hypotelorism, with a lowest observable effect level of 67mg/kg. Median forebrain deficiency characteristic of HPE was observed in severely affected animals, whereas all effective doses disrupted development of Shh-dependent transient forebrain structures that generate cortical interneurons. Normally silent heterozygous Shh null mutations exacerbated PBO teratogenicity at all doses tested, including 33mg/kg.Discussion:These findings demonstrate that prenatal PBO exposure can cause overt forebrain and face malformations or neurodevelopmental disruptions with subtle or no craniofacial dysmorphology in mice. By targeting Shh signaling as a sensitive mechanism of action and examining gene–environment interactions, this study defined a lowest observable effect level for PBO developmental toxicity in mice more than 30-fold lower than previously recognized. Human exposure to PBO and its potential contribution to etiologically complex birth defects should be rigorously examined. https://doi.org/10.1289/EHP5260
Orofacial clefts (OFCs) of the lip and/or palate are among the most common human birth defects. Current treatment strategies focus on functional and cosmetic repair but even when this care is available, individuals born with OFCs are at high risk for persistent neurobehavioral problems. In addition to learning disabilities and reduced academic achievement, recent evidence associates OFCs with elevated risk for a constellation of psychiatric outcomes including anxiety disorders, autism spectrum disorder, and schizophrenia. The relationship between these outcomes and OFCs is poorly understood and controversial. Recent neuroimaging studies in humans and mice demonstrate subtle morphological brain abnormalities that co-occur with OFCs but specific molecular and cellular mechanisms have not been investigated. Here, we provide the first evidence directly linking OFC pathogenesis to abnormal development of GABAergic cortical interneurons (cINs). Lineage tracing revealed that the structures that form the upper lip and palate develop in molecular synchrony and spatiotemporal proximity to cINs, suggesting these populations may have shared sensitivity to genetic and/or teratogenic insult. Examination of cIN development in a mouse model of nonsyndromic OFCs revealed significant disruptions in cIN proliferation and migration, culminating in misspecification of the somatostatin-expressing subgroup. These findings reveal a unified developmental basis for orofacial clefting and disrupted cIN development, and may explain the significant overlap in neurobehavioral and psychiatric outcomes associated with OFCs and cIN dysfunction. This emerging mechanistic understanding for increased prevalence of adverse neurobehavioral outcomes in OFC patients is the entry-point for developing evidence-based therapies to improve patient outcomes.
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