Disruption of the Hedgehog signaling pathway has been implicated as an important molecular mechanism in the pathogenesis of fetal alcohol syndrome. In severe cases, the abnormalities of the face and brain that result from prenatal ethanol exposure fall within the spectrum of holoprosencephaly. Single allele mutations in the Hh pathway genes Sonic Hedgehog (SHH) and GLI2 cause holoprosencephaly with extremely variable phenotypic penetrance in humans. Here, we tested whether mutations in these genes alter the frequency or severity of ethanol-induced dysmorphology in a mouse model. Timed pregnancies were established by mating Shh^+/− or Gli2^+/− male mice backcrossed to C57BL/6J strain, with wildtype females. On gestational day 7, dams were treated with two ip doses of 2.9 g/kg ethanol (or vehicle alone), administered four hrs apart. Fetuses were then genotyped and imaged, and the severity of facial dysmorphology was assessed. Following ethanol exposure, mean dysmorphology scores were increased by 3.2- and 6.6-fold in Shh^+/− and Gli2^+/− groups, respectively, relative to their wildtype littermates. Importantly, a cohort of heterozygous fetuses exhibited phenotypes not typically produced in this model but associated with severe holoprosencephaly, including exencephaly, median cleft lip, otocephaly, and proboscis. As expected, a correlation between the severity of facial dysmorphology and medial forebrain deficiency was observed in affected animals. While Shh^+/− and Gli2^+/− mice have been described as phenotypically normal, these results illustrate a functional haploinsufficiency of both genes in combination with ethanol exposure. By demonstrating an interaction between specific genetic and environmental risk factors, this study provides important insights into the multifactorial etiology and complex pathogenesis of fetal alcohol syndrome and holoprosencephaly.

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 15^th to 24^th 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.

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 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 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.

An essential aspect of goal-directed decision-making is selecting actions based on anticipated consequences, a process that involves the orbitofrontal cortex (OFC) and potentially, the plasticity of dendritic spines in this region. To investigate this possibility, we trained male and female mice to nose poke for food reinforcers, or we delivered the same number of food reinforcers non-contingently to separate mice. We then decreased the likelihood of reinforcement for trained mice, requiring them to modify action–outcome expectations. In a separate experiment, we blocked action–outcome updating via chemogenetic inactivation of the OFC. In both cases, successfully selecting actions based on their likely consequences was associated with fewer immature, thin-shaped dendritic spines and a greater proportion of mature, mushroom-shaped spines in the ventrolateral OFC. This pattern was distinct from spine loss associated with aging, and we identified no effects on hippocampal CA1 neurons. Given that the OFC is involved in prospective calculations of likely outcomes, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for solidifying durable expectations. To investigate causal relationships, we inhibited the RNA-binding protein fragile X mental retardation protein (encoded by Fmr1), which constrains dendritic spine turnover. Ventrolateral OFC-selective Fmr1 knockdown recapitulated the behavioral effects of inducible OFC inactivation (and lesions; also shown here), impairing action–outcome conditioning, and caused dendritic spine excess. Our findings suggest that a proper balance of dendritic spine plasticity within the OFC is necessary for one9s ability to select actions based on anticipated consequences. SIGNIFICANCE STATEMENT Navigating a changing environment requires associating actions with their likely outcomes and updating these associations when they change. Dendritic spine plasticity is likely involved, yet relationships are unconfirmed. Using behavioral, chemogenetic, and viral-mediated gene silencing strategies and high-resolution microscopy, we find that modifying action–outcome expectations is associated with fewer immature spines and a greater proportion of mature spines in the ventrolateral orbitofrontal cortex (OFC). Given that the OFC is involved in prospectively calculating the likely outcomes of one9s behavior, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for maintaining durable expectations.

Social experiences influence decision making, including decision making lacking explicit social content, yet mechanistic factors are unclear. We developed a new procedure, social incentivization of future choice (SIFC). Female mice are trained to nose poke for equally-preferred foods, then one food is paired with a novel conspecific, and the other with a novel object. Mice later respond more for the conspecific-associated food. Thus, prior social experience incentivizes later instrumental choice. SIFC is pervasive, occurring following multiple types of social experiences, and is not attributable to warmth or olfactory cues alone. SIFC requires the prelimbic prefrontal cortex (PL), but not the neighboring orbitofrontal cortex. Further, inputs from the basolateral amygdala to the PL and outputs to the nucleus accumbens are necessary for SIFC, but not memory for a conspecific. Basolateral amygdala→PL connections may signal the salience of social information, leading to the prioritization of coincident rewards via PL→nucleus accumbens outputs.