OBJECTIVE To assess the antinociceptive efficacy and safety of neuraxial morphine in inland bearded dragons (Pogona vitticeps). ANIMALS 10 healthy adult bearded dragons. PROCEDURES Animals were sedated with alfaxalone (15 mg/kg) SC prior to neuraxial injections. In a randomized, blinded, placebo-controlled, crossover design, animals received preservative-free morphine (0.5 mg/kg) combined with lidocaine (2 mg/kg) or lidocaine (2 mg/kg) only (control treatment). For both treatments, saline (0.9% NaCl) solution was used for dilution to a total volume of 0.3 mL/kg. If the initial injection did not result in motor block of the pelvic limbs or cloaca relaxation within 10 minutes, a second injection was performed. Measurements consisted of bilateral mechanical stimulation of the limbs and at 25%, 50%, and 75% of the trunk's length as well as cloacal tone to assess spread and duration of motor block. Pelvic limb withdrawal latencies in response to a thermal noxious stimulus were measured over a 48-hour period to assess antinociception. RESULTS Success rate following the first injection was 90% (18/20 injections) and increased to 100% following a second injection. Motor block occurred within 5 minutes with both treatments. Pelvic limb withdrawal latencies were significantly prolonged following neuraxial morphine versus control treatment for at least 12 hours after injection. By 24 hours, no effect of morphine on pelvic limb latencies was detectable. CLINICAL RELEVANCE These results demonstrated that neuraxial administration of morphine results in regional antinociceptive effects for at least 12 hours and has no clinically relevant adverse effects in healthy bearded dragons. This technique has potential for providing regional analgesia in this species.

Objective To assess the success rate, onset, duration and extent of motor/sensory block following neuraxial injection of two dosages of bupivacaine in bearded dragons (Pogona vitticeps). Study design Prospective, randomized, blinded, crossover experimental study. Animals A total of 10 adult bearded dragons (0.3 +/- 0.1 kg). Methods After sedation with alfaxalone (15 mg kg(-1) subcutaneously), neuraxial injections were performed with 1 or 2 mg kg(-1) bupivacaine hydrochloride (0.5%, treatments BUP-1 and BUP-2, respectively) in a randomized treatment sequence with a 7 day washout period. If the initial bupivacaine injection was not successful within 10 minutes, a second injection was performed at the same dose. Mechanical stimulation of limbs, 25%, 50%, 75% of the trunk's length and cloacal tone were assessed. Results Success rate following the first neuraxial injection was 95%, which increased to 100% after the second injection. Motor/sensory block were noted by 5 minutes after the injection of bupivacaine at either dose. BUP-2 was associated with more cranial spread. The median (range) duration of cloacal tone loss was longer following treatment BUP-2 [120 (75-225) minutes] than followed treatment BUP-1 [83 (25-135) minutes; p = 0.03]. Duration of pelvic limb motor block was comparable between both doses, lasting a median of 68 minutes in both treatments (p = 0.94). There was a transient, not clinically relevant increase from baseline in heart rate in treatment BUP-1 only. No significant difference from baseline in respiratory rate was noted in either treatment; however, two animals in treatment BUP-2 became apneic (10-20 minutes). Conclusions and clinical relevance Bupivacaine (1 mg kg(-1)) is recommended for neuraxial anesthesia in bearded dragons. In treatment BUP-2, extensive cranial spread resulted in apnea and motor block of the thoracic limb in several animals; therefore this dose is not recommended.

Tinidazole is a second-generation nitroimidazole compound that is used as an antimicrobial to treat anaerobic bacterial and protozoal infections in humans and, less frequently, in veterinary medicine. However, metronidazole, another secondgeneration nitroimidazole, is more commonly used. Nonetheless, tinidazole has proven to be a superior therapy for parasitic infections in humans, particularly in the treatment of giardiasis. Furthermore, in chinchillas, metronidazole has been shown to cause a clinically relevant reduction in food intake after oral administration at published dosages. This study's objective was to evaluate the effect of orally administered tinidazole on food intake in healthy chinchillas. In 2 randomized, placebocontrolled, blinded, crossover studies, tinidazole was evaluated at 2 single high doses (100 mg/kg and 200 mg/kg PO, n = 9) and a repeated dosing schedule at a lower dose (20 mg/kg PO q12h for 10 doses, n = 12). Food intake was measured over 24-h periods before and after drug administration. The single-dose treatment groups both displayed significantly reduced food intake (200 mg/kg: -26 ± 21%; 100 mg/kg: -9 ± 21%, P < 0.01) as compared with the control group during the first 24 h after drug administration. Food intake returned to pretreatment values within 4-5 d. Repeated administration at 20 mg/kg q12h was not associated with any significant changes in food intake. No other adverse effects were noted during this study. Tinidazole administration at single higher doses resulted in an acute self-limiting reduction in food intake. In comparison, repeated administration of lower doses (20 mg/kg PO q12h) had no significant effects on food intake in healthy chinchillas. Therefore, tinidazole may be a more suitable drug for treating Giardia and anaerobic bacterial infections in this species than the more commonly used metronidazole.

Background: Cisapride, a prokinetic drug, has been anecdotally recommended in chinchillas at a dose of 0.5 mg/kg PO q8-12h to treat gastrointestinal hypomotility. However, studies in other rodent species suggest that higher doses are necessary to be effective. Methods: In two randomized, placebo-controlled, blinded, crossover studies, the effects of cisapride (10 mg/kg PO q12h for four doses), administered with or without concurrent syringe feeding of a critical care formula (25 mL/kg PO q12h for two doses) were evaluated in chinchillas following recovery from sedation induced by alfaxalone-butorphanol. Food intake and fecal output were quantified to assess the effects of cisapride on these parameters. Results: Over the first 24 hours after recovery from sedation, animals that received cisapride and syringe feeding had the least reduction in fecal output (-27 +/- 23%) compared to the control treatment (-48 +/- 22%, P = 0.008) or to animals which received syringe feedings alone (-40 +/- 23%, P = 0.12). Cisapride administered without concurrent syringe feedings had no effect on fecal output. No adverse effects were recorded following the administration of cisapride. Conclusions and clinical relevance: The oral administration of cisapride at 10 mg/kg q12h in conjunction with syringe feeding resulted in a slight, but not clinically relevant, attenuation of fecal output reduction by similar to 13% compared to syringe feedings alone and by similar to 20% compared to no treatment. Without the concurrent administration of syringe feedings, cisapride had no effect on fecal output in chinchillas.

Background: The administration of metronidazole in chinchillas remains controversial and some sources raise concerns regarding drug-induced anorexia or hyporexia. Therefore, the objective of this study was to evaluate if the oral administration of oral metronidazole has a significant effect on food intake in clinically healthy chinchillas, and if drug-induced reduction of food intake differs between metronidazole and metronidazole benzoate as well as between different concentrations of metronidazole benzoate suspensions. Methods: In a series of randomized, blinded, crossover experiments, the effects of oral administration of metronidaole at 20 mg/kg was evaluated following q12h (for 3 days, n = 12) or q24h (for 5 days, n = 9) dosing using two different drug compounding protocols. Results: Following administration of metronidazole, food intake was significantly reduced within 24 hours of drug administration, regardless of drug formulation type, compounding protocol, or drug concentration. Variation in metronidazole-induced food intake reduction differed widely between individual chinchillas, with a reduction of >50% of food intake reported in 50% of the animals following q12h administration, and 33% of animals after q24h administration of metronidazole. The reduction in food intake was self-limiting and return to normal food intake occurred after discontinuation of metronidazole administration. Neurological adverse effects were observed in one animal following q12h administration. Conclusions and clinical relevance: The oral administration of metronidazole and metronidazole benzoate a published dosages can result in a marked reduction in food intake, and therefore, this drug should be used cautiously in chinchillas and food intake carefully monitored.

Amlodipine is a calcium channel blocker shown to be effective in lowering blood pressure with minimal adverse effects in mammals. To provide a retrospective evaluation of amlodipine use in psittacine birds, medical records were reviewed for all avian patients prescribed amlodipine for treatment for the presumptive diagnosis of hypertension, based on clinical signs and indirect blood pressure measurements. Five birds were treated with amlodipine between 2010 and 2018. The median age was 33 years (range, 22-37 years) and 3/5 birds presented for ataxia or seizures. The median indirect systolic blood pressure at diagnosis was 243 mm Hg (range, 200-275 mm Hg). In 3/5 birds, amlodipine was the only drug prescribed, whereas, in 2/5 birds, enalapril was also prescribed in addition to amlodipine. In addition to the prescription of enalapril, blood pressure measurements were obtained indirectly, which are variables to be considered in this report. The initial median dose of amlodipine prescribed was 0.4 mg/kg (range, 0.1-0.4 mg/kg) PO q24h. In 3/5 birds, amlodipine administration was increased either in dose or frequency. Median follow-up time was 25 months (range, 2-55 months) after the initiation of amlodipine treatment. Owners in all 5 cases reported improvement of clinical signs by a median time of 2 months (range, 1-15 months). Three of 5 birds (60%) demonstrated a decreasing trend in blood pressure during the first 6 months after treatment with amlodipine was started (average, ≥20% decrease). Prospective, controlled studies are needed to investigate the efficacy of amlodipine in psittacine birds.

Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity in vitro is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable in vitro approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed in vivo. Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development.

Sonic hedgehog (Shh) pathway disruption causes craniofacial malformations including orofacial clefts (OFCs) of the lip and palate. In normal craniofacial morphogenesis, Shh signals to multipotent cranial neural crest cells (cNCCs) and was recently discovered to regulate the angiogenic transcriptome, including expression markers of perivascular cells and pericytes. The mural cells of microvasculature, pericytes in the brain and face differentiate from cNCCs, but their role in facial development is not known. Here, we examined microvascular morphogenesis in a mouse model of Shh pathway antagonist-induced cleft lip and the impact of cNCC-specific Shh pathway activation in a cNCC-endothelial cell co-culture system. During cleft pathogenesis in vivo, disrupted microvascular morphogenesis localized with attenuated tissue outgrowth in the medial nasal processes that form the upper lip. In vitro, we found that human umbilical vein endothelial cell (HUVEC) cord formation was not affected by direct Shh pathway perturbation. However, in a co-culture system in which cNCCs directly interact with endothelial cells, cNCC-autonomous Shh pathway activity significantly prolonged endothelial cord network stability. Taken together, these findings support the premise that Shh pathway activation in cNCCs promotes pericyte-like function and microvascular stability. In addition to suggesting a previously unrecognized role for Shh signaling in facial development, these studies also identify perivascular differentiation and microvascular morphogenesis as new focuses for understanding normal and abnormal craniofacial development.

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.

Sonic Hedgehog (Shh) signaling plays key regulatory roles in embryonic development and postnatal homeostasis and repair. Modulation of the Shh pathway is known to cause malformations and malignancies associated with dysregulated tissue growth. However, our understanding of the molecular mechanisms by which Shh regulates cellular proliferation is incomplete. Here, using mouse embryonic fibroblasts, we demonstrate that the Forkhead box gene Foxd1 is transcriptionally regulated by canonical Shh signaling and required for downstream proliferative activity. We show that Foxd1 deletion abrogates the proliferative response to SHH ligand while FOXD1 overexpression alone is sufficient to induce cellular proliferation. The proliferative response to both SHH ligand and FOXD1 overexpression was blocked by pharmacologic inhibition of cyclin-dependent kinase signaling. Time-course experiments revealed that Shh pathway activation of Foxd1 is followed by downregulation of Cdkn1c, which encodes a cyclin-dependent kinase inhibitor. Consistent with a direct transcriptional regulation mechanism, we found that FOXD1 reduces reporter activity of a Fox enhancer sequence in the second intron of Cdkn1c. Supporting the applicability of these findings to specific biological contexts, we show that Shh regulation of Foxd1 and Cdkn1c is recapitulated in cranial neural crest cells and provide evidence that this mechanism is operational during upper lip morphogenesis. These results reveal a novel Shh-Foxd1-Cdkn1c regulatory circuit that drives the mitogenic action of Shh signaling and may have broad implications in development and disease.

OBJECTIVE To evaluate whether the sedative effects of a combination of dexmedetomidine and ketamine differed when it was administered IM in a hind limb versus a forelimb of leopard geckos (Eublepharis macularius). DESIGN Randomized crossover study. ANIMALS 9 healthy adult leopard geckos. PROCEDURES Each gecko received a combination of dexmedetomidine (0.1 mg/kg [0.045 mg/lb]) and ketamine (10 mg/kg [4.5 mg/lb]; DK), IM, in a forelimb and hind limb in a randomized order and with a 7-day interval between treatments. All geckos received atipamezole (1 mg/kg [0.45 mg/lb], SC) 45 minutes after DK administration. Palpebral and righting reflexes, jaw tone, and superficial pain and escape responses were each assessed on a 3-point scale, and the scores for those variables were summed to calculate a sedation score. Those variables and heart and respiratory rates were evaluated at predetermined times before and for 1 hour after DK administration. RESULTS For the forelimb treatment, mean sedation score was higher and mean heart rate was lower than the corresponding values for the hind limb treatment at most time points after DK administration. The righting reflex remained intact for all 9 geckos following the hind limb treatment but became absent in 7 geckos following the forelimb treatment. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the extent of DK-induced sedation was greater when the combination was injected IM in a forelimb versus a hind limb of leopard geckos, likely owing to a hepatic first-pass effect following hind limb injection. In reptiles, IM hind limb administration of drugs that undergo hepatic metabolism and excretion is not recommended.

BACKGROUND:The evolutionarily conserved Sonic Hedgehog (Shh) signaling pathway is essential for embryogenesis and orofacial development. SHH ligand secreted from the surface ectoderm activates pathway activity in the underlying cranial neural crest cell (cNCC)-derived mesenchyme of the developing upper lip and palate. Disruption of Shh signaling causes orofacial clefts, but the biological action of Shh signaling and the full set of Shh target genes that mediate normal and abnormal orofacial morphogenesis have not been described.RESULTS:Using comparative transcriptional profiling, we have defined the Shh-regulated genes of the cNCC-derived mesenchyme. Enrichment analysis demonstrated that in cultured cNCCs, Shh-regulated genes are involved in smooth muscle and chondrocyte differentiation, as well as regulation of the Forkhead family of transcription factors, G1/S cell cycle transition, and angiogenesis. Next, this gene set from Shh-activated cNCCs in vitro was compared to the set of genes dysregulated in the facial primordia in vivo during the initial pathogenesis of Shh pathway inhibitor-induced orofacial clefting. Functional gene annotation enrichment analysis of the 112 Shh-regulated genes with concordant expression changes linked Shh signaling to interdependent and unique biological processes including mesenchyme development, cell adhesion, cell proliferation, cell migration, angiogenesis, perivascular cell markers, and orofacial clefting.CONCLUSIONS:We defined the Shh-regulated transcriptome of the cNCC-derived mesenchyme by comparing the expression signatures of Shh-activated cNCCs in vitro to primordial midfacial tissues exposed to the Shh pathway inhibitor in vivo. In addition to improving our understanding of cNCC biology by determining the identity and possible roles of cNCC-specific Shh target genes, this study presents novel candidate genes whose examination in the context of human orofacial clefting etiology is warranted.

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.

OBJECTIVE To compare sedation in cockatiels (Nymphicus hollandicus) after intranasal administration of midazolam and midazolam-butorphanol. ANIMALS 9 healthy adult cockatiels. PROCEDURES A randomized, controlled, blinded, complete crossover study was conducted. Birds were assigned to 3 treatment groups. Midazolam (3 mg/kg), midazolam-butorphanol (3 mg/kg for each drug), or sterile saline (0.9% NaCl) solution (control treatment) was administered intranasally. Sedation quality was assessed at 3 time points by use of eye and body position; response to visual, auditory, and tactile stimulation; and response during manual restraint on the basis of eye position and struggling intensity. To evaluate attenuation of the manual restraint-induced stress response, heart rate, respiratory rate, and cloacal temperature were measured over a 15-minute period. Treatments were repeated after a minimum washout period of 7 days. RESULTS Median onset of first sedation effects was 85 seconds (range, 60 to 120 seconds) for midazolam and 90 seconds (range, 45 to 180 seconds) for midazolam-butorphanol. Midazolam-butorphanol resulted in significantly less vigorous struggling during restraint than did midazolam or the control treatment. Heart rate did not differ significantly among treatments. The stress-induced increase in respiratory rate was significantly attenuated by midazolam and midazolam-butorphanol, whereas the increase in cloacal temperature was not attenuated by midazolam or midazolam-butorphanol. CONCLUSIONS AND CLINICAL RELEVANCE Intranasal administration of midazolam and midazolam-butorphanol resulted in a rapid onset of sedation in cockatiels. Midazolam-butorphanol resulted in deeper sedation in both restrained and unrestrained birds than did midazolam alone. Midazolam and midazolam-butorphanol both provided safe and effective sedation in cockatiels.

OBJECTIVE:To compare dexmedetomidine-midazolam with alfaxalone-midazolam for sedation in leopard geckos (Eublepharis macularius). STUDY DESIGN:Prospective, randomized, blinded, complete crossover study. ANIMALS:Nine healthy adult leopard geckos. METHODS:Geckos were administered a combination of dexmedetomidine (0.1 mg kg-1) and midazolam (1.0 mg kg-1; treatment D-M) or alfaxalone (15 mg kg-1) and midazolam (1.0 mg kg-1; treatment A-M) subcutaneously craniodorsal to a thoracic limb. Heart rate (HR), respiratory rate (fR), righting reflex, palpebral reflex, superficial and deep pain reflexes, jaw tone and escape response were assessed every 5 minutes until reversal. Conditions for intubation and response to needle prick were evaluated. Antagonist drugs [flumazenil (0.05 mg kg-1) ± atipamezole (1.0 mg kg-1)] were administered subcutaneously, craniodorsal to the contralateral thoracic limb, 45 minutes after initial injection, and animals were monitored until recovery. RESULTS:HR, but not fR, decreased significantly over time in both treatments. HR was significantly lower than baseline at all time points in D-M and for all but the 5 and 10 minute time points in A-M. HR was significantly higher in A-M at all time points after drug administration when compared with D-M. Sedation scores between protocols were similar for most time points. All animals in A-M lost righting reflex compared with seven out of nine (78%) geckos in D-M. Geckos in A-M lost righting reflex for significantly longer time. Mean ± standard deviation time to recovery after antagonist administration was 6.1 ± 2.2 minutes for D-M and 56 ± 29 minutes for A-M, and these times were significantly different. CONCLUSIONS AND CLINICAL RELEVANCE:Combination D-M or A-M provided sedation of a level expected to allow physical examinations and venipuncture in leopard geckos. A-M provided a faster onset of sedation compared with D-M. Recovery was significantly faster following antagonist reversal of D-M, compared with A-M.

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.

Human embryonic stem cell line WA01 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC+ endothelial cells express tdTomato and CD43+ hematopoietic progenitors express eGFP.