Causative Effects of Genetically Determined High Maternal/Fetal Endothelin-1 on Preeclampsia-Like Conditions in Mice.

Endothelin-1 (ET-1) is implicated in the pathophysiology of preeclampsia. An association between an EDN1 gene polymorphism with high ET-1 and preeclampsia was reported in humans, but their cause and effect relationships have not been defined. We examined the pregnancy effects in mice with a modified Edn1 allele that increases mRNA stability and thus ET-1 production. Heterozygous Edn1H/+ females showed no obvious abnormalities before pregnancy, but when mated with wild-type (WT) males developed a full spectrum of preeclampsia-like phenotypes, including increased systolic blood pressure, proteinuria, glomerular endotheliosis, and intrauterine fetal growth restriction. At 7.5 days post-coitus, the embryos from Edn1H/+ dams, regardless of their Edn1 genotype, lagged 12 hours in development compared with embryos from WT dams, had disoriented ectoplacental cones, and retained high E-cadherin expression. In contrast, WT females mated with Edn1H/+ males, which also carried half of the fetuses with Edn1H/+ genotype, showed a mild systolic blood pressure increase only. These WT dams had 2× higher plasma soluble fms-like tyrosine kinase-1 than WT dams mated with WT males. In human first trimester trophoblast cells, pharmacological doses of ET-1 increased the cellular sFlt1 transcripts and protein secretion via both type A and B ET-1 receptors. Our data demonstrate that high maternal ET-1 production causes preeclampsia-like phenotypes during pregnancy, affecting both initial stage of trophoblast differentiation/invasion and maternal peripheral vasculature during late gestation. High fetal ET-1 production, however, could cause increased soluble fms-like tyrosine kinase-1 in the maternal circulation and contribute to blood pressure elevation.

Human germline hedgehog pathway mutations predispose to fatty liver.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. METHODS: Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2+/-) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. RESULTS: Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2+/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2+/- mice exposed to a high-fat diet. CONCLUSIONS: Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous buildup (scar tissue) and inflammation of the liver tissue. For the first time patients with holoprosencephaly, a disease caused by SHH signaling mutations, are shown to have increased liver steatosis independent of obesity. This observation was recapitulated in a mouse model of attenuated SHH signaling that also showed increased liver steatosis but with decreased fibrosis and inflammation. While SHH inhibition is associated with a good NAFLD prognosis, this increase in liver fat accumulation in the context of SHH signaling inhibition must be studied prospectively to evaluate its long-term effects, especially in individuals with Western-type dietary habits.

Genetic vulnerabilities to prenatal alcohol exposure: Limb defects in sonic hedgehog and GLI2 heterozygous mice.

BACKGROUND: Genetic factors influence the physical and neurobehavioral manifestations of prenatal alcohol exposure (PAE). Animal models allow the investigation of specific genes that confer vulnerability to, or protection from, birth defects associated with fetal alcohol spectrum disorders (FASDs). The objective of the present experiments was to determine if genetic alterations in the Sonic Hedgehog (Shh) signaling pathways affect the vulnerability to PAE-induced skeletal defects involving the forelimbs and/or hindlimbs. METHOD: Wild-type C57BL/6J female mice were bred with males in which one copy of the Shh or Gli2 genes had been knocked out, to produce litters with both wild-type (+/+) and heterozygous (+/-) embryos. Alcohol doses (two injections of 2.9 g/kg, 4 hours apart) or vehicles were administered starting at gestational day (GD) 9.25, 9.5, or 9.75, a critical exposure time for inducing limb defects. Limb defects were examined at GD 17 using a dysmorphology scale based on abnormalities ranging from increased interdigital spacing to the deletion of multiple fingers and the ulna. RESULTS: Alcohol treatment caused a high incidence of forelimb defects, particularly on the right side, that was higher in Shh+/- and Gli2+/- fetuses compared to wild-type fetuses. Dysmorphology scores were also significantly higher in the Shh+/- and Gli2+/- mice. CONCLUSIONS: These results extend previous findings demonstrating enhanced sensitivity to PAE-induced craniofacial dysmorphology and support the hypothesis that genetic alterations in the Shh signaling pathway influences the vulnerability to alcohol-induced birth defects. Moreover, these results emphasize the importance of understanding the interactions between genes and prenatal exposure to alcohol or other teratogens. Birth Defects Research 109:860-865, 2017. © 2017 Wiley Periodicals, Inc.

Preaxial polydactyly following early gestational exposure to the smoothened agonist, SAG, in C57BL/6J mice.

BACKGROUND: While pharmacological activation of the Hedgehog (HH) signaling pathway may have therapeutic benefits for developmental and adult diseases, its teratogenic potential is of concern. The membrane molecule Smoothened (SMO) transduces HH signaling and can be acutely modulated by antagonists and agonists. The objective of the current experiments was to determine how maternal treatment with the Smo agonist, SAG, affects the developing limb. METHODS: Pregnant C57BL/6J mice received a single injection of SAG (15, 17, or 20 mg/kg, i.p.) or its vehicle on gestational day (GD) 9.25, the time of limb bud induction. Embryos were examined on GD 15 for gross dysmorphology and skeletal staining was performed to visualize the number and type of digits on the fore- and hindlimbs. Additionally, in situ hybridization was performed 4 hr after GD 9.25 SAG administration to determine SAG's effects on Gli1 and Gli2 mRNA expression. RESULTS: The most prevalent effect of SAG was the dose-dependent induction of pre-axial polydactyly; defects ranged from a broad thumb to the duplication of two finger-like digits on the preaxial side of the thumb. The highest SAG dose was effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated. CONCLUSION: Preaxial polydactyly can be caused in the developing embryo by acute maternal administration of a Smo agonist that activates HH signaling. These results are consistent with the preaxial polydactyly induced in developmental disorders associated with mutations in HH signaling genes.Birth Defects Research 109:49-54, 2017. © 2016 Wiley Periodicals, Inc.

Dose-dependent teratogenicity of the synthetic cannabinoid CP-55,940 in mice.

Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n>10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose-response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB.

Hypothalamic-pituitary-adrenal axis and behavioral dysfunction following early binge-like prenatal alcohol exposure in mice.

The range of defects that fall within fetal alcohol spectrum disorder (FASD) includes persistent behavioral problems, with anxiety and depression being two of the more commonly reported issues. Previous studies of rodent FASD models suggest that interference with hypothalamic-pituitary-adrenal (HPA) axis structure and/or function may be the basis for some of the prenatal alcohol (ethanol) exposure (PAE)-induced behavioral abnormalities. Included among the previous investigations are those illustrating that maternal alcohol treatment limited to very early stages of pregnancy (i.e., gestational day [GD]7 in mice; equivalent to the third week post-fertilization in humans) can cause structural abnormalities in areas such as the hypothalamus, pituitary gland, and other forebrain regions integral to controlling stress and behavioral responses. The current investigation was designed to further examine the sequelae of prenatal alcohol insult at this early time period, with particular attention to HPA axis-associated functional changes in adult mice. The results of this study reveal that GD7 PAE in mice causes HPA axis dysfunction, with males and females showing elevated corticosterone (CORT) and adrenocorticotropic hormone (ACTH) levels, respectively, following a 15-min restraint stress exposure. Males also showed elevated CORT levels following an acute alcohol injection of 2.0 g/kg, while females displayed blunted ACTH levels. Furthermore, analysis showed that anxiety-like behavior was decreased after GD7 PAE in female mice, but was increased in male mice. Collectively, the results of this study show that early gestational alcohol exposure in mice alters long-term HPA axis activity and behavior in a sexually dimorphic manner.

Fetal alcohol spectrum disorder: pathogenesis and mechanisms.

This chapter provides an overview of animal model-based studies that have generated information critical to our understanding of the pathogenesis and mechanisms underlying alcohol-induced birth defects, in particular those involving the brain. Focus is placed on the developing organism itself, rather than the mother, placenta, or other extraembryonic tissues. Components of the cascades of alcohol-induced damage that are considered herein are excessive cell death, changes in the cell cycle and proliferation, cell migration, cell morphogenesis, and gene expression as well as free radical damage and interference with cell signaling. The roles played by one or more of these various factors in the genesis of structural and functional birth defects are dependent upon alcohol exposure patterns and dosage, the involved tissue, and the prenatal stage(s) at the time of exposure. Technologic advances and rapidly increasing knowledge in the fields of genetics, cell, developmental, and neurobiology are critical to accurately piecing together experimental evidence in refining our understanding of the genesis of alcohol-induced birth defects, to the planning and execution of future studies, and to applying the knowledge gained to diminish the severity or occurrence of fetal alcohol spectrum disorder.

Prenatal alcohol exposure reduces magnetic susceptibility contrast and anisotropy in the white matter of mouse brains.

Prenatal alcohol exposure can result in long-term cognitive and behavioral deficits. Fetal alcohol spectrum disorder (FASD) refers to a range of permanent birth defects caused by prenatal alcohol exposure, and is the most common neurodevelopmental disorder in the US. Studies by autopsy and conventional structural MRI indicate that the midline structures of the brain are particularly vulnerable to prenatal alcohol exposure. Diffusion tensor imaging (DTI) has shown that abnormalities in brain white matter especially the corpus callosum are very common in FASD. Quantitative susceptibility mapping (QSM) is a novel technique that measures tissue's magnetic property. Such magnetic property is affected by tissue microstructure and molecular composition including that of myelin in the white matter. In this work, we studied three major white matter fiber bundles of a mouse model of FASD and compared it to control mice using both QSM and DTI. QSM revealed clear and significant abnormalities in anterior commissure, corpus callosum, and hippocampal commissure, which were likely due to reduced myelination. Our data also suggested that QSM may be even more sensitive than DTI for examining changes due to prenatal alcohol exposure. Although this is a preclinical study, the technique of QSM is readily translatable to human brain.

Characterization of subtle brain abnormalities in a mouse model of Hedgehog pathway antagonist-induced cleft lip and palate.

Subtle behavioral and cognitive deficits have been documented in patient cohorts with orofacial clefts (OFCs). Recent neuroimaging studies argue that these traits are associated with structural brain abnormalities but have been limited to adolescent and adult populations where brain plasticity during infancy and childhood may be a confounding factor. Here, we employed high resolution magnetic resonance microscopy to examine primary brain morphology in a mouse model of OFCs. Transient in utero exposure to the Hedgehog (Hh) signaling pathway antagonist cyclopamine resulted in a spectrum of facial dysmorphology, including unilateral and bilateral cleft lip and palate, cleft of the secondary palate only, and a non-cleft phenotype marked by midfacial hypoplasia. Relative to controls, cyclopamine-exposed fetuses exhibited volumetric differences in several brain regions, including hypoplasia of the pituitary gland and olfactory bulbs, hyperplasia of the forebrain septal region, and expansion of the third ventricle. However, in affected fetuses the corpus callosum was intact and normal division of the forebrain was observed. This argues that temporally-specific Hh signaling perturbation can result in typical appearing OFCs in the absence of holoprosencephaly--a condition classically associated with Hh pathway inhibition and frequently co-occurring with OFCs. Supporting the premise that some forms of OFCs co-occur with subtle brain malformations, these results provide a possible ontological basis for traits identified in clinical populations. They also argue in favor of future investigations into genetic and/or environmental modulation of the Hh pathway in the etiopathogenesis of orofacial clefting.

Dysmorphogenic effects of first trimester-equivalent ethanol exposure in mice: a magnetic resonance microscopy-based study.

BACKGROUND: The first trimester of human development and the equivalent developmental period in animal models is a time when teratogenic ethanol (EtOH) exposure induces the major structural birth defects that fall within fetal alcohol spectrum disorder (FASD). Previous FASD research employing an acute high dose maternal intraperitoneal EtOH treatment paradigm has identified sensitive periods for a number of these defects. Extending this work, this investigation utilized high resolution magnetic resonance microscopy (MRM)-based analyses to examine the dysmorphology resulting from maternal dietary EtOH intake occurring during selected first trimester-equivalent time periods. METHODS: Female C57Bl/6J mice were acclimated to a liquid 4.8% EtOH (v/v)-containing diet, then bred while on standard chow. Dams were again provided the EtOH-containing liquid diet for a period that extended either from the beginning of gestational day (GD) 7 to the end of GD 11 or from the beginning of GD 12 to the end of GD 16. On GD 17, a subset of fetuses was selected for MRM-based analyses. Group comparisons were made for litter characteristics and gross dysmorphology, as well as whole and regional brain volumes. RESULTS: EtOH-induced stage of exposure-dependent structural brain abnormalities were observed. The GD 7 to 11 EtOH-exposed group presented with a significant decrease in cerebellar volume and an increase in septal volume, while GD 12 to 16 EtOH treatment resulted in a reduction in right hippocampal volume accompanied by enlarged pituitaries. Additionally, the GD 12 to 16 EtOH exposure caused a high incidence of edema/fetal hydrops. CONCLUSIONS: These results illustrate the teratogenic impact of maternal dietary EtOH intake occurring at time periods approximately equivalent to weeks 3 through 6 (GD 7 to 11 in mice) and weeks 7 through 12 (GD 12 to 16 in mice) of human gestation, further documenting EtOH's stage of exposure-dependent neuroteratogenic end points and highlighting the vulnerability of selected brain regions during the first trimester. Additionally they suggest that clinical attention should be paid to fetal hydrops as a likely component of FASD.

The teratogenic effects of prenatal ethanol exposure are exacerbated by Sonic Hedgehog or GLI2 haploinsufficiency in the mouse.

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 i.p. 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.

Cleft Palate in a Mouse Model of SOX2 Haploinsufficiency.

OBJECTIVE: While SEX-determining region Y-Box 2 (SOX2) mutations are typically recognized as yielding ocular and central nervous system abnormalities, they have also been associated with other craniofacial defects. To elucidate the genesis of the latter, Sox2 hypomorphic (Sox2(HYP)) mice were examined, with particular attention to secondary palatal development. RESULTS: Clefts of the secondary palate were found to be highly penetrant in Sox2(HYP) mice. The palatal clefting occurred in the absence of mandibular hypoplasia and resulted from delayed or failed shelf elevation. CONCLUSIONS: Sox2 hypomorphism can result in clefting of the secondary palate, an effect that appears to be independent of mandibular hypoplasia and is thus expected to result from an abnormality that is inherent to the palatal shelves and/or their progenitor tissues. Further clinical attention relative to SOX2 mutations as a basis for secondary palatal clefts appears warranted.

Magnetic resonance microscopy-based analyses of the neuroanatomical effects of gestational day 9 ethanol exposure in mice.

Animal model-based studies have shown that ethanol exposure during early gestation induces developmental stage-specific abnormalities of the face and brain. The exposure time-dependent variability in ethanol's teratogenic outcomes is expected to contribute significantly to the wide spectrum of effects observed in humans with fetal alcohol spectrum disorder (FASD). The work presented here employs a mouse FASD model and magnetic resonance microscopy (MRM; high resolution magnetic resonance imaging) in studies designed to further our understanding of the developmental stage-specific defects of the brain that are induced by ethanol. At neurulation stages, i.e. at the beginning of gestational day (GD) 9 and again 4 hours later, time-mated C57Bl/6J dams were intraperitoneally administered 2.9 g/kg ethanol or vehicle. Ethanol-exposed fetuses were collected on GD 17, processed for MRM analysis, and results compared to comparably staged controls. Linear and volume measurements as well as shape changes for numerous individual brain regions were determined. GD 9 ethanol exposure resulted in significantly increased septal region width, reduction of cerebellar volume, and enlargement of all of the ventricles. Additionally, the results of shape analyses showed that many areas of the ethanol-exposed brains including the cerebral cortex, hippocampus and right striatum were significantly misshapen. These data demonstrate that ethanol can induce dysmorphology that may not be obvious based on volumetric analyses alone, highlight the asymmetric aspects of ethanol-induced defects, and add to our understanding of ethanol's developmental stage-dependent neuroteratogenesis.

Mitogen-activated protein kinase modulates ethanol inhibition of cell adhesion mediated by the L1 neural cell adhesion molecule.

There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise, leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain, did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity.

Ethanol-induced face-brain dysmorphology patterns are correlative and exposure-stage dependent.

Prenatal ethanol exposure is the leading preventable cause of congenital mental disability. Whereas a diagnosis of fetal alcohol syndrome (FAS) requires identification of a specific pattern of craniofacial dysmorphology, most individuals with behavioral and neurological sequelae of heavy prenatal ethanol exposure do not exhibit these defining facial characteristics. Here, a novel integration of MRI and dense surface modeling-based shape analysis was applied to characterize concurrent face-brain phenotypes in C57Bl/6J fetuses exposed to ethanol on gestational day (GD)7 or GD8.5. The facial phenotype resulting from ethanol exposure depended upon stage of insult and was predictive of unique patterns of corresponding brain abnormalities. Ethanol exposure on GD7 produced a constellation of dysmorphic facial features characteristic of human FAS, including severe midfacial hypoplasia, shortening of the palpebral fissures, an elongated upper lip, and deficient philtrum. In contrast, ethanol exposure on GD8.5 caused mild midfacial hypoplasia and palpebral fissure shortening, a shortened upper lip, and a preserved philtrum. These distinct, stage-specific facial phenotypes were associated with unique volumetric and shape abnormalities of the septal region, pituitary, and olfactory bulbs. By demonstrating that early prenatal ethanol exposure can cause more than one temporally-specific pattern of defects, these findings illustrate the need for an expansion of current diagnostic criteria to better capture the full range of facial and brain dysmorphology in fetal alcohol spectrum disorders.

SOX2 hypomorphism disrupts development of the prechordal floor and optic cup.

Haploinsufficiency for the HMG-box transcription factor SOX2 results in abnormalities of the human ventral forebrain and its derivative structures. These defects include anophthalmia (absence of eye), microphthalmia (small eye) and hypothalamic hamartoma (HH), an overgrowth of the ventral hypothalamus. To determine how Sox2 deficiency affects the morphogenesis of the ventral diencephalon and eye, we generated a Sox2 allelic series (Sox2(IR), Sox2(LP), and Sox2(EGFP)), allowing for the generation of mice that express germline hypomorphic levels (<40%) of SOX2 protein and that faithfully recapitulate SOX2 haploinsufficient human phenotypes. We find that Sox2 hypomorphism significantly disrupts the development of the posterior hypothalamus, resulting in an ectopic protuberance of the prechordal floor, an upregulation of Shh signaling, and abnormal hypothalamic patterning. In the anterior diencephalon, both the optic stalks and optic cups (OC) of Sox2 hypomorphic (Sox2(HYP)) embryos are malformed. Furthermore, Sox2(HYP) eyes exhibit a loss of neural potential and coloboma, a common phenotype in SOX2 haploinsufficient humans that has not been described in a mouse model of SOX2 deficiency. These results establish for the first time that germline Sox2 hypomorphism disrupts the morphogenesis and patterning of the hypothalamus, optic stalk, and the early OC, establishing a model of the development of the abnormalities that are observed in SOX2 haploinsufficient humans.

Regional brain volume reductions relate to facial dysmorphology and neurocognitive function in fetal alcohol spectrum disorders.

Individuals with heavy prenatal alcohol exposure can experience significant deficits in cognitive and psychosocial functioning and alterations in brain structure that persist into adulthood. In this report, data from 99 participants collected across three sites (Los Angeles and San Diego, California, and Cape Town, South Africa) were analyzed to examine relationships between brain structure, neurocognitive function, facial morphology, and maternal reports of quantities of alcohol consumption during the first trimester. Across study sites, we found highly significant volume reductions in the FASD group for all of the brain regions evaluated. After correcting for scan location, age, and total brain volume, these differences remained significant in some regions of the basal ganglia and diencephalon. In alcohol-exposed subjects, we found that smaller palpebral fissures were significantly associated with reduced volumes in the ventral diencephalon bilaterally, that greater dysmorphology of the philtrum predicted smaller volumes in basal ganglia and diencephalic structures, and that lower IQ scores were associated with both smaller basal ganglia volumes and greater facial dysmorphology. In subjects from South Africa, we found a significant negative correlation between intracranial volume and total number of drinks per week in the first trimester. These results corroborate previous reports that prenatal alcohol exposure is particularly toxic to basal ganglia and diencephalic structures. We extend previous findings by illustrating relationships between specific measures of facial dysmorphology and the volumes of particular subcortical structures, and for the first time show that continuous measures of maternal alcohol consumption during the first trimester relates to overall brain volume reduction.

Callosal thickness reductions relate to facial dysmorphology in fetal alcohol spectrum disorders.

BACKGROUND: Structural abnormalities of the corpus callosum (CC), such as reduced size and increased shape variability, have been documented in individuals with fetal alcohol spectrum disorders (FASD). However, the regional specificity of altered CC structure, which may point to the timing of neurodevelopmental disturbances and/or relate to specific functional impairments, remains unclear. Furthermore, associations between facial dysmorphology and callosal structure remain undetermined. METHODS: One hundred and fifty-three participants (age range 8 to 16) including 82 subjects with FASD and 71 nonexposed controls were included in this study. The structural magnetic resonance imaging data of these subjects was collected at 3 sites (Los Angeles and San Diego, California, and Cape Town, South Africa) and analyzed using classical parcellation schemes, as well as more refined surface-based geometrical modeling methods, to identify callosal morphological alterations in FASD at high spatial resolution. RESULTS: Reductions in callosal thickness and area, specifically in the anterior third and the splenium, were observed in FASD compared with nonexposed controls. In addition, reduced CC thickness and area significantly correlated with reduced palpebral fissure length. CONCLUSIONS: Consistent with previous reports, findings suggest an adverse effect of prenatal alcohol exposure on callosal growth and further indicate that fiber pathways connecting frontal and parieto-occipital regions in each hemisphere may be particularly affected. Significant associations between callosal and facial dysmorphology provide evidence for a concurrent insult to midline facial and brain structural development in FASD.

Abnormal cortical thickness alterations in fetal alcohol spectrum disorders and their relationships with facial dysmorphology.

Accumulating evidence from structural brain imaging studies on individuals with fetal alcohol spectrum disorder (FASD) has supported links between prenatal alcohol exposure and brain morphological deficits. Although global and regional volumetric reductions appear relatively robust, the effects of alcohol exposure on cortical thickness and relationships with facial dysmorphology are not yet known. The structural magnetic resonance imaging data from 69 children and adolescents with FASD and 58 nonexposed controls collected from 3 sites were examined using FreeSurfer to detect cortical thickness changes across the entire brain in FASD and their associations with facial dysmorphology. Controlling for brain size, subjects with FASD showed significantly thicker cortices than controls in several frontal, temporal, and parietal regions. Analyses conducted within site further revealed prominent group differences in left inferior frontal cortex within all 3 sites. In addition, increased inferior frontal thickness was significantly correlated with reduced palpebral fissure length. Consistent with previous reports, findings of this study are supportive of regional increases in cortical thickness serving as a biomarker for disrupted brain development in FASD. Furthermore, the significant associations between thickness and dysmorphic measures suggest that the severity of brain anomalies may be reflected by that of the face.

Magnetic resonance-based imaging in animal models of fetal alcohol spectrum disorder.

Magnetic resonance imaging (MRI) techniques, such as magnetic resonance microscopy (MRM), diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS), have recently been applied to the study of both normal and abnormal structure and neurochemistry in small animals. Herein, findings from studies in which these methods have been used for the examination of animal models of Fetal Alcohol Spectrum Disorder (FASD) are discussed. Emphasis is placed on results of imaging studies in fetal and postnatal mice that have highlighted the developmental stage dependency of prenatal ethanol exposure-induced CNS defects. Consideration is also given to the promise of methodological advances to allow in vivo studies of aberrant brain and behavior relationships in model animals and to the translational nature of this work.