The impact of prenatal alcohol, synthetic cannabinoid and co-exposure on behavioral adaptations in adolescent offspring and alcohol self-administration in adulthood.

Prenatal exposure to alcohol or cannabinoids can produce enduring neurobiological, cognitive, and behavioral changes in the offspring. Furthermore, prenatal co-exposure to alcohol and cannabinoids induces malformations in brain regions associated with reward and stress-related circuitry. This study examined the effects of co-exposure to alcohol and the synthetic cannabinoid (SCB) CP55,940 throughout gastrulation and neurulation in rats on basal corticosterone levels and a battery of behavioral tests during adolescence and alcohol self-administration in adulthood. Importantly, we find that prenatal alcohol exposure (PAE) caused lower baseline corticosterone levels in adolescent males and females. Co-exposure to alcohol + CP produced hyperactivity during open field test in males, but not females. During the two-bottle choice alcohol-drinking procedure, prenatal cannabinoid exposed male and female adolescent rats drank more alcohol than their vehicle-exposed controls. In adulthood, female rats treated with prenatal cannabinoid exposure (PCE), showed an overall total increase in alcohol intake during alcohol self-administration; but this was not found in males. When the reinforcer was changed to a 1% sucrose solution, male rats exposed to PCE, showed a reduced self-administration compared to vehicle-exposed males, potentially indicative of an anhedonic response. This lower self-administration persisted when 20% alcohol was reintroduced to the sucrose solution. Lastly, following an abstinence period, there were no changes due to prenatal drug exposure in either males or females. Overall, these data suggest lasting consequences of prenatal alcohol and cannabinoid exposure during adolescence and adulthood in male and female rats.

Gastrulation-stage alcohol exposure induces similar rates of craniofacial malformations in male and female C57BL/6J mice.

BACKGROUND: Prenatal alcohol exposure during gastrulation (embryonic day [E] 7 in mice, ~3rd week of human pregnancy) impairs eye, facial, and cortical development, recapitulating birth defects characteristic of Fetal Alcohol Syndrome (FAS). However, it is not known whether the prevalence or severity of craniofacial features associated with FAS is affected by biological sex. METHODS: The current study administered either alcohol (2.9 g/kg, two i.p. doses, 4 hr apart) or vehicle to pregnant C57BL/6J females on E7, prior to gonadal sex differentiation, and assessed fetal morphology at E17. RESULTS: Whereas sex did not affect fetal size in controls, alcohol-exposed females were smaller than both control females and alcohol-treated males. Alcohol exposure increased the incidence of eye defects to a similar degree in males and females. Together, these data suggest that females might be more sensitive to the general developmental effects of alcohol, but not effects specific to the craniofacies. Whole transcriptomic analysis of untreated E7 embryos found 214 differentially expressed genes in females vs. males, including those in pathways related to cilia and mitochondria, histone demethylase activity, and pluripotency. CONCLUSION: Gastrulation-stage alcohol induces craniofacial malformations in male and female mouse fetuses at similar rates and severity, though growth deficits are more prevalent females. These findings support the investigation of biological sex as a contributing factor in prenatal alcohol studies.

The impact of prenatal alcohol and synthetic cannabinoid exposure on behavioral adaptations in adolescent offspring and alcohol self-administration in adulthood.

Prenatal exposure to alcohol or cannabinoids can produce enduring neurobiological, cognitive, and behavioral changes in the offspring. Furthermore, prenatal co-exposure to alcohol and cannabinoids induces malformations in brain regions associated with reward and stress-related circuitry. This study examined the effects of co-exposure to alcohol and the synthetic cannabinoid (SCB) CP55,940 throughout gastrulation and neurulation in rats on basal corticosterone levels and a battery of behavioral tests during adolescence and alcohol self-administration in adulthood. Importantly, we find that prenatal alcohol exposure (PAE) caused lower baseline corticosterone levels in adolescent males and females. Co-exposure to alcohol + CP produced hyperactivity during open field test in males, but not females. During the two-bottle choice alcohol-drinking procedure, prenatal cannabinoid exposed male and female adolescent rats drank more alcohol than their vehicle-exposed controls. In adulthood, female rats treated with prenatal cannabinoid exposure (PCE), showed an overall total increase in alcohol intake during alcohol self-administration; but this was not found in males. When the reinforcer was changed to a 1% sucrose solution, male rats exposed to PCE, showed a reduced self-administration compared to vehicle-exposed males, potentially indicative of an anhedonic response. This lower self-administration persisted when 20% alcohol was reintroduced to the sucrose solution. Lastly, following an abstinence period, there were no changes due to prenatal drug exposure in either males or females. Overall, these data suggest lasting consequences of prenatal alcohol and cannabinoid exposure during adolescence and adulthood in male and female rats.

Short-term transcriptomic changes in the mouse neural tube induced by an acute alcohol exposure.

Alcohol exposure during the formation and closure of the neural tube, or neurulation (embryonic day [E] 8-10 in mice; ∼4th week of human pregnancy), perturbs development of midline brain structures and significantly disrupts gene expression in the rostroventral neural tube (RVNT). Previously, alcohol exposure during neurulation was found to alter gene pathways related to cell proliferation, p53 signaling, ribosome biogenesis, immune signaling, organogenesis, and cell migration 6 or 24 h after administration. Our current study expands upon this work by investigating short-term gene expression changes in the RVNT following a single binge-like alcohol exposure during neurulation. Female C57BL/6J mice were administered a single dose of 2.9 g/kg alcohol or vehicle on E9.0 to target mid-neurulation. The RVNTs of stage-matched embryos were collected 2 or 4 h after exposure and processed for RNA-seq. Functional profiling was performed with g:Profiler, as well as with the CiliaCarta and DisGeNet databases. Two hours following E9.0 alcohol exposure, 650 genes in the RVNT were differentially expressed. Functional enrichment analysis revealed that pathways related to cellular metabolism, gene expression, cell cycle, organogenesis, and Hedgehog signaling were down-regulated, and pathways related to cellular stress response, p53 signaling, and hypoxia were up-regulated by alcohol. Four hours after alcohol exposure, 225 genes were differentially expressed. Biological processes related to metabolism, RNA binding, ribosome biogenesis, and methylation were down-regulated, while protein localization and binding, autophagy, and intracellular signaling pathways were up-regulated. Two hours after alcohol exposure, the differentially expressed genes were associated with disease terms related to eye and craniofacial development and anoxia. These data provide further information regarding the biological functions targeted by alcohol exposure during neurulation in regions of the neural tube that give rise to alcohol-sensitive midline brain structures. Disruption of these gene pathways contributes to the craniofacial and brain malformations associated with prenatal alcohol exposure.

The pro-apoptotic Bax gene modifies susceptibility to craniofacial dysmorphology following gastrulation-stage alcohol exposure.

BACKGROUND: During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol-induced cell death is the pro-apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation-stage alcohol exposure. METHODS: Male and female Bax+/- mice mated to produce embryos with full (-/- ) or partial (+/- ) Bax deletions, or Bax+/+ wild-type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS-like facial features. RESULTS: Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol-induced defects. An RNA-seq analysis of GD 7 Bax+/+ and Bax-/- embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis. CONCLUSIONS: Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation-stage alcohol exposure.

Pharmacological activation of the Sonic hedgehog pathway with a Smoothened small molecule agonist ameliorates the severity of alcohol-induced morphological and behavioral birth defects in a zebrafish model of fetal alcohol spectrum disorder.

Ethanol exposure during the early stages of embryonic development can lead to a range of morphological and behavioral differences termed fetal alcohol spectrum disorders (FASDs). In a zebrafish model, we have shown that acute ethanol exposure at 8-10 hr postfertilization (hpf), a critical time of development, produces birth defects similar to those clinically characterized in FASD. Dysregulation of the Sonic hedgehog (Shh) pathway has been implicated as a molecular basis for many of the birth defects caused by prenatal alcohol exposure. We observed in zebrafish embryos that shh expression was significantly decreased by ethanol exposure at 8-10 hpf, while smo expression was much less affected. Treatment of zebrafish embryos with SAG or purmorphamine, small molecule Smoothened agonists that activate Shh signaling, ameliorated the severity of ethanol-induced developmental malformations including altered eye size and midline brain development. Furthermore, this rescue effect of Smo activation was dose dependent and occurred primarily when treatment was given after ethanol exposure. Markers of Shh signaling (gli1/2) and eye development (pax6a) were restored in embryos treated with SAG post-ethanol exposure. Since embryonic ethanol exposure has been shown to produce later-life neurobehavioral impairments, juvenile zebrafish were examined in the novel tank diving test. Our results further demonstrated that in zebrafish embryos exposed to ethanol, SAG treatment was able to mitigate long-term neurodevelopmental impairments related to anxiety and risk-taking behavior. Our results indicate that pharmacological activation of the Shh pathway at specific developmental timing markedly diminishes the severity of alcohol-induced birth defects.

Prenatal alcohol exposure disrupts Sonic hedgehog pathway and primary cilia genes in the mouse neural tube.

Neurulation-stage alcohol exposure (NAE; embryonic day [E] 8-10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert syndrome. We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Female C57BL/6 J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on E9. Embryos were collected 6, 12, or 24 h later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). Within the first 24 h post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 forms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 h. In addition, altered expression of multiple cilia-related genes was observed at 6 h post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to alcohol-sensitive brain structures and identifies disruption of primary cilia function, or a "transient ciliopathy", as a possible cellular mechanism of prenatal alcohol pathogenesis.

Loss of tumor protein 53 protects against alcohol-induced facial malformations in mice and zebrafish.

BACKGROUND: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. METHODS: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. RESULTS: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. CONCLUSIONS: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

Programmed suppression of oxidative phosphorylation and mitochondrial function by gestational alcohol exposure correlate with widespread increases in H3K9me2 that do not suppress transcription.

BACKGROUND: A critical question emerging in the field of developmental toxicology is whether alterations in chromatin structure induced by toxicant exposure control patterns of gene expression or, instead, are structural changes that are part of a nuclear stress response. Previously, we used a mouse model to conduct a three-way comparison between control offspring, alcohol-exposed but phenotypically normal animals, and alcohol-exposed offspring exhibiting craniofacial and central nervous system structural defects. In the cerebral cortex of animals exhibiting alcohol-induced dysgenesis, we identified a dramatic increase in the enrichment of dimethylated histone H3, lysine 9 (H3K9me2) within the regulatory regions of key developmental factors driving histogenesis in the brain. However, whether this change in chromatin structure is causally involved in the development of structural defects remains unknown. RESULTS: Deep-sequencing analysis of the cortex transcriptome reveals that the emergence of alcohol-induced structural defects correlates with disruptions in the genetic pathways controlling oxidative phosphorylation and mitochondrial function. The majority of the affected pathways are downstream targets of the mammalian target of rapamycin complex 2 (mTORC2), indicating that this stress-responsive complex plays a role in propagating the epigenetic memory of alcohol exposure through gestation. Importantly, transcriptional disruptions of the pathways regulating oxidative homeostasis correlate with the emergence of increased H3K9me2 across genic, repetitive, and non-transcribed regions of the genome. However, although associated with gene silencing, none of the candidate genes displaying increased H3K9me2 become transcriptionally repressed, nor do they exhibit increased markers of canonical heterochromatin. Similar to studies in C. elegans, disruptions in oxidative homeostasis induce the chromatin looping factor SATB2, but in mammals, this protein does not appear to drive increased H3K9me2 or altered patterns of gene expression. CONCLUSIONS: Our studies demonstrate that changes in H3K9me2 associate with alcohol-induced congenital defects, but that this epigenetic change does not correlate with transcriptional suppression. We speculate that the mobilization of SATB2 and increased enrichment of H3K9me2 may be components of a nuclear stress response that preserve chromatin integrity and interactions under prolonged oxidative stress. Further, we postulate that while this response may stabilize chromatin structure, it compromises the nuclear plasticity required for normal differentiation.

Transcriptomic analyses of gastrulation-stage mouse embryos with differential susceptibility to alcohol.

Genetics are a known contributor to differences in alcohol sensitivity in humans with fetal alcohol spectrum disorders (FASDs) and in animal models. Our study profiled gene expression in gastrulation-stage embryos from two commonly used, genetically similar mouse substrains, C57BL/6J (6J) and C57BL/6NHsd (6N), that differ in alcohol sensitivity. First, we established normal gene expression patterns at three finely resolved time points during gastrulation and developed a web-based interactive tool. Baseline transcriptional differences across strains were associated with immune signaling. Second, we examined the gene networks impacted by alcohol in each strain. Alcohol caused a more pronounced transcriptional effect in the 6J versus 6N mice, matching the increased susceptibility of the 6J mice. The 6J strain exhibited dysregulation of pathways related to cell death, proliferation, morphogenic signaling and craniofacial defects, while the 6N strain showed enrichment of hypoxia and cellular metabolism pathways. These datasets provide insight into the changing transcriptional landscape across mouse gastrulation, establish a valuable resource that enables the discovery of candidate genes that may modify alcohol susceptibility that can be validated in humans, and identify novel pathogenic mechanisms of alcohol. This article has an associated First Person interview with the first author of the paper.

Transcriptome-Wide Regulation of Key Developmental Pathways in the Mouse Neural Tube by Prenatal Alcohol Exposure.

BACKGROUND: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation. METHODS: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA-seq. RESULTS: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration. CONCLUSIONS: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation-stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol's actions.

Cannabinoids Exacerbate Alcohol Teratogenesis by a CB1-Hedgehog Interaction.

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ9-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.

Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development.

3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in three genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5ß, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5ß. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism.

Knockdown of Mns1 Increases Susceptibility to Craniofacial Defects Following Gastrulation-Stage Alcohol Exposure in Mice.

BACKGROUND: MNS1 (meiosis-specific nuclear structural protein 1) is necessary for motile cilia function, such as sperm flagella or those found in the embryonic primitive node. While little is known regarding the function or expression pattern of MNS1 in the embryo, co-immunoprecipitation experiments in sperm have determined that MNS1 interacts with ciliary proteins, which are also important during development. Establishment of morphogenic gradients is dependent on normal ciliary motion in the primitive node beginning during gastrulation (gestational day [GD] 7 in the mouse, second-third week of pregnancy in humans), a critical window for face, eye, and brain development and particularly susceptible to perturbations of developmental signals. The current study investigates the role of Mns1 in craniofacial defects associated with gastrulation-stage alcohol exposure. METHODS: On GD7, pregnant Mns1+/- dams were administered 2 doses of ethanol (5.8 g/kg total) or vehicle 4 hours apart to target gastrulation. On GD17, fetuses were examined for ocular defects by scoring each eye on a scale from 1 to 7 (1 = normal, 2 to 7 = defects escalating in severity). Craniofacial and brain abnormalities were also assessed. RESULTS: Prenatal alcohol exposure (PAE) significantly increased the rate of defects in wild-type fetuses, as PAE fetuses had an incidence rate of 41.18% compared to a 10% incidence rate in controls. Furthermore, PAE interacted with genotype to significantly increase the defect rate and severity in Mns1+/- (64.29%) and Mns1-/- mice (92.31%). PAE Mns1-/- fetuses with severe eye defects also presented with craniofacial dysmorphologies characteristic of fetal alcohol syndrome and midline tissue loss in the brain, palate, and nasal septum. CONCLUSIONS: These data demonstrate that a partial or complete knockdown of Mns1 interacts with PAE to increase the susceptibility to ocular defects and correlating craniofacial and brain anomalies, likely though interaction of alcohol with motile cilia function. These results further our understanding of genetic risk factors that may underlie susceptibility to teratogenic exposures.

Combined Face-Brain Morphology and Associated Neurocognitive Correlates in Fetal Alcohol Spectrum Disorders.

BACKGROUND: Since the 1970s, a range of facial, neurostructural, and neurocognitive adverse effects have been shown to be associated with prenatal alcohol exposure. Typically, these effects are studied individually and not in combination. Our objective is to improve the understanding of the teratogenic effects of prenatal alcohol exposure by simultaneously considering face-brain morphology and neurocognitive measures. METHODS: Participants were categorized as control (n = 47), fetal alcohol syndrome (FAS, n = 22), or heavily exposed (HE) prenatally, but not eligible for a FAS diagnosis (HE, n = 50). Structural brain MRI images and high-resolution 3D facial images were analyzed using dense surface models of features of the face and surface shape of the corpus callosum (CC) and caudate nucleus (CN). Asymmetry of the CN was evaluated for correlations with neurocognitive measures. RESULTS: (i) Facial growth delineations for FAS, HE, and controls are replicated for the CN and the CC. (ii) Concordance of clinical diagnosis and face-based control-FAS discrimination improves when the latter is combined with specific brain regions. In particular, midline facial regions discriminate better when combined with a midsagittal profile of the CC. (iii) A subset of HE individuals was identified with FAS-like CN dysmorphism. The average of this HE subset was FAS-like in its facial dysmorphism. (iv) Right-left asymmetry found in the CNs of controls is not apparent for FAS, is diminished for HE, and correlates with neurocognitive measures in the combined FAS and HE population. CONCLUSIONS: Shape analysis which combines facial regions with the CN, and with the CC, better identify those with FAS. CN asymmetry was reduced for FAS compared to controls and is strongly associated with general cognitive ability, verbal learning, and recall in those with prenatal alcohol exposure. This study further extends the brain-behavior relationships known to be vulnerable to alcohol teratogenesis.

A novel method for determining sex in late term gestational mice based on the external genitalia.

In many experiments using fetal mice, it is necessary to determine the sex of the individual fetus. However, other than genotyping for sex-specific genes, there is no convenient, reliable method of sexing mice between gestational day (GD) 16.5 and GD 18.0. We designed a rapid, relatively simple visual method to determine the sex of mouse fetuses in the GD 16.5-GD 18.0 range that can be performed as part of a routine morphological assessment. By examining the genitalia for the presence or absence of key features, raters with minimal experience with the method were able to correctly identify the sex of embryos with 99% accuracy, while raters with no experience were 95% accurate. The critical genital features include: the presence or absence of urethral seam or proximal urethral meatus; the shape of the genitalia, and the presence or absence of an area related to the urethral plate. By comparing these morphological features of the external genitalia, we show a simple, accurate, and fast way to determine the sex of late stage mouse fetuses. Integrating this method into regular morphological assessments will facilitate the determination of sex differences in fetuses between GD 16.5 and GD 18.0.

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.

The contributions of Dr. Kathleen K. Sulik to fetal alcohol spectrum disorders research and prevention.

Dr. Kathleen Sulik (Kathy) has spent 35 years studying fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD). Beginning with her landmark 1981 Science paper describing the early gestational window when alcohol can cause the craniofacial malformations characteristic of FAS, Kathy has contributed a vast amount of research furthering our knowledge of FASD. After her seminal work that definitively demonstrated that alcohol is the causative factor in FAS, she and her lab went on to explore and define the stage-dependent effects of early gestational alcohol exposure on the face and brain in numerous different ways throughout her career. She explored and discovered numerous mechanisms of alcohol's effects on the embryo, as well as describing several genetic factors that can modify susceptibility to developmental alcohol exposure. She did not restrict her research to the face and brain; her lab described in intricate detail the effects of developmental alcohol exposure on many different organs, including the heart, ears, kidneys, and limbs. In addition to her research, and in conjunction with NIAAA and the National Organization on Fetal Alcohol Syndrome (NOFAS), Kathy developed several FASD prevention curricula that are still in use today. Finally, as part of her drive to eradicate FAS and FASD, Kathy labored tirelessly with public policy makers to change how FASD is viewed by the public, how FASD is identified in affected individuals, and how FASD is studied by researchers. While no article could fully cover Kathy's contributions to FASD research and prevention, or her other contributions to embryology and teratology, this review will attempt to illustrate some of the highlights of Kathy's remarkable career.

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.