A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Association of embryonic inositol status with susceptibility to neural tube defects, metabolite profile, and maternal inositol intake.

Maternal nutrition contributes to gene-environment interactions that influence susceptibility to common congenital anomalies such as neural tube defects (NTDs). Supplemental myo-inositol (MI) can prevent NTDs in some mouse models and shows potential for prevention of human NTDs. We investigated effects of maternal MI intake on embryonic MI status and metabolism in curly tail mice, which are genetically predisposed to NTDs that are inositol-responsive but folic acid resistant. Dietary MI deficiency caused diminished MI in maternal plasma and embryos, showing that de novo synthesis is insufficient to maintain MI levels in either adult or embryonic mice. Under normal maternal dietary conditions, curly tail embryos that developed cranial NTDs had significantly lower MI content than unaffected embryos, revealing an association between diminished MI status and failure of cranial neurulation. Expression of inositol-3-phosphate synthase 1, required for inositol biosynthesis, was less abundant in the cranial neural tube than at other axial levels. Supplemental MI or d-chiro-inositol (DCI) have previously been found to prevent NTDs in curly tail embryos. Here, we investigated the metabolic effects of MI and DCI treatments by mass spectrometry-based metabolome analysis. Among inositol-responsive metabolites, we noted a disproportionate effect on nucleotides, especially purines. We also found altered proportions of 5-methyltetrahydrolate and tetrahydrofolate in MI-treated embryos suggesting altered folate metabolism. Treatment with nucleotides or the one-carbon donor formate has also been found to prevent NTDs in curly tail embryos. Together, these findings suggest that the protective effect of inositol may be mediated through the enhanced supply of nucleotides during neural tube closure.

Overexpression of Grainyhead-like 3 causes spina bifida and interacts genetically with mutant alleles of Grhl2 and Vangl2 in mice.

The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)-mediated transgenesis, prevents SB in Grhl3-null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss-of-function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3-null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3-transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

Neural tube closure: cellular, molecular and biomechanical mechanisms.

Neural tube closure has been studied for many decades, across a range of vertebrates, as a paradigm of embryonic morphogenesis. Neurulation is of particular interest in view of the severe congenital malformations - 'neural tube defects' - that result when closure fails. The process of neural tube closure is complex and involves cellular events such as convergent extension, apical constriction and interkinetic nuclear migration, as well as precise molecular control via the non-canonical Wnt/planar cell polarity pathway, Shh/BMP signalling, and the transcription factors Grhl2/3, Pax3, Cdx2 and Zic2. More recently, biomechanical inputs into neural tube morphogenesis have also been identified. Here, we review these cellular, molecular and biomechanical mechanisms involved in neural tube closure, based on studies of various vertebrate species, focusing on the most recent advances in the field.

Biomechanical coupling facilitates spinal neural tube closure in mouse embryos.

Neural tube (NT) formation in the spinal region of the mammalian embryo involves a wave of "zippering" that passes down the elongating spinal axis, uniting the neural fold tips in the dorsal midline. Failure of this closure process leads to open spina bifida, a common cause of severe neurologic disability in humans. Here, we combined a tissue-level strain-mapping workflow with laser ablation of live-imaged mouse embryos to investigate the biomechanics of mammalian spinal closure. Ablation of the zippering point at the embryonic dorsal midline causes far-reaching, rapid separation of the elevating neural folds. Strain analysis revealed tissue expansion around the zippering point after ablation, but predominant tissue constriction in the caudal and ventral neural plate zone. This zone is biomechanically coupled to the zippering point by a supracellular F-actin network, which includes an actin cable running along the neural fold tips. Pharmacologic inhibition of F-actin or laser ablation of the cable causes neural fold separation. At the most advanced somite stages, when completion of spinal closure is imminent, the cable forms a continuous ring around the neuropore, and simultaneously, a new caudal-to-rostral zippering point arises. Laser ablation of this new closure initiation point causes neural fold separation, demonstrating its biomechanical activity. Failure of spinal closure in pre-spina bifida Zic2Ku mutant embryos is associated with altered tissue biomechanics, as indicated by greater neuropore widening after ablation. Thus, this study identifies biomechanical coupling of the entire region of active spinal neurulation in the mouse embryo as a prerequisite for successful NT closure.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure.

During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Arachidonic acid-dependent gene regulation during preadipocyte differentiation controls adipocyte potential.

Arachidonic acid (AA) is a major PUFA that has been implicated in the regulation of adipogenesis. We examined the effect of a short exposure to AA at different stages of 3T3-L1 adipocyte differentiation. AA caused the upregulation of fatty acid binding protein 4 (FABP4/aP2) following 24 h of differentiation. This was mediated by the prostaglandin F(2α) (PGF(2α)), as inhibition of cyclooxygenases or PGF(2α) receptor signaling counteracted the AA-mediated aP2 induction. In addition, calcium, protein kinase C, and ERK are all key elements of the pathway through which AA induces the expression of aP2. We also show that treatment with AA during the first 24 h of differentiation upregulates the expression of the transcription factor Fos-related antigen 1 (Fra-1) via the same pathway. Finally, treatment with AA for 24 h at the beginning of the adipocyte differentiation is sufficient to inhibit the late stages of adipogenesis through a Fra-1-dependent pathway, as Fra-1 knockdown rescued adipogenesis. Our data show that AA is able to program the differentiation potential of preadipocytes by regulating gene expression at the early stages of adipogenesis.

Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice.

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a "brite" transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with "browning," as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

Spinal neural tube closure depends on regulation of surface ectoderm identity and biomechanics by Grhl2.

Lack or excess expression of the surface ectoderm-expressed transcription factor Grainyhead-like2 (Grhl2), each prevent spinal neural tube closure. Here we investigate the causative mechanisms and find reciprocal dysregulation of epithelial genes, cell junction components and actomyosin properties in Grhl2 null and over-expressing embryos. Grhl2 null surface ectoderm shows a shift from epithelial to neuroepithelial identity (with ectopic expression of N-cadherin and Sox2), actomyosin disorganisation, cell shape changes and diminished resistance to neural fold recoil upon ablation of the closure point. In contrast, excessive abundance of Grhl2 generates a super-epithelial surface ectoderm, in which up-regulation of cell-cell junction proteins is associated with an actomyosin-dependent increase in local mechanical stress. This is compatible with apposition of the neural folds but not with progression of closure, unless myosin activity is inhibited. Overall, our findings suggest that Grhl2 plays a crucial role in regulating biomechanical properties of the surface ectoderm that are essential for spinal neurulation.

Vangl2 disruption alters the biomechanics of late spinal neurulation leading to spina bifida in mouse embryos.

Human mutations in the planar cell polarity component VANGL2 are associated with the neural tube defect spina bifida. Homozygous Vangl2 mutation in mice prevents initiation of neural tube closure, precluding analysis of its subsequent roles in neurulation. Spinal neurulation involves rostral-to-caudal 'zippering' until completion of closure is imminent, when a caudal-to-rostral closure point, 'Closure 5', arises at the caudal-most extremity of the posterior neuropore (PNP). Here, we used Grhl3Cre to delete Vangl2 in the surface ectoderm (SE) throughout neurulation and in an increasing proportion of PNP neuroepithelial cells at late neurulation stages. This deletion impaired PNP closure after the ∼25-somite stage and resulted in caudal spina bifida in 67% of Grhl3Cre/+Vangl2Fl/Fl embryos. In the dorsal SE, Vangl2 deletion diminished rostrocaudal cell body orientation, but not directional polarisation of cell divisions. In the PNP, Vangl2 disruption diminished mediolateral polarisation of apical neuroepithelial F-actin profiles and resulted in eversion of the caudal PNP. This eversion prevented elevation of the caudal PNP neural folds, which in control embryos is associated with formation of Closure 5 around the 25-somite stage. Closure 5 formation in control embryos is associated with a reduction in mechanical stress withstood at the main zippering point, as inferred from the magnitude of neural fold separation following zippering point laser ablation. This stress accommodation did not happen in Vangl2-disrupted embryos. Thus, disruption of Vangl2-dependent planar-polarised processes in the PNP neuroepithelium and SE preclude zippering point biomechanical accommodation associated with Closure 5 formation at the completion of PNP closure.

Maternal cholestasis during pregnancy programs metabolic disease in offspring.

The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

Absence of RIP140 reveals a pathway regulating glut4-dependent glucose uptake in oxidative skeletal muscle through UCP1-mediated activation of AMPK.

Skeletal muscle constitutes the major site of glucose uptake leading to increased removal of glucose from the circulation in response to insulin. Type 2 diabetes and obesity are often associated with insulin resistance that can be counteracted by exercise or the use of drugs increasing the relative proportion of oxidative fibers. RIP140 is a transcriptional coregulator with a central role in metabolic tissues and we tested the effect of modulating its level of expression on muscle glucose and lipid metabolism in two mice models. Here, we show that although RIP140 protein is expressed at the same level in both oxidative and glycolytic muscles, it inhibits both fatty acid and glucose utilization in a fiber-type dependent manner. In RIP140-null mice, fatty acid utilization increases in the extensor digitorum longus and this is associated with elevated expression of genes implicated in fatty acid binding and transport. In the RIP140-null soleus, depletion of RIP140 leads to increased GLUT4 trafficking and glucose uptake with no change in Akt activity. AMPK phosphorylation/activity is inhibited in the soleus of RIP140 transgenic mice and increased in RIP140-null soleus. This is associated with increased UCP1 expression and mitochondrial uncoupling revealing the existence of a signaling pathway controlling insulin-independent glucose uptake in the soleus of RIP140-null mice. In conclusion, our findings reinforce the participation of RIP140 in the maintenance of energy homeostasis by acting as an inhibitor of energy production and particularly point to RIP140 as a promising therapeutic target in the treatment of insulin resistance.

The nuclear receptor cofactor receptor-interacting protein 140 is a positive regulator of amphiregulin expression and cumulus cell-oocyte complex expansion in the mouse ovary.

The nuclear receptor cofactor receptor-interacting protein 140 (RIP140) is essential for cumulus cell-oocyte complex (COC) expansion, follicular rupture, and oocyte release during ovulation. The expression of many genes necessary for COC expansion is impaired in the absence of RIP140, but the studies herein document that their expression can be restored and COC expansion rescued by treatment with the epidermal growth factor (EGF)-like factor amphiregulin (AREG) both in vitro and in vivo. We demonstrate by several approaches that RIP140 is required for the expression of the EGF-like factors in granulosa cells, but the dependence of genes involved in cumulus expansion, including Ptgs2 Has2, Tnfaip6, and Ptx3, is indirect because they are induced by AREG. Treatment of granulosa cells with forskolin to mimic the effects of LH increases AREG promoter activity in a RIP140-dependent manner that 1) requires an intact cAMP response element in the proximal promoter region of the Areg gene and 2) involves its actions as a coactivator for cAMP response element-binding protein/c-Jun transcription factors. Although human chorionic gonadotropin and AREG coadministration is sufficient to restore ovulation fully in RIP140 heterozygous mice in vivo, both follicular rupture and ovulation remain impaired in the RIP140 null mice. Thus, we conclude that although the level of RIP140 expression in the ovary is a crucial factor required for the transient expression of EGF-like factors necessary for cumulus expansion, it also plays a role in other signaling pathways that induce follicular rupture.