Glycine Cleavage System H Protein Is Essential for Embryonic Viability, Implying Additional Function Beyond the Glycine Cleavage System

Glycine cleavage system H protein (GCSH) is a component of the glycine cleavage system (GCS), a conserved protein complex that acts to decarboxylate glycine. Mutation of AMT or GLDC, encoding the GCS components aminomethyltransferase and glycine decarboxylase, can cause malformations of the developing CNS (neural tube defects (NTDs) and ventriculomegaly) as well as a post-natal life-limiting neurometabolic disorder, Non-Ketotic Hyperglycinemia. In contrast, it is unclear whether mutation of GCSH contributes to these conditions and we therefore investigated GCSH loss of function in mice. Mice that were heterozygous for a Gcsh null allele were viable and did not exhibit elevated plasma glycine. Moreover, heterozygous mutation of Gcsh did not increase the frequency of NTDs in Gldc mutant embryos. Homozygous Gcsh null mice were not recovered at post-natal stages. Analysis of litters at E8.5-10.5, revealed the presence of homozygous null embryos which were much smaller than littermates and had failed to develop beyond early post-implantation stages with no visible somites or head-folds. Hence, unlike null mutations of Gldc or Amt, which are compatible with embryonic survival despite the presence of NTDs, loss of Gcsh causes embryonic death prior to mid-gestation. Maternal supplementation with formate did not restore embryonic development beyond E7.5, suggesting that the primary cause of lethality was not loss of glycine cleavage activity or suppression of folate one-carbon metabolism. These findings suggest that GCSH has additional roles beyond function in the glycine cleavage system. We hypothesize that GCSH potentially acts in lipoylation of 2-oxoacid dehydrogenase proteins, as reported in bacteria

Cellular mechanisms underlying Pax3-related neural tube defects and their prevention by folic acid

Neural tube defects (NTDs), including spina bifida and anencephaly, are among the most common birth defects worldwide but the underlying genetic and cellular causes are not well understood. Some NTDs are preventable by supplemental folic acid. However, the protective mechanism is unclear despite widespread use of folic acid supplements and implementation of food fortification in many countries. Pax3 mutant (splotch; Sp 2H ) mice provide a model in which NTDs are preventable by folic acid and exacerbated by maternal folate deficiency. Here, we found that cell proliferation was diminished in the dorsal neuroepithelium of mutant embryos, corresponding to the region of abolished Pax3 function. This was accompanied by premature neuronal differentiation in the prospective midbrain. Contrary to previous reports, we did not find evidence that increased apoptosis could underlie failed neural tube closure in Pax3 mutant embryos, nor did inhibition of apoptosis prevent NTDs. These findings suggest that Pax3 functions to maintain the neuroepithelium in a proliferative, undifferentiated state allowing neurulation to proceed. NTDs in Pax3 mutants were not associated with abnormal abundance of specific folates, nor prevented by formate, a one-carbon donor to folate metabolism. Supplemental folic acid restored proliferation in the cranial neuroepithelium. This effect was mediated by enhanced progression of the cell cycle from S- to G2-phase, specifically in the Pax3-mutant dorsal neuroepithelium. We propose that the cell cycle-promoting effect of folic acid compensates for loss of Pax3 and thereby prevents cranial NTDs

Regulation of glycine metabolism by the glycine cleavage system and conjugation pathway in mouse models of Non-Ketotic Hyperglycinemia

Glycine abundance is modulated in a tissue-specific manner by use in biosynthetic reactions, catabolism by the glycine cleavage system (GCS) and excretion via glycine conjugation. Dysregulation of glycine metabolism is associated with multiple disorders including epilepsy, developmental delay and birth defects. Mutation of the GCS component glycine decarboxylase (GLDC) in Non-Ketotic Hyperglycinemia (NKH) causes accumulation of glycine in body fluids, but there is a gap in our knowledge regarding the effects on glycine metabolism in tissues. Here, we analysed mice carrying mutations in Gldc that result in severe or mild elevations of plasma glycine and model NKH. Liver of Gldc-deficient mice accumulated glycine and numerous glycine derivatives, including multiple acylglycines, indicating increased flux through reactions mediated by enzymes including glycine-N-acyltransferase and arginine:glycine amidinotransferase. Levels of dysregulated metabolites increased with age and were normalised by liver-specific rescue of Gldc expression. Brain tissue exhibited increased abundance of glycine, as well as derivatives including guanidinoacetate, which may itself be epileptogenic. Elevation of brain tissue glycine occurred even in the presence of only mildly elevated plasma glycine in mice carrying a missense allele of Gldc. Treatment with benzoate enhanced hepatic glycine conjugation thereby lowering plasma and tissue glycine. Moreover, administration of a glycine conjugation pathway intermediate, cinnamate, similarly achieved normalisation of liver glycine derivatives and circulating glycine. Although exogenous benzoate and cinnamate impact glycine levels via activity of glycine-N-acyltransferase, that is not expressed in brain, they are sufficient to lower levels of glycine and derivatives in brain tissue of treated Gldc-deficient mice

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

Over-expression 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, is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of spina bifida and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by BAC-mediated transgenesis, prevents spina bifida in Grhl3 null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably however, further increase in expression of Grhl3 causes highly penetrant spina bifida. Grhl3 over-expression 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 homo- and heterodimers, suggesting a possible model in which defects arising from over-expression 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 over-expressing embryo. Instead we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe spina bifida arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 over-expression also interacted with the Vangl2Lp allele to cause spina bifida, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs

Folate metabolite profiling of different cell types and embryos suggests variation in folate one-carbon metabolism, including developmental changes in human embryonic brain

Folates act as co-factors for transfer of one-carbon units for nucleotide production, methylation and other biosynthetic reactions. Comprehensive profiling of multiple folates can be achieved using liquid chromatography tandem mass spectrometry, enabling determination of their relative abundance that may provide an indication of metabolic differences between cell types. For example, cell lines exposed to methotrexate showed a dose-dependent elevation of dihydrofolate, consistent with inhibition of dihydrofolate reductase. We analysed the folate profile of E. coli sub-types as well as cell lines and embryonic tissue from both human and mouse. The folate profile of bacteria differed markedly from those of all the mammalian samples, most notably in the greater abundance of formyl tetrahydrofolate. The overall profiles of mouse and human fibroblasts and mid-gestation mouse embryos were broadly similar, with specific differences. The major folate species in these cell types was 5-methyl tetrahydrofolate, in contrast to lymphoblastoid cell lines in which the predominant form was tetrahydrofolate. Analysis of embryonic human brain revealed a shift in folate profile with increasing developmental stage, with a decline in relative abundance of dihydrofolate and increase in 5-methyl tetrahydrofolate. These cell type-specific and developmental changes in folate profile may indicate differential requirements for the various outputs of folate metabolism

Diffusion Microscopic MRI of the Mouse Embryo: Protocol and Practical Implementation in the splotch Mouse Model

PURPOSE: Advanced methodologies for visualizing novel tissue contrast are essential for phenotyping the ever-increasing number of mutant mouse embryos being generated. Although diffusion microscopic MRI (μMRI) has been used to phenotype embryos, widespread routine use is limited by extended scanning times, and there is no established experimental procedure ensuring optimal data acquisition. METHODS: We developed two protocols for designing experimental procedures for diffusion μMRI of mouse embryos, which take into account the effect of embryo preparation and pulse sequence parameters on resulting data. We applied our protocols to an investigation of the splotch mouse model as an example implementation. RESULTS: The protocols provide DTI data in 24 min per direction at 75 μm isotropic using a three-dimensional fast spin-echo sequence, enabling preliminary imaging in 3 h (6 directions plus one unweighted measurement), or detailed imaging in 9 h (42 directions plus six unweighted measurements). Application to the splotch model enabled assessment of spinal cord pathology. CONCLUSION: We present guidelines for designing diffusion μMRI experiments, which may be adapted for different studies and research facilities. As they are suitable for routine use and may be readily implemented, we hope they will be adopted by the phenotyping community