Nicholas D.E. Greene

The genetic basis of mammalian neurulation

More than 80 mutant mouse genes disrupt neurulation and allow an in-depth analysis of the underlying developmental mechanisms. Although many of the genetic mutants have been studied in only rudimentary detail, several molecular pathways can already be identified as crucial for normal neurulation. These include the planar cell-polarity pathway, which is required for the initiation of neural tube closure, and the sonic hedgehog signalling pathway that regulates neural plate bending. Mutant mice also offer an opportunity to unravel the mechanisms by which folic acid prevents neural tube defects, and to develop new therapies for folate-resistant defects.

Metformin Retards Aging in C. elegans by Altering Microbial Folate and Methionine Metabolism

Summary The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy—and also to its side effects, which include folate deficiency and gastrointestinal upset. PaperClip

Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure

Planar-cell-polarity (PCP) signalling is necessary for initiation of neural tube closure in higher vertebrates. In mice with PCP gene mutations, a broad embryonic midline prevents the onset of neurulation through wide spacing of the neural folds. In order to evaluate the role of convergent extension in this defect, we vitally labelled the midline of loop-tail (Lp) embryos mutant for the PCP gene Vangl2. Injection of DiI into the node, and electroporation of a GFP expression vector into the midline neural plate, revealed defective convergent extension in both axial mesoderm and neuroepithelium, before the onset of neurulation. Chimeras containing both wild-type and Lp-mutant cells exhibited mainly wild-type cells in the midline neural plate and notochordal plate, consistent with a cell-autonomous disturbance of convergent extension. Inhibitor studies in whole-embryo culture demonstrated a requirement for signalling via RhoA-Rho kinase, but not jun N-terminal kinase, in convergent extension and the onset of neural tube closure. These findings identify a cell-autonomous defect of convergent extension, requiring PCP signalling via RhoA-Rho kinase, during the development of severe neural tube defects in the mouse.

Abnormalities of floor plate, notochord and somite differentiation in the loop-tail (Lp) mouse: a model of severe neural tube defects

Mouse embryos homozygous for the loop-tail (Lp) mutation fail to initiate neural tube closure at E8.5, leading to a severe malformation in which the neural tube remains open from midbrain to tail. During initiation of closure, the normal mouse neural plate bends sharply in the midline, at the site of the future floor plate. In contrast, Lp/Lp embryos exhibit a broad region of flat neural plate in the midline, displacing the sites of neuroepithelial bending to more lateral positions. Sonic hedgehog (Shh) and Netrin1 are expressed in abnormally broad domains in the ventral midline of the E9.5 Lp/Lp neural tube, suggesting over-abundant differentiation of the floor plate. The notochord is also abnormally broad in Lp/Lp embryos with enlarged domains of Shh and Brachyury expression. The paraxial mesoderm shows evidence of ventralisation, with increased expression of the sclerotomal marker Pax1, and diminished expression of the dermomyotomal marker Pax3. While the expression domain of Pax3 does not differ markedly from wild-type, there is a dorsal shift in the domain of Pax6 expression in the neural tube at caudal levels of Lp/Lp embryos. We suggest that the Lp mutation causes excessive differentiation of floor-plate and notochord, with over-production of Shh from these midline structures causing ventralisation of the paraxial mesoderm and, to a lesser extent, the neural tube. Comparison with other mouse mutants suggests that the enlarged floor plate may be responsible for the failure of neural tube closure in Lp/Lp embryos.

Neural tube defects.

Neural tube defects (NTDs), including spina bifida and anencephaly, are severe birth defects of the central nervous system that originate during embryonic development when the neural tube fails to close completely. Human NTDs are multifactorial, with contributions from both genetic and environmental factors. The genetic basis is not yet well understood, but several nongenetic risk factors have been identified as have possibilities for prevention by maternal folic acid supplementation. Mechanisms underlying neural tube closure and NTDs may be informed by experimental models, which have revealed numerous genes whose abnormal function causes NTDs and have provided details of critical cellular and morphological events whose regulation is essential for closure. Such models also provide an opportunity to investigate potential risk factors and to develop novel preventive therapies.

Mouse models of neural tube defects: Investigating preventive mechanisms

Neural tube defects (NTD), including anencephaly and spina bifida, are a group of severe congenital abnormalities in which the future brain and/or spinal cord fail to close. In mice, NTD may result from genetic mutations or knockouts, or from exposure to teratogenic agents, several of which are known risk factors in humans. Among the many mouse NTD models that have been identified to date, a number have been tested for possible primary prevention of NTD by exogenous agents, such as folic acid. In genetic NTD models such as Cart1, splotch, Cited2, and crooked tail, and NTD induced by teratogens including valproic acid and fumonisins, the incidence of defects is reduced by maternal folic acid supplementation. These folate‐responsive models provide an opportunity to investigate the possible mechanisms underlying prevention of NTD by folic acid in humans. In another group of mouse models, that includes curly tail, axial defects, and the Ephrin‐A5 knockout, NTD are not preventable by folic acid, reflecting the situation in humans in which a subset of NTD appear resistant to folic acid therapy. In this group of mutants alternative preventive agents, including inositol and methionine, have been shown to be effective. Overall, the data from mouse models suggests that a broad‐based in utero therapy may offer scope for prevention of a greater proportion of NTD than is currently possible.

Neural tube defects—disorders of neurulation and related embryonic processes

Neural tube defects (NTDs) are severe congenital malformations affecting 1 in every 1000 pregnancies. ‘Open’ NTDs result from failure of primary neurulation as seen in anencephaly, myelomeningocele (open spina bifida), and craniorachischisis. Degeneration of the persistently open neural tube in utero leads to loss of neurological function below the lesion level. ‘Closed’ NTDs are skin‐covered disorders of spinal cord structure, ranging from asymptomatic spina bifida occulta to severe spinal cord tethering, and usually traceable to disruption of secondary neurulation. ‘Herniation’ NTDs are those in which meninges, with or without brain or spinal cord tissue, become exteriorized through a pathological opening in the skull or vertebral column (e.g., encephalocele and meningocele). NTDs have multifactorial etiology, with genes and environmental factors interacting to determine individual risk of malformation. While over 200 mutant genes cause open NTDs in mice, much less is known about the genetic causation of human NTDs. Recent evidence has implicated genes of the planar cell polarity signaling pathway in a proportion of cases. The embryonic development of NTDs is complex, with diverse cellular and molecular mechanisms operating at different levels of the body axis. Molecular regulatory events include the bone morphogenetic protein and Sonic hedgehog pathways which have been implicated in control of neural plate bending. Primary prevention of NTDs has been implemented clinically following the demonstration that folic acid (FA), when taken as a periconceptional supplement, can prevent many cases. Not all NTDs respond to FA, however, and adjunct therapies are required for prevention of this FA‐resistant category. WIREs Dev Biol 2013, 2:213–227. doi: 10.1002/wdev.71

Mutations in ZMYND10, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms in Humans and Flies, Cause Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.

Apoptosis is not required for mammalian neural tube closure

Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube closure. In mice, excessive apoptosis occurs in association with failure of neural tube closure in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube closure in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube closure. We confirm the presence of apoptosis in the neural tube before and during closure, and identify a correlation with 3 main events: bending and fusion of the neural folds, postfusion remodeling of the dorsal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos exhibit severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In contrast, the mutant embryos fail to complete neural tube closure in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-α to neurulation-stage embryos in culture suppresses apoptosis but does not prevent initiation or progression of neural tube closure along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube closure in the mouse embryo.

Developmental expression of palmitoyl protein thioesterase in normal mice

Deficiency in palmitoyl protein thioesterase (PPT) results in the rapid death of neocortical neurons in human. Very little is known about the developmental and cell-specific expression of this lysosomal enzyme. Here we show that PPT is expressed as a major 2.65 kb and a minor 1.85 kb transcript in the mouse brain. Transcript levels gradually increase between postnatal days 10 and 30. In situ hybridization analysis revealed that PPT transcripts are found widely but not homogeneously in the brain. The most intense signal was detected in the cerebral cortex (layers II, IV-V), hippocampal CA1-CA3 pyramidal cells, dentate gyrus granule cells and the hypothalamus. Immunostaining of PPT was localized in the cell soma, axons and dendrites, especially in the pyramidal and granular cells of the hippocampus, correlating well, both spatially and temporally, with the immunoreactivity of a presynaptic vesicle membrane protein, synaptophysin. In whole embryos, at embryonic day 8, the PPT mRNA expression was most apparent throughout the neuroepithelium, and from day 9 onwards it was seen in all tissues. The expression pattern of PPT suggests its general significance for the brain cells and reflects the response to maturation and growth of the neural networks. Strong PPT immunoreactivity in the axons and dentrites would imply that PPT may not be exclusively a lysosomal enzyme. A notable correlation with synaptophysin would suggest that PPT may have a role in the function of the synaptic machinery.