Henny W. M. van Straaten

Folate, Homocysteine and Neural Tube Defects: An Overview

Folate administration substantially reduces the risk on neural tube defects (NTD). The interest for studying a disturbed homocysteine (Hcy) metabolism in relation to NTD was raised by the observation of elevated blood Hcy levels in mothers of a NTD child. This observation resulted in the examination of enzymes involved in the folate-dependent Hcy metabolism. Thus far, this has led to the identification of the first and likely a second genetic risk factor for NTD. The C677T and A1298C mutations in the methylenetetrahydrofolate reductase (MTHFR) gene are associated with an increased risk of NTD and cause elevated Hcy concentrations. These levels can be normalized by additional folate intake. Thus, a dysfunctional MTHFR partly explains the observed elevated Hcy levels in women with NTD pregnancies and also, in part, the protective effect of folate on NTD. Although the MTHFR polymorphisms are only moderate risk factors, population-wide they may account for an important part of the observed NTD prevalence.

Angiotensin II stimulates angiogenesis in the chorio-allantoic membrane of the chick embryo.

Angiotensin II was applied daily in doses of 67 or 670 ng to a section of the chick embryo chorio-allantoic membrane from day 7 to day 14 after fertilization of the eggs. During this one-week period, it caused a significant, dose-dependent increase in the vascular density index. The increase obtained with 670 ng daily was comparable to that after daily administration of 1.7 micrograms adenosine, a known stimulator of angiogenesis. The data suggest a possible role for angiotensin II as a mediator of vascular growth.

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 closure in the chick embryo is multiphasic

Progression of neurulation in the chick embryo has not been well documented. To provide a detailed description, chick embryos were stained in ovo after the least manipulation possible to avoid distortion of the neural plate and folds. This allowed a morphological and morphometric description of the process of neurulation in relatively undisturbed chick embryos. Neurulation comprises several specific phases with distinct closure patterns and closure rates. The first closure event occurs, de novo, in the future mesencephalon at the 4–6 somite stage (sst 4–6). Soon afterwards, at sst 6–7, de novo closure is seen at the rhombocervical level in the form of multisite contacts of the neural folds. These contacts occur in register with the somites, suggesting that the somites may play a role in forcing elevation and apposition of the neural folds. The mesencephalic and rhombocervical closure events define an intervening rhombencephalic neuropore, which is present for a brief period before it closes. The remaining pear‐shaped posterior neuropore (PNP) narrows and displaces caudally, but its length remains constant in embryos with seven to ten somites, indicating that the caudal extension of the rhombocervical closure point and elongation of the caudal neural plate are keeping pace with each other. From sst 10 onward, the tapered cranial portion of the PNP closes fast in a zipper‐like manner, and, subsequently, the wide caudal portion of the PNP closes rapidly as a result of the parallel alignment of its folds, with numerous button‐like temporary contact points. A role for convergent extension in this closure event is suggested. The final remnant of the PNP closes at sst 18. Thus, as in mammals, chick neurulation involves multisite closure and probably results from several different development mechanisms at varying levels of the body axis.

Essential role of TGF‐β in the natural resistance to experimental allergic encephalomyelitis in rats

Experimental allergic encephalomyelitis (EAE) is a T cell‐mediated autoimmune disease induced in susceptible rat strains by a single immunization with myelin basic protein (MBP). The Lewis (LEW) strain is susceptible to disease induction while the Brown Norway (BN) strain is resistant. This resistance involves non‐MHC genes since congenic BN‐1L rats, with LEW MHC on a BN‐derived background, are also resistant. In the present study we show that, upon immunization with MBP, the non‐MHC‐encoded resistance to develop clinical EAE in BN‐1L rats is associated with a decreased production of IFN‐γ. This may be due to a difference between LEW and BN‐1L rats in their ability to produce regulatory cytokines such as IL‐4, IL‐10 and TGF‐β. In comparison to LEW rats, immune lymph node cells from BN‐1L rats express an increased amount of IL‐4 mRNA but produce less IL‐10. Furthermore, the sera from BN‐1L rats contain higher amounts of active TGF‐β1. Therefore, we have investigated the involvement of IL‐4 and TGF‐β in the resistance of BN‐1L rats to develop EAE using neutralizing mAb. Neutralization of TGF‐β, but not IL‐4, renders BN‐1L rats susceptible to clinical EAEwithout affecting the proliferation or the cytokine repertoire of immune lymph node cells. With respect to the origin of the endogenous TGF‐β production, we excluded the involvement of CD8 T cells and discuss a possible role of platelets and of CD4 T cells exhibiting the CD45RClow phenotype.

Over-expression of Grhl2 causes spina bifida in the Axial defects mutant mouse

Cranial neural tube defects (NTDs) occur in mice carrying mutant alleles of many different genes, whereas isolated spinal NTDs (spina bifida) occur in fewer models, despite being common human birth defects. Spina bifida occurs at high frequency in the Axial defects (Axd) mouse mutant but the causative gene is not known. In the current study, the Axd mutation was mapped by linkage analysis. Within the critical genomic region, sequencing did not reveal a coding mutation whereas expression analysis demonstrated significant up-regulation of grainyhead-like 2 (Grhl2) in Axd mutant embryos. Expression of other candidate genes did not differ between genotypes. In order to test the hypothesis that over-expression of Grhl2 causes Axd NTDs, we performed a genetic cross to reduce Grhl2 function in Axd heterozygotes. Grhl2 loss of function mutant mice were generated and displayed both cranial and spinal NTDs. Compound heterozygotes carrying both loss (Grhl2 null) and putative gain of function (Axd) alleles exhibited normalization of spinal neural tube closure compared with Axd/+ littermates, which exhibit delayed closure. Grhl2 is expressed in the surface ectoderm and hindgut endoderm in the spinal region, overlapping with grainyhead-like 3 (Grhl3). Axd mutants display delayed eyelid closure, as reported in Grhl3 null embryos. Moreover, Axd mutant embryos exhibited increased ventral curvature of the spinal region and reduced proliferation in the hindgut, reminiscent of curly tail embryos, which carry a hypomorphic allele of Grhl3. Overall, our data suggest that defects in Axd mutant embryos result from over-expression of Grhl2.

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos

Neural tube defects, including spina bifida, develop in the curly tail mutant mouse as a result of delayed closure of the posterior neuropore at 10.5 days of gestation. Affected embryos are characterized by increased ventral curvature of the caudal region. To determine whether closure of the neuropore could be affected by this angle of curvature, we experimentally enhanced the curvature of non-mutant embryos. The amnion was opened in 9.5 day embryos; after 20 h of culture, a proportion of the embryos exhibited a tightly wrapped amnion with enhanced curvature of the caudal region compared with the control embryos in which the opened amnion remained inflated. Enhanced curvature correlated with a higher frequency of embryos with an open posterior neuropore, irrespective of developmental stage within the range, 27–32 somites. Thus, within this somite range, caudal curvature is a more accurate determinant for normal spinal neurulation than the exact somite stage. Enhanced ventral curvature of the curly tail embryo correlates with an abnormal growth difference between the neuroepithelium and ventral structures (the notochord and hindgut). We experimentally corrected this imbalance by culturing under conditions of mild hyperthermia and subsequently determined whether the angle of curvature would also be corrected. The mean angle of curvature and length of the posterior neuropore were both reduced in embryos cultured at 40.5°C by comparison with control embryos cultured at 38°C. We conclude that the sequence of morphogenetic events leading to spinal neural tube defects in curly tail embryos involves an imbalance of growth rates, which leads to enhanced ventral curvature that, in turn, leads to delayed closure of the posterior neuropore.

Deceleration and acceleration in the rate of posterior neuropore closure during neurulation in the curly tail (ct) mouse embryo

SummaryCurly tail (ct) is a mouse mutant producing spinal neural tube defects as a result of delayed closure of the posterior neuropore (PNP). The purpose of the present study was to determine in ct/ct embryos the time of onset of the delay in PNP closure, and the pattern of this closure, as well as to study the possibility that reopening of the neural tube occurs. Normal spinal neurulation was studied in non-mutant Swiss (Sw) embryos. In the latter, the average PNP length diminished steadily between the 7- and 25-somite stages, and then decreased more rapidly, indicating an acceleration of closure rate, until the 30- to 32-somite stage, when all PNPs closed. PNP width decreased steadily between the stages of 7 and 30 somites. In ct/ct embryos the average PNP length showed a slight increase between the stage of 23 to 28 somites, indicating a temporary deceleration of closure rate, and the range of PNP sizes increased markedly. This was followed by a decrease in PNP length until the 37-somite stage, indicating an acceleration of closure rate. From the stage of 32 somites onwards, the proportion of embryos with closed PNPs gradually increased to 90%. The population of ct/ct embryos was subdivided. Embryos with large PNPs showed a marked deceleration of closure rate during a period of 11 somite stages, followed by a brief but very high acceleration of closure rate. This resulted in closure of the PNP in a proportion of these embryos, while in the remainder of the embryos the deceleration phase had been too enhanced to allow complete catch up of closure during the acceleration phase; these embryos would develop spina bifida. Embryos with relative small PNPs also showed a deceleration of closure rate, but only during a period of four somite stages. This was followed by an acceleration, resulting in closure of all PNPs at the stage of 32 to 33 somites. The enlargement of the PNP in ct/ct embryos was not due to re-opening of a closed neural tube, but resulted from a sharp decline in the rate of PNP closure combined with a normal rate of caudal elongation of the embryo. It is concluded that the ct strain forms a homogeneous population, with a large variation of its specific phenotype: deceleration of PNP closure during a restricted period. The disturbance of spinal neurulation in ct/ct embryos takes the form of a deceleration/acceleration pattern, resulting in a net delay of closure. It is suggested that, due to the ct mutation, forces are generated in the embryonic axis which oppose a normal neurulation process at a specific stage of development.

Role of differential cell proliferation in the tail bud in aberrant mouse neurulation

In the mouse mutant curly tail, the phenotypes spina bifida and curled tail result from a delay in closure of the posterior neuropore (PNP). At the developmental stage when this delay can first be recognized, the caudal region of the embryo demonstrates a transiently enhanced curvature of the body axis which likely inhibits elevation, convergence, and fusion of the neural folds. The enhanced curvature is thought to be the result of a decreased proliferation in the ventrally located gut endoderm and notochord, together with a normal proliferation of the overlying neuroepithelium of the PNP. However, the proliferation defect and the enhanced curvature were originally demonstrated at the same developmental stage, while it is expected that reduced proliferation should precede enhanced curvature and delayed PNP closure. The caudal region originates from the tail bud and we therefore propose that the enhanced curvature is induced by a disturbed dorso‐ventral proliferation pattern in the tail bud. Using flow cytometry, proliferation patterns were determined separately for the dorsal and ventral halves of the tail bud of curly tail and of control embryos as well as of recombinant embryos having the curly tail phenotype with a genetic background which is matched to the BALB/c control strain. In general, it appeared that about half of the cell cycle duration in tail bud cells was occupied by S phase, about 40% by G0/G1 and the rest by G2/M. For the control embryos, no dorso‐ventral differences in relative phase duration were demonstrated. However, curly tail and recombinant embryos at the 21–25 somite stage, prior to the onset of enhanced curvature, exhibited ventrally a higher proportion of G0/G1 phase cells than dorsally, and a complementary relationship for S phase cells. We interpret these observations as indicating a prolonged G1 phase at the ventral side of the tail bud, resulting in a prolongation of the cell cycle and thus a decreased proliferation. In 26–30 somite stage embryos, prior to the normalization of curvature in curly tail embryos, the dorso‐ventral proliferation balance was re‐established. We conclude that a reduced proliferation in the ventral part of the tail bud of the curly tail embryo precedes both the onset of enhanced curvature and the previously observed reduction in proliferation of the hindgut and notochord, and is a likely candidate for an early event in the pathogenetic sequence leading to the curly tail phenotype. Dev. Dyn. 1998;211:382‐389.

Multistep role for actin in initial closure of the mesencephalic neural groove in the chick embryo

In a previous study, we have demonstrated that initial closure of the mesencephalic neural groove in the chick embryo is different from neurulation elsewhere. The neural groove invaginates, the walls appose and make contact in a ventrodorsal direction, and subsequently separate ventrally, forming an incipient neural tube lumen, which finally widens into a definitive lumen. In this study, a role for actin in the processes of this initial mesencephalic closure is studied. Based on rhodamine‐phalloidin–stained sections, three distinct actin distribution patterns emerged, and time‐lapse video microscopy revealed cytochalasin‐D–reversible neurulation movements. We propose that actin is involved in formation and stabilization of the neural groove hinge point, in invagination of dorsal neuroepithelial cells into the neural groove, in the origin of the incipient lumen and the reinforcement of adhesion of the dorsal neural folds, and finally in the development of a wide lumen. Such a multifunctional effect of actin microfilaments within a narrow time window and at specific sites has not been reported yet.