Inhibition of transmethylation disturbs neurulation in chick embryos.

Periconceptional folic acid supplementation can reduce the occurrence of neural tube defects. A low folate status will result in reduced remethylation of homocysteine (Hcy) to methionine and, subsequently, in a rise of Hcy levels. Indeed, elevated Hcy concentrations have been reported in mothers of children with neural tube defects. In our previous study, we showed that treatment of chick embryos with Hcy resulted in a delay of neural tube closure in an in vitro model. In the present study, we examined whether this effect of Hcy is due to inhibition of transmethylation via elevation of S-adenosylhomocysteine (AdoHcy). Transmethylation involves methylation of DNA, RNA and proteins by donation of a methyl group from S-adenosylmethionine (AdoMet). After application of inhibitors of S-adenosylhomocysteine hydrolase and of methionine adenosyltransferase, a delay of anterior neuropore closure, comparable to that observed after Hcy treatment, was observed. The changes in AdoMet and AdoHcy concentrations confirmed the inhibition of S-adenosylhomocysteine hydrolase or methionine adenosyltransferase, respectively, and the AdoMet/AdoHcy ratio was decreased in all cases, indicating reduced transmethylation. Moreover, the inhibition of methionine adenosyltransferase was prevented by pretreatment with methionine. This study, therefore, indicates that the Hcy-induced delay of the neural tube closure is caused by the inhibition of transmethylation via elevation of AdoHcy levels and a reduction of the AdoMet/AdoHcy ratio.

Morphogenetic movements during cranial neural tube closure in the chick embryo and the effect of homocysteine.

In order to unravel morphogenetic mechanisms involved in neural tube closure, critical cell movements that are fundamental to remodelling of the cranial neural tube in the chick embryo were studied in vitro by quantitative time-lapse video microscopy. Two main directions of movements were observed. The earliest was directed medially; these cells invaginated into a median groove and were the main contributors to the initial neural tube closure. Once the median groove was completed, cells changed direction and moved anteriorly to contribute to the anterior neural plate and head fold. This plate developed into the anterior neuropore, which started to close from the 4-somite stage onwards by convergence of its neural folds. Posteriorly, from the initial closure site onwards, the posterior neuropore started to close almost instantaneously by convergence of its neural folds. Homocysteine is adversely involved in human neural tube closure defects. After application of a single dose of homocysteine to chick embryos, a closure delay at the initial closure site and at the neuropores, flattening of the head fold and neural tube, and a halt of cell movements was seen. A possible interference of Hcy with actin microfilaments is discussed.

Hyaluronan Disappears Intercellularly and Appears at the Basement Membrane Region during Formation of Embryonic Epithelia: (mouse embryo/hyaluronan/epithelium/basement membrane/development).

The distribution of tissue hyaluronan has been assessed in the neuraxial region of 8.5 to 10.5 day mouse embryos using a fragment of bovine nasal cartilage proteoglycan that binds specifically to hyaluronan. Hyaluronan is abundant in all mesenchymal tissues, predominantly intercellularly, but markedly diminishes when mesenchymal cells organize into epithelia, as in the formation of somites. Hyaluronan reappears in abundance when epithelia (e.g. sclerotome) disperse into mesenchyme. Hyaluronan is present between cells of early epithelia (e.g. neural plate), but is lost during their subsequent development when it becomes abundant at their basement membrane regions. These results show for the first time changes in hyaluronan distribution during the development of embryonic epithelia. The hyaluronan distribution found is consistent with the functions proposed for hyaluronan in embryonic mesenchyme: intercellular hyaluronan would allow the epithelial cells to move and reduced hyaluronan would allow the cells to associate. The absence of intercellular hyaluronan in later epithelia would allow increased membrane contacts that lead to the formation of intercellular junctions. The restriction of hyaluronan to basement membrane regions in later epithelia further substantiates the suggestion that hyaluronan is a bona fide component of the basal lamina and that it is involved in maintaining epithelial morphology.

Analysis of the embryonic phenotype of Bent tail, a mouse model for X-linked neural tube defects.

Neural tube defects, mostly believed to result from closure defects of the neural tube during embryonic development, are frequently observed congenital malformations in humans. Since the etiology of these defects is not well understood yet, many animal models for neural tube defects, either arising from spontaneous mutations or generated by gene targeting, are being studied. The Bent tail mouse is a model for X-linked neural tube defects. This mutant has a characteristic short and kinked tail. Exencephaly occurs in Bent tail embryos with a frequency of 11-16%. Laterality defects also belong to the phenotypic spectrum. In this study, we analyzed the embryonic phenotype in further detail using scanning electron microscopy during the stages of neurulation. We observed a number of defects in both wild type and Bent tail embryos, including a kinked neural tube, tight amnion, delay in axial rotation and even malrotation. The severity or frequency of most defects, the delay in axial rotation excluded, was significantly higher in Bent tail embryos compared to wild type embryos. Other abnormalities were seen in Bent tail embryos only. These defects were related to anterior and posterior neural tube closure and resulted in exencephaly and a closure delay of the posterior neuropore, respectively. The exencephalic phenotype was further analyzed by light microscopy in ED14 embryos, showing disorganization and overgrowth in the mesencephalon and rhombencephalon. In conclusion, the anterior and posterior neural tube closure defects in the Bent tail are strictly linked to the genetic defect in this mouse. Other phenotypic features described in this study also occur in the wild type genetic background of the Bent tail strain. Apparently, the genetic background contains elements conducive to these developmental abnormalities.

Spinal congenital dermal sinus in a chick embryo model. Laboratory investigation.

OBJECT: The origin of spinal congenital dermal sinuses is not known. A local nondisjunction of the closing neural tube and the epidermal ectoderm is thought to be the cause of this malformation. In this experimental study, a nondisjunction was mimicked in chick embryos to create an animal model for the dermal sinus. METHODS: A piece of amniotic tissue was implanted in the closing neural tube in ovo in chick embryos at 2 days of incubation. A total of 50 embryos were manipulated. After a further incubation time of 2-7 days, the embryos were macroscopically and histologically evaluated. RESULTS: Dermal sinus-like anomalies were induced in 24 embryos. The induced abnormalities varied from superficial, epidermal lesions to epidermal dimples continuing as a strand of tissue toward the neural tube. This strand invariably was of nonneuronal origin. Additionally, in 3 embryos a split cord malformation was noted, most likely caused by damage to the neural tube during implantation. CONCLUSIONS: Implantation of donor amniotic tissue in the closing chick neural tube does result in a dimple, from which a strand of tissue continues to the neural tube in various cases, indicating that formation of a dermal sinus-like anomaly can be successfully induced by experimental continuation of the connection between neural tube and surface ectoderm. This finding strengthens the hypothesis that a human dermal sinus arises after nondisjunction of neural tube and surface ectoderm.

Ciliation and gene expression distinguish between node and posterior notochord in the mammalian embryo.

The mammalian node, the functional equivalent of the frog dorsal blastoporal lip (Spemann's organizer), was originally described by Viktor Hensen in 1876 in the rabbit embryo as a mass of cells at the anterior end of the primitive streak. Today, the term "node" is commonly used to describe a bilaminar epithelial groove presenting itself as an indentation or "pit" at the distal tip of the mouse egg cylinder, and cilia on its ventral side are held responsible for molecular laterality (left-right) determination. We find that Hensen's node in the rabbit is devoid of cilia, and that ciliated cells are restricted to the notochordal plate, which emerges from the node rostrally. In a comparative approach, we use the organizer marker gene Goosecoid (Gsc) to show that a region of densely packed epithelium-like cells at the anterior end of the primitive streak represents the node in mouse and rabbit and is covered ventrally by a hypoblast (termed "visceral endoderm" in the mouse). Expression of Nodal, a gene intricately involved in the determination of vertebrate laterality, delineates the wide plate-like posterior segment of the notochord in the rabbit and mouse, which in the latter is represented by the indentation frequently termed "the node." Similarly characteristic ciliation and nodal expression exists in Xenopus neurula embryos in the gastrocoel roof plate (GRP), i.e., at the posterior end of the notochord anterior to the blastoporal lip. Our data suggest that (1) a posterior segment of the notochord, here termed PNC (for posterior notochord), is characterized by features known to be involved in laterality determination, (2) the GRP in Xenopus is equivalent to the mammalian PNC, and (3) the mammalian node as defined by organizer gene expression is devoid of cilia and most likely not directly involved in laterality determination.

Cellular concentrations of glutamine synthetase in murine organs.

Glutamine synthetase (GS) is the only enzyme that can synthesize glutamine, but it also functions to detoxify glutamate and ammonia. Organs with high cellular concentrations of GS appear to function primarily to remove glutamate or ammonia, whereas those with a low cellular concentration appear to primarily produce glutamine. To validate this apparent dichotomy and to clarify its regulation, we determined the GS concentrations in 18 organs of the mouse. There was a >100-fold difference in GS mRNA, protein, and enzyme-activity levels among organs, whereas there was only a 20-fold difference in the GS protein:mRNA ratio, suggesting extensive transcriptional and posttranscriptional regulation. In contrast, only small differences in the GS enzyme activity : protein ratio were found, indicating that posttranslational regulation is of minor importance. The cellular concentration of GS was determined by relating the relative differences in cellular GS concentration, detected using image analysis of immunohistochemically stained tissue sections, to the biochemical data. There was a >1000-fold difference in cellular concentrations of GS between GS-positive cells in different organs, and cellular concentrations were up to 20x higher in subpopulations of cells within organs than in whole organs. GS activity was highest in pericentral hepatocytes (approximately 485 micromol.g(-1).min-(1), followed in descending order by epithelial cells in the epididymal head, Leydig cells in the testicular interstitium, epithelial cells of the uterine tube, acid-producing parietal cells in the stomach, epithelial cells of the S3 segment of the proximal convoluted tubule of the kidney, astrocytes of the central nervous tissue, and adipose tissue. GS activity in muscle amounted to only 0.4 micromol.g(-1).min(-1). Our findings confirmed the postulated dichotomy between cellular concentration and GS function.

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.