Experimental manipulation and morphometric analysis of neural tube development.
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Animals↗
Biomedical subjects
M E Desmond
Publications and source records attributed to M E Desmond.
Isolation of neuroepithelium and formation of minispheres.
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Animals↗
No turning, a mouse mutation causing left-right and axial patterning defects.
Patterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was named no turning (nt) because the mutant embryos fail to undergo embryonic turning. nt embryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, in nt embryos, the direction of heart looping was randomized. Nodal and lefty, two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm. Lefty, which is normally also expressed in the floorplate, is not found in the prospective floor plate of nt embryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination of sonic hedgehog, Brachyury, and HNF-3 beta gene expression. These studies showed that the notochord is present in the early nt embryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest that nt may participate in this process by modulating the notochordal expression of HNF-3 beta.
Animals↗
The region encoded by the alternatively spliced exon IIIA in mesenchymal fibronectin appears essential for chondrogenesis at the level of cellular condensation.
Fibronectin in the extracellular matrix of tissues acts as a substrate for cell adhesion and migration during development. Heterogeneity in the structure of fibronectin is largely due to the alternative splicing of at least three exons (IIIB, IIIA, and V) during processing of a single primary transcript. Fibronectin mRNA alternative splicing patterns change from B+A+V+ to B+A-V+ during chondrogenesis. In this report, immunohistochemical analysis demonstrates that while fibronectin protein containing the region encoded by exon IIIB is present throughout the limb at all stages of development, fibronectin protein containing the region encoded by exon IIIA disappears from cartilaginous regions just after condensation in vivo and in high-density mesenchymal micromass cultures in vitro. Treatment of mesenchymal micromass cultures prior to condensation with an antibody specific for the region encoded by exon IIIA disrupts the formation of cellular condensations and inhibits subsequent chondrogenesis in a dose- and time-dependent manner. Furthermore, microinjection of the exon IIIA antibody into embryonic chick limb primordia in vivo results in malformations characterized by smaller limbs and loss of limb skeletal elements. These results strongly suggest that the presence of the region encoded by exon IIIA in mesenchymal fibronectin is necessary for the condensation event that occurs during chondrogenesis.
Alternative Splicing↗
Second messenger regulation of occlusion of the spinal neurocoel in the chick embryo.
We know that, once rostral neurulation is completed in the neuroaxis of the chick embryo, the caudal neurocoel becomes occluded and the brain rapidly expands. However, very little is known about the mechanisms maintaining occlusion. Studies had shown that occluded neurocoels reopened in embryos treated with chelators of cations, but the reasons remained unclear and the cations unidentified. To begin defining the role of cations, this study explored the effect of Ca2+, calmodulin, and cAMP on maintaining the occluded neurocoel. Chick embryos during the natural phase of neurocoel occlusion (stage 12) were cultured in vitro with drugs known to modulate Ca2+ transport, to inhibit calmodulin activity, or to elevate cAMP levels. To test if occlusion is a Ca(2+)-dependent process, embryos were treated with verapamil and ionophore A23187. To test if occlusion requires calmodulin, embryos were treated with antipsychotic agents. To test if occlusion is cAMP dependent, embryos were treated with methylisobutylxanthine (MIX), forskolin (FOR), or dibutyl cyclic adenosine (DbC). Following each treatment, occlusion of the neurocoel was tested by injecting dye into the midbrain. All treatments resulted in a predominant number of precocious reopenings of the occluded neurocoels. MIX-treated, naked neural tubes had a four-fold increase in cAMP, whereas FOR- and DbC-treated neural tubes showed ten- and 14-fold increases, respectively. The presence of calmodulin in the cells of the neural tube was confirmed by fluorescent tagging and 3H-chlorpromazine labelling. The combined results of this study show that occlusion of the spinal neurocoel depends on exogenous Ca2+, requires calmodulin, and is cAMP sensitive.
Animals↗
Evaluation of neural fold fusion and coincident initiation of spinal cord occlusion in the chick embryo.
Although it is known that rapid expansion of the vertebrate brain begins near the time that the spinal neurocoel is occluded, it still remains unknown when occlusion occurs in relation to neurulation. Since both morphogenetic events are critical for normal brain growth, it is important to decipher the temporal relationship between the two processes. This study assessed the temporal relationship of the two events with the rationale that if it could be demonstrated that occlusion occurs coincident with the completion of neurulation, then it could be argued that factors shown to direct neurulation could also initiate occlusion. Nearly 600 chick embryos (stages 9- through 12+) were cultured atop egg-agar, the caudal extent of neurulation determined, the cranial five pairs of somites removed and the neurocoels assessed for occlusion. In stage 9- through 10- chicks, neurulation of the spinal cord is incomplete. Stages 10 through 12+ exhibit neurulation and occlusion from the 8th to 19th somites. When lateral tissues were removed in embryos 8 through 10-, the neural folds became dysraphic whereas in embryos stage 10 and older, the folds remained fused dorsomedially and occluded. The only surgical manipulation that was found to prevent occlusion was elimination of the lateral tissues responsible for elevation and closure of the neural folds. Analysis of particular components of the lateral tissues essential for convergence, by treating embryos (n = 75) with chemicals known to degrade tissue-tissue bonds or specific components of the perineural matrix, indicated that more than 75% of the embryos treated with EDTA, EDTA plus Ca2+, trypsin, collagenase, or hyaluronidase exhibited little or no effect on convergence, dorsomedial fusion, and concomitant occlusion.
Animals↗
Timing and positioning of reopening of the occluded spinal neurocele in the chick embryo.
Occlusion and reopening of the spinal neurocele are important morphogenetic events occurring during rapid brain enlargement in amphibians, birds, and mammals. The purpose of the present study, a companion to a previous one on occlusion, is to examine reopening. We determined when reopening occurred and at what frequency, whether it occurred suddenly or gradually, the extents of the neurocele that were patent at particular stages of reopening, and the morphological pattern of reopening. Reopening begins at stages 14+ to 15 and is virtually complete at stages 16-17. Thus, reopening of the spinal neurocele occurs suddenly and occlusion is a relatively short-lived developmental event. Because rapid brain enlargement still occurs after reopening, occlusion probably functions principally in the initial phases of enlargement prior to closure of the posterior neuropore. Further studies on the mechanisms of occlusion and reopening are under way.
Animals↗
Quantification of the initial phases of rapid brain enlargement in the chick embryo.
Rapid brain enlargement requires a hydraulic mechanism in the chick embryo. Such a mechanism involves a closed, fluid-filled system that generates positive pressure. For the chick embryo this study determined when rapid brain enlargement begins, assessed the relative contributions of cavity expansion and tissue growth to overall brain enlargement, and evaluated mathematical models of overall brain enlargement and expansion and growth of the component parts. Three to five embryos were collected at each Hamburger and Hamilton state (11, 12, 14, 16, and 18) and processed for paraffin serial sectioning. Brain growth was determined over a 24-hr period (stages 11-18) by calculating volumes from area measurements of sections of brains from individual embryos by using a computerized image-analysis system. Statistical analysis indicated that a linear model adequately described cavity expansion, and a linear model was rejected for the description of tissue growth and total brain enlargement. At the onset of brain enlargement, the cavity expands faster than the tissue grows; but after 12 hr the reverse is true. Initially (i.e., at stage 11), the cavity accounts for 60% of the total brain volume and tissue for 40%. At stages 12-16, cavity and tissue contribute 50% each. Finally at stage 18, cavity accounts for 55% and tissue for 45%. In order to better distinguish changes in cavity expansion and tissue growth over the 24-hr period studied, this period was divided into four intervals (I-IV). The rates of both cavity expansion and tissue growth increase between intervals I and II, decrease between intervals II and III, and increase between intervals III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Identification of glycosaminoglycans in the chondrocranium of the chick embryo before and at the onset of chondrogenesis.
It appears that hyaluronate is associated with cell migration and the chondroitin sulphates with differentiation during morphogenesis of the chick embryo. The aim of this study was to see if such a correlation could be made for chondrocranium morphogenesis. Specifically, the purpose of this study was to determine the proportion of extracellular matrix (ECM) to cell area and total head mesenchymal area during chondrocranium morphogenesis; and to identify the location, types, and relative amounts of glycosaminoglycans (GAG) being synthesized in the presumptive chondrocranium at the onset of chondrogenesis and prior to this time. Morphometric analyses were made on median and parasagittal sections of heads of stage-24 and -33 embryos in order to determine relative contributions of cells and ECM to the total area of head mesenchyme at these stages. Presumptive chondrocrania (heads minus eyes) of these stage embryos were also analysed histochemically and biochemically in order to identify the GAGs present in the ECM. Sections of whole heads were stained with alcian blue at low and high pH as well as digested prior to staining with hyaluronidase (Streptomyces and testicular). Identification of GAGs was done by pulse labelling embryos with [3H]glucosamine, digesting homogenates with hyaluronidase (Streptomyces or testicular), precipitating the undigested GAGs with cetylpyridinium chloride and counting the dissolved precipitates using scintillation spectrophotometry. The types and relative amounts of GAGs present in the presumptive chondrocranium were determined by comparing the amount of radioactivity in the precipitates of the non-digested GAG with the counts in the precipitates of the predigested GAGs. This study reports that chondrogenesis begins in the presumptive chondrocranium of the chick embryo at stage 33 and that the area of the head mesenchyme increases 60-fold between stages 24 and 33. Little change in cell density and individual cell area as well as in the relative proportion of total area allocated to cells and ECM occurs. GAGs are localized exclusively in the presumptive chondrocranium. These GAGs are restricted to the ventral half of the presumptive chondrocranium. Within this region, the GAGs are further localized to the presumptive facial area, perichordal region, ethmoid, sphenoid and periotic regions. The types of GAG being synthesized in the head mesenchyme of both stage-24 and -33 embryos are hyaluronate, the chondroitins and unidentified sulphated GAGs (dermatan, keratin, heparin and heparan sulphate).(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Evaluation of the roles of intrinsic and extrinsic factors in occlusion of the spinal neurocoel during rapid brain enlargement in the chick embryo.
The spinal neurocoel normally occludes during the second day of chick embryogenesis as the lateral walls of the spinal cord become apposed closely in the midline. Concomitantly, the brain initiates its rapid and substantial enlargement. Occlusion, although short-lived, might play a major role in brain enlargement. As a result of occlusion, the brain ventricles are sealed off from the external milieu prior to closure of the posterior neuropore, establishing a closed fluid-filled system. The present study focuses on the mechanisms of occlusion of the spinal neurocoel. We tested two postulated intrinsic factors (microtubule-mediated neuroepithelial cell elongation and microfilament-mediated apical neuroepithelial cell constriction) and five extrinsic factors (three mediad pushing forces generated by the somites, perineural extracellular matrix and expanding surface ectoderm; and two stretching forces generated either vertically by pulling of the elongated notochord or longitudinally by elongation of the embryo) in maintaining occlusion. Our results suggest that occlusion is maintained by other, untested intrinsic factors and/or by forces generated within a perineural collar, composed of cellular and extracellular materials, intimately associated with the basal aspects of the spinal cord. Cytoskeletal-mediated changes in cell shapes, pushing forces and vertical and longitudinal tensions are not involved. Further studies are needed to examine the intrinsic properties of the neuroepithelium and the factors initiating occlusion and reopening.
Animals↗
Timing and positioning of occlusion of the spinal neurocele in the chick embryo.
The timing and positioning of occlusion of the spinal neurocele were studied in living and serially sectioned chick embryos at stages 8-14. Occlusion occurs in three phases: preocclusion, incipient occlusion, and definitive occlusion. Preocclusion occurs at stage 8. At this time, the neural groove of the spinal cord has not yet closed to form a neural tube. Incipient occlusion begins as early as stage 9 and lasts until stage 11. The neural groove of the cranial spinal cord closes during these stages and incipient occlusion occurs concomitantly with this closure. Seventy-eight percent of the embryos exhibit incipient occlusion. Incipient occlusion extends along the mid-somitic region of the neuroaxis, occupying about one-half the length of the spinal cord. Injection of the brains of living embryos with dyes often reopens incipient occluded areas. Definitive occlusion occurs at stages 11-14 and is present in 89% of the embryos. Definitive occlusion is restricted to mid-somitic regions, as was incipient occlusion, but it extends approximately two-thirds the length of the spinal cord. Injection of the brains of living embryos with dyes rarely reopens definitive occluded areas, even when injection pressures are maximal. Six morphological types of definitive occlusion can be identified on the basis of the relative proportions and locations of total and partial occlusion. Closure of the cranial neuropore and roof plate of the hindbrian occurs near the end of the incipient occlusion phase, whereas closure of the caudal neuropore occurs well after definitive occlusion is initiated.(ABSTRACT TRUNCATED AT 250 WORDS)
Age Factors↗
Reduced number of brain cells in so-called neural overgrowth.
Patten described as neural overgrowth a folding of brain tissue into the ventricles in abnormal human embryos. Intubation of chick embryo brains release cerebrospinal fluid pressure resulting in a similar folding. These folded brains have 2.5 times less tissue volume than control embryos of identical stage. This study shows, with total DNA measurements, that brain cell number is 50% less in folded brains (intubated embryos) as compared to control brain of identical staged embryos.
Animals↗
Descriptive studies of occlusion and reopening of the spinal canal of the early chick embryo.
Occlusion and reopening of the lumen of the spinal cord, two processes believed to be involved in early brain enlargement, were examined in chick embryos to determine what morphological features characterize these events. Occlusion begins at a particular craniocaudal level near the time that the neural folds become apposed in the dorsal midline and blocklike somites form from the segmental plates. During occlusion, the apical sides of the lateral walls of the neural tube are in close apposition. Interdigitating apical surface protrusions, cross-luminal intercellular junctions, and abundant cell-surface materials are lacking. Reopening has occurred by about stage 20 throughout most of the craniocaudal extent of the spinal cord. A lumen suddenly appears during this process, but correlated structural changes that might account for such a dramatic change in morphology were undetectable. Reopening involves the release of the forces that previously maintained occlusion, or the generation of new forces that overcome those causing occlusion, but what these forces are remains to be determined. Observations suggest that forces generated outside of the neural tube might be largely responsible for occlusion, and experiments are in progress to test this possibility.
Animals↗
Neural tube occlusion precedes rapid brain enlargement.
Histological examination of early vertebrate embryos during rapid brain enlargement (an event partially driven by fluid pressure) reveals that the spinal cord lumen is occluded. Occlusion (if it is not merely a fixation artifact) may confine neural tube fluid to brain regions and seal off the ventricles before posterior neuropore closure. We injected neural tubes of living chick embryos with dyes, asking (1) is occlusion real; and (2) does occlusion precede brain enlargement? Both questions were answered affirmatively. Experimental analyses of occlusion and brain enlargement are in progress.
Animals↗
The effects of low dosages of trichloroethylene on chick development.
This study reports the effects of low dosages of trichloroethylene (TCE) (1-25 mumol/egg) on chick development when embryos were exposed directly to TCE on days 1 and 2 and examined at day 14 of embryogenesis. The results indicate that in terms of embryotoxicity, growth defects and morphological anomalies, the treated embryos differed significantly from the controls. The low doses of TCE tested produced 50% mortalities. Survivors in all instances exhibited these developmental defects: evisceration; subcutaneous edema; light pigmentation of the epidermis; beak malformations; club foot and patchy feathering. The incidence of these malformations varied depending on the day of exposure to TCE except for embryos treated with 1 mumol. Irrespective of the day of injection or concentration, the highest proportion of malformations were those of light pigmentation and edema. Neither controls (uninjected embryos, O) nor sham controls (mineral oil injected embryos OMO) exhibited any malformations. Differences in measurements of crown-rump (CR), leg, wing, toe and beak lengths between treated embryos and controls were significant at the P less than 0.05 level of confidence. However, there was no significant difference between the weights of livers of treated embryos and controls nor was any pathology noted for the livers. The embryotoxicity of TCE is described by comparing the average percentage of deaths caused by all concentrations of TCE injected on both days 1 and 2 of embryogenesis. For TCE injected embryos this average was 2.5 times higher than the average for sham controls and 6 times higher than for uninjected controls.
Abnormalities, Drug-Induced↗
Description of the occlusion of the spinal cord lumen in early human embryos.
Previous studies of both chick and human embryos have shown that the brain enlarges rapidly once the neural tube becomes a closed, fluid-filled system. Prior to such enlargement, the medial walls of the spinal cord appear fused, occluding the lumen. This study describes occlusion of the lumen in terms of its incidence, location along the neuroaxis, time of occurrence, duration of occurrence, and morphology in human embryos. Eighty-two human embryos (stages 9-15) from the Carnegie Collection were analyzed. Occlusion first occurs (and is most prevalent) in stage 11 embryos and is absent in embryos older than stage 13. In all cases examined, the neuroaxis demonstrated uninterrupted occlusion from the level of the third pair of somites to at least the ninth pair (i.e., approximately 60% of the neuroaxis was occluded). The appearance of the occluded neural tube in cross sections is similar to that of a soda straw that has been pinched between one's fingers.
Embryo, Mammalian↗
The growth of the human brain during the embryonic period proper. 1. Linear axes.
Linear axes of the brain were measured in 143 human embryos from Carnegie stages 11 to 23 (3 1/2-8 postovulatory weeks). The embryos ranged from 3 to 30 mm in C.-R. length. Both Born reconstructions and serial sections of the central nervous system were used. The brain axes included were the fronto-occipital diameter, bitemporal diameter, and length and width of both the mesencephalon and cerebellum. A least squares line was fitted to the set of data points corresponding to each brain axis measured, and a t test verified that a linear model was an appropriate representation of the data. Based on these linear measurements it can be concluded that for forebrain grows more rapidly than the rest of the brain at the onset of tubular brain enlargement. Furthermore, as seen by comparing growth along two dimensions, the forebrain and midbrain grow at the same rate, whereas the cerebellum grows at different rates along the length and height axes. In addition, the cerebellum begins to grow later than the rostral part of the brain. Covariance analysis of the data points of the embryonic brain axes with data points of identical brain axes of the fetus showed that the measurements from the embryonic and fetal brain axes cannot be represented by a single regression line.
Anthropometry↗
Embryonic brain enlargement requires cerebrospinal fluid pressure.
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Animals↗