Alterations in cranial morphogenesis in the Lp mutant mouse.

The effects of exencephaly on cranial morphogenesis were studied at 10 to 12 days of gestation in the loop-tail (Lp) mutant mouse in which the hindbrain and spinal cord fail to close. At the level of the hindbrain, the otocysts became displaced ventrally in abnormal (Lp/Lp) embryos, and the everted neuroepithelium showed a diminished "luminal" reaction to the lectins WGA and Con A, as compared with normal embryos. Also, occasional clusters of rounded cells that resembled presumptive neural crest cells and that labeled with WGA, Con A, and anti-N-CAM were observed at the everted tips of the open neural folds. By 12 days' gestation, there was a loss of integrity in some areas of the neuroepithelium. However, despite the topographic and neuroepithelial distortions, normal differentiation of a roof plate-like neuroepithelium occurred at the ends of the everted neural folds. In addition, the mesenchyme showed normal condensations that labeled with WGA, Con A, and anti-N-CAM in the perinotochordal basicranium and periotic regions. Thus, in this mutant mouse model of neural dysraphism, some features of cranial morphogenesis and differentiation appear to be dependent on timely and proper closure of the cranial neural tube, whereas other aspects may proceed independently of neural closure.

Patterns of lectin binding during mammalian neurogenesis.

Temporospatial changes in surface carbohydrates of neuroepithelial cells were analysed by means of lectin histochemistry in normal mouse embryos subsequent to closure of the neural tube. The lectins used were concanavalin A (con A), soybean (SBA), Maclura pomifera (MPA), peanut (PNA), wheatgerm (WGA), succinylated wheatgerm (sWGA) and Limax flavus (LFA). Although labelling was obtained with all of the lectins, the most striking temporospatial differences occurred with con A which in the early embryos (9-10 somites) labelled the basal and intercellular surfaces, but not the luminal surfaces of the neuroepithelial cells, whereas in the older embryos (26-30 somites), con A showed light luminal surface labelling. A midventral wedge of cells in the floor of the neural tube in the older embryos also exhibited more intense labelling with con A, WGA, and sWGA than with the other lectins. In addition, comparisons of lectin localisation were made between the closed neural tube in normal embryos and the open neural folds in the loop-tail (Lp) mutant mouse in which the neural tube fails to close. Although similar temporospatial patterns in lectin localisation occurred as in normal embryos, the retention of lectin labelling associated with rounded putative neural crest cells that remained sequestered in the apices of the open neural folds, along with an attenuation of the luminal reaction in the older abnormal embryos, suggest that during normal mammalian development closure of the spinal neural folds may be important for the timely exit of neural crest cells as well as for eliciting changes in the luminal surfaces of the neuroepithelial cells.

Immunofluorescent analysis of the spinal cord in dysraphic mice.

Development of the spinal cord was analyzed immunocytochemically at 10-12 days of gestation in normal and dysraphic embryos of the loop-tail mutant mouse, using an anti-neural cell adhesion molecule (NCAM) and the lectin concanavalin A (Con A) as histological markers for evaluating neural cell organization and distribution. The normal and abnormal embryos showed similar patterns of reactivity to anti-NCAM and Con A, even though the neural folds were open and everted in the abnormal embryos, with displacement of the dorsal root ganglia. In the abnormal embryos the floor plate was similar to that in normal embryos, as evidenced by its increased anti-NCAM and Con A labeling relative to that in the rest of the neuroepithelium. Moreover, each lateral end of the everted abnormal neuroepithelium developed an attenuated 'roof plate' that appeared to be structurally similar to the normal roof plate. However, some of these 'roof plates' exhibited prominent clusters of labeled and nonlabeled cells, especially in the 10-day embryos. In addition, whereas normal embryos showed strong luminal labeling of the neuroepithelial cells with Con A, comparable regions in the abnormals were spotty and poorly defined except for the 'roof plate' and floor plate. The results indicate that dorsoventral polarity in the spinal cord, as assessed structurally and histochemically, develops essentially normally in abnormal dysraphic embryos, even though the topographic relationships of the abnormal neural tube are disturbed.

Analysis of neurulation in a mouse model for neural dysraphism.

Elongation of the neuraxis was analyzed quantitatively with respect to the subpopulation of longitudinally oriented mitoses in the neuroepithelium in homozygous embryos of the loop-tail (Lp) mutant mouse, which is characterized by failure of fusion of the neural folds from the midbrain to the tail, as well as a shortening of the neuraxis. Correlations were made with mitotic cell orientation in the underlying gut and notochord, which are likewise shortened. In the abnormal dysraphic embryos at the 7- to 11-somite stage, the percentage of longitudinally oriented mitotic spindles in the neuroepithelium was significantly less than in normal embryos. In contrast, significant differences were not obtained with respect to the orientation of mitotic spindles in the gut or notochord. At the 15- to 20-somite stage, significant differences in mitotic orientation in the neuroepithelium, gut, or notochord did not occur between dysraphic and normal embryos. The results suggest that during elevation and fusion of the neural folds, a decrease in the percentage of longitudinally arranged spindles in the neuroepithelium of Lp/Lp embryos may contribute to the disturbance in neuraxial elongation and possibly closure failure, but that the shortened gut and notochord that also characterize this mutant may not result from defective orientation of mitotic spindles.

Immunocytochemical effects of thyroxine stimulation on the adenohypophysis of dwarf (dw) mutant mice.

The effects of dietary thyroxine on the immunoreactivity of cells in the pars distalis of the adenohypophysis in dwarf (dw/dw) mice were determined by ultrastructural immunocytochemistry. In nontreated dwarfs only adrenocorticotropic hormone (ACTH) cells and luteinizing hormone (LH) cells showed positive reactions to their respective antibodies, whereas no cells showed immunoreactivity to antibodies to growth hormone (GH), thyroid-stimulating hormone (TSH), or prolactin (Prl). In dwarfs supplemented postnatally with dietary thyroxine for 9 wks, the treatment failed to produce immunoreactive GH, TSH or Prl cells. However, LH cells became more prominent and fully developed, with denser concentrations of immunoreactive particles overlying the secretory granules than occurred in nontreated dwarfs. In thyroxine-treated dwarfs, ACTH cells were similar in ultrastructural features and immunoreactivity to those in nontreated dwarfs.

Immunocytochemistry of ambiguous cells in adult and embryonic dwarf (dw) mouse pituitaries.

In the pars distalis of the pituitary gland in adult and embryonic dwarf (dw/dw) mutant mice, ambiguous cells exhibiting ultrastructural features common to growth hormone (GH) cells and prolactin (Prl) cells were analyzed by means of colloidal gold ultrastructural immunocytochemistry in order to define the functional nature of these peculiar cells. Adult and 18-day embryonic pituitaries from normal (+/+; dw/+) and dwarf (dw/dw) mice were processed with antibodies to GH, Prl, TSH (thyroid-stimulating hormone), ACTH (adrenocorticotropic hormone), LH (luteinizing hormone), FSH (follicle-stimulating hormone), and HCG (chorionic gonadotropic hormone). In the adult and embryonic dwarf pituitaries, the ambiguous cells reacted negatively to all of the antibodies except for anti-ACTH, which labeled them well. In addition, the ACTH-positive cells showed a much wider variety of shapes and granule size and distribution, as compared with normal adults. In the embryos, this variability in ACTH cell morphology occurred not only in dwarf embryos, but in their normal counterparts as well. The results thus suggest that adult dwarf pituitaries may retain an embryonic or incompletely differentiated form of ACTH cells.

In vitro expression of neural tube pathology in the vl mutant mouse.

The pathogenesis of lumbosacral spinal dysraphism was analyzed in vitro in early homozygous embryos of the vl (vacuolated lens) mutant mouse, and comparisons were made between the lag in closure of the posterior neuropore at the beginning of culture and the degree of severity in the spinal defect at the end of culture for each embryo. In the majority of abnormal (vl/vl) embryos, the neural tube closed completely in culture, although mild defects were observed in the spinal roofplate comparable to those that occur in vivo. Although some abnormal embryos did exhibit small openings in the caudal neural tube at the end of the culture period, in none of the cultured embryos was the extent and severity of the defect as great as that observed in some abnormal embryos of comparable age obtained in vivo. Moreover, the degree of delay in closure of the posterior neuropore at the outset of culture did not necessarily correlate with the severity of the defect obtained at the end of culture. Thus, the expression of the neural tube defect in this mutant appears to be modulated and attenuated by biochemical and/or mechanical factors which may be peculiar to the culture milieu.

Adrenocorticotropic cell distribution in adult and embryonic pituitaries of the little (lit) mutant mouse.

Immunofluorescence and colloidal gold immunocytochemistry were used to analyze the regional distribution of adrenocorticotropic (ACTH) cells and their ultrastructural relationship with growth hormone (GH) cells in adult and 17-day embryonic little (lit/lit) mice that exhibit GH cell defects. Adult lit/lit pituitaries lack the distinctive regionalization of ACTH cells that characterizes normal pituitaries, although typical ultrastructural relationships occur between some ACTH and GH cells. In 17-day embryos, normal and lit/lit pituitaries show similar distributions of ACTH cells. However, in lit/lit pituitaries the pars distalis cells are more loosely arranged, with poorly defined clusters and cords, than in normal glands. The results indicate that whereas the lack of ACTH cell regionalization in adult glands may be a secondary effect of the GH cell defect, the differences in overall integrity of the 17-day embryonic glands suggest the possibility of a developmental disturbance during early stages of gestation in this mutant.

Histopathology of the pituitary gland in neonatal little (lit) mutant mice.

The pituitary gland in the little (lit) mutant mouse was analyzed with respect to the cytoarchitecture of the pars distalis and the volumetric density of immunoreactive growth hormone (GH) cell granules in neonatal lit/lit and normal C57BL mice. At 8 days postnatally the volume of GH granules/total tissue was significantly less in the lit/lit pars distalis, and the cells were loosely arranged, as compared with the normal pars distalis. In newborn mice a statistically significant difference could not be detected between normal and lit/lit mice with respect to the volumetric density of GH granules; however, differences occurred in the cytoarchitectural organization of the pars distalis. These differences included prominent vascular channels and well-defined cords and clusters of cells in the normal newborn mice, in contrast to indistinct vascular elements and a more diffuse arrangement of cells in lit/lit.

Aberrant convergence of the neural folds in the mouse mutant vl.

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.

Abnormal elevation of the neural folds in the loop-tail mutant mouse.

The processes of elevation and convergence of the spinal neural folds were analyzed in normal (+/+; Lp/+) and abnormal (Lp/Lp) embryos of the loop-tail mutant mouse in order to determine possible mechanisms underlying the dysraphic defect characterized by a failure of the neural fold to close in this mutant. The results indicate that the neural folds are already defective during very early phases of elevation, with greater distances between the apical points of the paired walls of the neural groove, larger ventral angles and higher ratios of luminal/basal linear distances occurring in the abnormal embryos relative to those in normal embryos. The cross-sectional area of the neuroepithelium is also greater in abnormals, suggesting that faulty elongation of the neuraxis may contribute to the dysraphic condition.

Ultrastructural analysis of the midaxial extracellular matrix in spinal dysraphism.

Ultrastructural aspects of the extracellular matrix (ECM) in the midaxial region of dysraphic embryos of the loop-tail (Lp) mutant mouse were analyzed by means of electron microscopy. In 17-23 somite embryos, ultrastructural differences in the ECM occurred with respect to the presence of a pair of long trailing basal laminar strands extending continuously from the ventral notochordal cells to the gut in abnormal (Lp/Lp) embryos, in contrast to short, ragged, discontinuous strands in normal (+/+; Lp/+) embryos. The ultrastructural localization and configuration of fibronectin (FN) and laminin (L) associated with these strands, however, were similar in normals and abnormals. In addition, FN occurred over interstitial bodies, fibrils, and sporadically along the basal laminae of the neural tube (or folds), notochord, gut, and vessels, whereas L was largely confined to the basal laminae. The results indicate that although the ultrastructural pattern of FN and L reactivity are similar in normal and abnormal embryos, a disturbance in the manner whereby the notochord detaches from the gut in dysraphic embryos may be of causal significance in the etiology of dysraphism in this mutant.

Cranial effects of retinoic acid in the loop-tail (Lp) mutant mouse.

The effects of retinoic acid (RA) on the manifestation and nature of neural tube defects (NTD) in heterozygous embryos of mutant mice carrying the gene loop-tail (Lp) and in normal (+/+) littermates and embryos from normal homozygous matings were compared with NTD that occur in untreated abnormal homozygous (Lp/Lp) embryos. A single intraperitoneal dose (5 mg/kg) of RA administered at 9 AM or 3 PM on day 8 of gestation induced NTD in +/+ as well as Lp/+ embryos removed on day 12 of gestation. All of the NTD were confined to the brain and consisted of exencephaly involving the diencephalon, mesencephalon, and metencephalon. In neither phenotype (Lp/+; +/+) was the massive exencephaly and myeloschisis characteristic of untreated Lp/Lp embryos produced; thus, it is possible that the teratogenic mechanisms of RA-induced defects and of Lp-induced defects may differ.

Immunofluorescent analysis of fibronectin and laminin distribution in the vl mutant mouse.

The distribution of fibronectin and laminin was determined in the basement membrane surrounding the caudal neural tube and at the site of initial apposition of the caudal neural folds by means of indirect immunofluorescence histochemistry on 9.0- to 10.5-day mouse embryos fixed in Carnoy's solution and serially sectioned in paraffin. At early phases of development of normal (+/+) and abnormal (vl/vl) embryos the dorsolateral neural basement membrane overlying putative neural crest cells caudal to the hindlimb shows a patchy fibronectin reaction, with laminin virtually absent. In older embryos, both components are present but are discontinuous overlying the neural crest. The results suggest that since discontinuities occur in the basement membrane of abnormal as well as normal embryos, the neutral crest cells are not prevented from emigrating from the abnormal neural tube; thus the faulty neural fold fusion that characterizes vl/vl embryos does not appear to be due to a suppression of emigration by the basement membrane. The results also demonstrate the advantages and reliability of embedding in paraffin for analysis of serially sectioned pathological material by means of indirect immunofluorescence, provided that normal controls and abnormals are processed simultaneously.

Axial interactions during neurogenesis in dysraphic mice.

The spatial relationships of midline axial structures were compared in normal and dysraphic embryos of the loop-tail (Lp) mutant mouse at 9 days of gestation, and the distribution of two glycoproteins, fibronectin and laminin, was determined in this region by means of immunofluorescence histochemistry. At various levels of the trunk in abnormal embryos, the notochord, ventral midline of the neural folds, dorsal aorta(e) and/or gut were variably separated from one another, in contrast to the closer contact that occurred among these midaxial structures at comparable trunk levels in normal embryos. Positive anti-fibronectin and anti-laminin reactions were noted within areas of contact in the normal embryos and at the interfaces of tissue separation in the abnormal embryos. Although the intensity of the immunofluorescent reaction could not be assessed or compared quantitatively, the presence and distribution of fibronectin and laminin in the abnormal embryos suggest that these two glycoproteins may not be causally or fundamentally involved in producing the abnormal spatial relationships and separation of the midaxial structures.

Deposition of laminin and fibronectin in dysraphic mutant mice: an immunofluorescent study.

Abnormal loop-tail (Lp/Lp) mutant mouse embryos exhibiting severe exencephaly and myeloschisis were analyzed and compared with their normal (+/+; Lp/+) littermates by means of immunofluorescence histochemistry to determine regional differences in the distribution of laminin (L) and fibronectin (FN). In the neural basement membrane and adjacent mesenchymal cell matrix of the abnormal embryos, regional differences in the deposition of L and FN were similar to those in normal littermates. Moreover, most of the putative neural crest (NC) cells appeared to emigrate normally in terms of their site of detachment and migration pathways, despite the severe topographic distortions and loss of neuroepithelial integrity. However, some putative NC cells projected incorrectly from the 'luminal' surface of the neuroepithelium, suggesting that some of the NC may be abnormal or sequestered and prevented from appropriate detachment and emigration from the neural tube.

Ultrastructural defects in the apical neural folds in mutant embryos with spina bifida.

Ultrastructural pathology in the apical neural folds was analyzed by means of tannic acid (TA) and ruthenium red (RR) cytochemistry in abnormal (vl/vl) mutant mouse embryos ranging in age from 17-35 somites. At lumbosacral levels of the spinal cord where closure fails to occur, as well as at more cranial levels where closure occurs but results in dorsal midline abnormalities, normal deposition of TA-positive and RR-positive material occurred in the space that develops between the overlying surface ectoderm (SE) and neuroepithelium (NE). However, in lumbosacral regions, pleomorphic excrescences projected abnormally from the apices of the transitional zone cells between SE and NE cells of the open neural folds. These abnormal projections consisted of enlarged cytoplasmic blebs, as well as entire cells. The cells were not necrotic nor did they show evidence of incipient degeneration. However, it is possible that they represent aberrant putative neural crest cells, as indicated by their location in the transitional zone and by the filopodia and lamellipodia projecting from their luminal surfaces.

Cytochemical analysis of neural dysraphism in the vl mutant mouse.

Closure of the posterior neuropore was analyzed by means of ultrastructural cytochemistry in ten-day dysraphic mouse embryos homozygous for the mutant gene vl, and comparisons were made with normal embryos in terms of convergence, apposition and fusion of the apices of the neural folds. In abnormal embryos, regional differences in the distribution of the surface coat were comparable to those in normal embryos. However, there was an abnormally acute medial bending of the neural folds, as well as a delay in closure of the posterior neuropore. In closed areas of the abnormal embryos the dorsum also showed an erratic knot of disorganized cells. Thus, the pathogenetic mechanism in this mutant appears to involve not only a failure in apposition in open areas, as well as an inappropriate association of cells in areas which do fuse, but possibly also a failure of proper alignment of neural fold apices prior to apposition and fusion.

Immunofluorescent analysis of somatotroph distribution in the adenohypophysis of developing lit/lit mice.

Regional and sexual patterns in the distribution and density of somatotroph cells exposed to anti-growth hormone serum were analysed by means of immunofluorescence histochemistry in adenohypophyses of normal C57BL mice and abnormal (lit/lit) mutant mice, which exhibit postnatal growth deficits. In adult (3-4 months) lit/lit mice, the regional distribution of somatotrophs both in males and females was normal; however, there was a sparsity of somatotrophs, relative to the normal condition, in the lateral wings of the pars distalis, and sexual differences in the concentration of immunoreactive cells were not as prominent as in the normal mice. In the midline region of the pars distalis a cranioventral zone virtually devoid of somatotrophs occurred in lit/lit as well as in normal mice, especially in the females, though it was not as well defined in lit/lit because of the overall sparsity of somatotrophs. In normal immature mice at 8 and 14 days after birth, the lateral wings did not show the striking sexual differences in density of somatotroph distribution as they did in the normal adults, and at 8 days they were more sparsely populated with somatotrophs than at 14 days. In 14 day lit/lit mice, the lateral wings were less densely populated with somatotrophs than their normal counterparts, but at 8 days these differences were not detectable. In both normal and abnormal 8 day mice, the medial and midline regions of the pars distalis contained less intensely immunoreactive somatotrophs than did the lateral wings.(ABSTRACT TRUNCATED AT 250 WORDS)