Darlene P. Wyatt

Pathogenesis of Neural Dysraphism in the Mouse Mutant Vacuolated Lens (vl)

Abstract The pathogenesis and expression of caudal neural dysraphism were studied by means of tritiated thymidine autoradiography in the mouse mutant vacuolated lens (vl) at 11–13 days of gestation. In homozygous litters, all of the embryos microscopically exhibited spinal cord defects, even though dysraphism could be detected grossly only in the most severely affected ones. The defects ranged from extreme eversion of widely open neural folds to a dorsally expanded neurocoel with an attenuated or distorted roof plate. Incorporation of tritiated thymidine occurred in cells in the defective roof plates, in severely distorted areas of the neural tube where cellular disaggregation occurred, and in isolated areas within the developing intermediate layer, as well as in normal sites of cellular proliferation. The frequently observed central mass of distorted but proliferating neural tissue associated with bilateral attenuations of the roof plate suggests a failure of, or defect in, the normal process of apposition and fusion of the neural folds, rather than a failure in opposition. The range in severity and the gross and microscopic manifestations of neuropathological lesions in the homozygous vl embryos indicate that this heretofore unexplored mutant has considerable potential as an experimental model for analyzing the origins and mechanisms of human neural dysraphism.

Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse.

Growth hormone (GH) cells were analyzed by means of ultrastructural morphometry in the pars distalis of pituitary glands from male adult and immature normal (C57BL) and homozygous little (lit/lit) mutant mice. Thin sections were exposed to anti-GH serum and processed immunocytochemically with the colloidal-gold technique. In the pars distalis of adult lit/lit mice, the mean volume density of GH cells/total tissue was 24% of the normal value, granules/GH cells was 58% of normal, and granules/total tissue was only 12% of normal. Deficits in all of these parameters likewise occurred in immature glands, though to a lesser extent than in the adults. The results indicate that the GH deficiency in this mutant reflects quantitative deficits in both the secretory granule content of GH cells, as well as the GH cell content of the gland, with the latter being the more severely affected.

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.

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.

Immunocytochemistry of TSH cells during development of the dwarf mutant mouse

SummaryThe functional status of thyrotrope (TSH) cells was evaluated by means of ultrastructural immunocytochemistry in the adult, early postnatal, and prenatal pars distalis of the pituitary gland in the dwarf (dw/dw) mutant mouse. In the normal (+/+;dw/+) pars distalis, thyrotropes were immunoreactive to anti-TSH serum at all stages studied, although the reaction at 17- and 18 days of gestation was much les intense than that at 26 days postnatally and in the adult. In the dwarf, cells exhibiting ultrastructural features resembling those of thyrotropes were observed, but their secretory granules were unreactive with anti-TSH. At early postnatal stages, thyrotrope-like cells were likewise unreactive in the dwarf, as were the poorly granulated cells present prenatally. Thus, the results indicate that although cells resembling thyrotropes are present in the dwarf, they are functionally abnormal not only in the adult, but also during development.

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.

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 produced 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.

Immunofluorescent analysis of fibronectin and laminin distribution in thevl mutant mouse

SummaryThe 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 Carnoys 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 neural crest cells are not prevented from emigrating from the abnormal neural tube; thus the faulty neural fold fusion that characterizesvl/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.

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

SummaryImmunofluorescence 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.

Ultrastructural analysis of the midaxial extracellular matrix in spinal dysraphism

SummaryUltrastructural 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 (+ /+;Lpj +) 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.