Journal of embryology and experimental morphology最新文献

A quantitative electrophoretic analysis of glucose phosphate isomerase (GPI-1) allozymes produced by heterozygous Gpi-1sa/Gpi-1sb mouse embryos has enabled us to estimate separately the contributions of GPI-1 enzyme that were oocyte coded, encoded by the embryonic, maternally derived Gpi-1sa allele and encoded by the embryonic, paternally derived Gpi-1sb allele. The oocyte-coded GPI-1 activity is stable until 2 1/2 days and then declines and is exhausted by 5 1/2 to 6 1/2 days post coitum (p.c.). The maternally and paternally derived Gpi-1s alleles are probably usually activated synchronously but several possible exceptions were observed. This activation was first detected in 2 1/2-day embryos. Total GPI-1 activity falls to a minimum around 3 1/2 to 4 1/2 days, even though embryonic gene expression has already begun. The profile of oocyte-coded GPI-1 activity is consistent with the suggestion (Harper & Monk, 1983) that there is a mechanism for the removal of oocyte-coded gene products at around 2 1/2 days p.c. The method of analysis described is applicable to other dimeric enzymes with electrophoretic variants.

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.

Small eyes (Sey) is a semidominant, homozygous lethal mutation in the mouse (Roberts, 1967). It is allelic with SeyH, a radiation-induced homozygous prenatal lethal which has been mapped on chromosome 2. The effect of the Sey mutation is apparently limited to the growth and differentiation of the presumptive lens and nasal placodes. Homozygous Sey/Sey embryos can be distinguished as early as 10.5 days post coitum (p.c.); the optic vesicles grow out, but the ectoderm does not give rise to a lens and nasal pits never form. Immunohistochemical studies show that the distribution of the extracellular matrix glycoprotein laminin is not significantly different in the cephalic region of Sey/Sey versus Sey/+ or +/+ embryos. Sey/Sey embryos develop to term but without eyes or nose, and die soon after birth. Further analysis of Sey/Sey embryos may throw light on the mechanisms underlying morphogenesis of craniofacial structures in mammals.

The otocyst is the epithelial anlage of the membranous labyrinth which interacts with surrounding cephalic mesenchyme to form an otic capsule. A series of in vitro studies was performed to gain a better understanding of the epithelial-mesenchymal interactions involved in this process. Parallel series of otocyst/mesenchyme (O/M) and isolated periotic mesenchyme (M) explants provided morphological and biochemical data to define the role of the otocyst in organizing and directing formation of its cartilaginous otic capsule. Explants were made from mouse embryos ranging in age from 10 to 14 days of gestation, and organ cultured under identical conditions until the chronological equivalent of 16 days of gestation. Expression of chondrogenesis was determined by both histology and biochemistry. The in vitro behaviour of periotic mesenchyme explanted either with or without an otocyst supports several hypotheses that explain aspects of otic capsule development. The results indicate that prior to embryonic day 12 the otocyst alone is not sufficient to stimulate chondrogenesis of the otic capsule within O/M explants; the otocyst acts as an inductor of capsule chondrogenesis within O/M explants between embryonic days 12 to 13; isolated mesenchyme within M explants taken from 13-day-old embryos are capable of initiating in vitro chondrogenesis, but without expressing capsule morphology in the absence of the otocyst; and the isolated mesenchyme of M explants obtained from 14-day-old embryos expresses both chondrogenesis and otic capsule morphology in the absence of the otocyst. These findings suggest that the otocyst acts as an inductor of chondrogenesis of periotic mesenchyme tissue between embryonic days 11 to 13, and controls capsular morphogenesis between embryonic days 13 to 14 in the mouse embryo.

The electrophysiological properties of the epithelium of the otic vesicle were studied in the chick embryo using conventional microelectrode techniques. A preparation is described that allows continuous recording of transmural potential and resistance during changes in the composition of the bathing fluid. Vesicles in stages 18 to 22 showed a spontaneous transmural potential (ET) that ranged from 2 to 6 mV, inner positive. This electrical potential difference was abolished after 2 h incubation in K+-free strophantidin (10(-4) M) and it increased by about twofold immediately after addition of the cation ionophore Amphotericin B (250 microM) to the bath. The specific resistance of the wall (RT) was about 80 omega cm2 between stages 18 and 22 indicating a low-resistance, noncellular, permeation pathway for current flow. The short-circuit current, calculated from ET and RT was about 50 X 10(-6) A cm-2 throughout this period. This corresponds to a net flux of 187 X 10(-8) mol cm-2 h-1 of a single cation pumped towards the towards the vesicular cavity. Diffusion potentials (salt gradients and single-ion substitutions) showed a selectivity ratio PK:PNa:PCl = 1:0.9:0.7, which is that of a weakly charged aqueous pathway. Measurements of vesicular volume and surface area showed an increase by a factor of ten in the size of the vesicle with maximal rates of change in volume of 5 microliter cm-2 h-1. The electrical properties reported here for the epithelium of the otic vesicle resemble very much those of 'leaky' epithelia which are known to transport ions and water at a very high rate.

The application of retinoic acid (RA) to the developing chick limb bud causes 6-digit double posterior limbs to form instead of the normal 3-digit limb. As an attempt to begin a molecular analysis of this phenomenon we have identified and characterized a soluble cytoplasmic receptor for RA, namely cytoplasmic retinoic acid-binding protein (CRABP), from the cells of the chick limb bud. It is present from stages 20-35 at similar levels and has an apparent Kd of 140-280 nM. In competition experiments with other retinoids Ro 13-7410 was found to be the most effective at competing for sites on CRABP followed by all-trans-RA, 13-cis-RA, Ro 10-1670 and retinal. Retinol, retinyl palmitate, retinyl acetate, etretinate and arotinoid showed low or no affinity for CRABP. Specificity for binding was thus demonstrated since analogues with an acid end group competed effectively, the aldehyde competed less effectively and the ester or alcohol groups did not compete. At the concentration of RA that needs to be administered to cause duplications in the pattern of the limb bud, we estimate that 4% of the CRABP present in the limb bud has RA bound. The similarities between steroid receptors in the mediation of steroid hormone action and CRABP in the mediation of RA action is discussed. In this regard we note that while there are 10(4) steroid receptors per cell in other cell types we estimate that there are about 10(5) RA receptors per cell in the chick limb bud.

Vital dye staining and cell lineage tracers were used to mark superficial cells of early Ambystoma mexicanum gastrulae. Superficial marks placed between the equator and the blastopore, on the dorsal midline, stained notochord, whereas marks or injections made at similar animal-vegetal levels but 90 degrees to either side of the dorsal midline were later found in somitic mesoderm. Notochord marks remained on the dorsal surface of the archenteron throughout gastrulation, though they became elongate and narrow by the morphogenetic movements of extension and convergence. Marked somitic mesoderm disappeared from the superficial epithelial layer soon after passing over the blastoporal lip and could not be found on the archenteron surface. A possible mechanism for this de-epithelialization is proposed on the basis of correlated SEM. The significance of a method of gastrulation so distinctly different from that of certain other amphibians is discussed in terms of amphibian phylogeny.

Ultimobranchial bodies (UBBs) were dissected from 17-day-old chick embryos and grafted onto the chorioallantoic membrane of 8-day-old embryos. The embryos with UBB grafts as well as sham-grafted controls were injected on the 10th day of incubation with 100 ng 1,25(OH)2D3 dissolved in ethyl alcohol or with an equal volume of ethyl alcohol alone; embryos were sacrificed on the 13th day. Grafted UBBs showed ultrastructural characteristics typical of actively secreting glands. A histological study of the tibiae from all embryos showed that while the grafted embryos responded to the injection of 1,25(OH)2D3 with a peripheral rim of undermineralized bone trabeculae, sham-grafted embryos never did so. These results confirm the original hypothesis that the presence of differentiated UBBs is a precondition for the production of undermineralized bone (osteoid) by 1,25(OH)2D3. In a second series of experiments, similarly treated embryos were sacrificed on the 10th, 11th, 12th and 13th day; the levels of calcium and inorganic phosphate were determined in their blood. The injection of 1,25(OH)2D3 produced in all embryos hypercalcaemia and hypophosphataemia. However, the hypophosphataemic response was more prolonged in the embryos with UBB grafts than in sham-grafted ones. These results suggest that the grafted UBBs prolonged the hypophosphataemic response, probably by secreting calcitonin and thus reducing the rate of bone resorption. It is also probable that the prolonged hypophosphataemia produced or contributed to the undermineralization of the peripheral (subperiosteal) trabeculae.

Neural crest-derived pigment cells form species-specific patterns of pigmentation in amphibian embryos. We have characterized the appearance and changes in pigment cell distribution in the embryos of the California newt, Taricha torosa. Black melanophores first appear scattered over the surface of the somites intermingled with yellow xanthophores in stage 34/35 embryos. The melanophores then migrate either dorsally to form a dorsal stripe at the apex of the somites or ventrally along the intersomitic furrows to form a midbody stripe at the somite-lateral plate mesoderm border. Xanthophores remain between the two melanophore stripes and are also found in the dorsal fin and head. The formation of the dorsal stripe coincides with a change in melanophore tissue affinity from the surface of the somites to the subectodermal extracellular matrix (ECM). The latter substratum is the location of the cue used to organize the dorsal stripe. In addition, melanophores become elongate and highly arborized, which would allow them to extend to the region where the dorsal stripe forms. In contrast, xanthophores do not form long processes in vitro. This suggests that the ability of melanophores but not xanthophores to search for a cue at the apex of the somites may account in part for the segregation of these cells types. Melanophores and xanthophores are trapped to form the midbody stripe by the pronephric duct, which is located just beneath the ectoderm at the bases of the intersomitic furrows. Ablation of the duct prevents formation of the midbody stripe, although melanophores and xanthophores still fail to migrate ventrally over the lateral plate mesoderm. Melanophores grafted to the ventral midline fail to leave the confines of the donor tissue. This suggests that a factor in the lateral plate mesoderm in addition to the pronephric duct is inhibiting further ventral migration. There is no gross morphological difference in the organization of the subectodermal ECM dorsal and ventral to the pronephric duct as revealed by alcian blue, ruthenium red and staining with antibodies to fibronectin. We also conclude that the directed dispersal of the neural crest into the space between the somites and ectoderm is due to contact inhibition of cell movement, since T. torosa neural crest cells demonstrate contact inhibition in vitro and there are enough cells in the lateral migratory spaces to make contact events likely during dispersal.

Retention of the capacity to induce the growth of hair by cultured adult rat vibrissa dermal papilla cells has been investigated. Small pellets of serially cultured papilla cells were implanted into the bases of the exposed follicular epidermis of amputated adult rat vibrissa follicles. Amputated follicles that received no cell implants or implants of cultured dorsal skin fibroblasts were used as controls. Over 50% of follicles implanted with cultured papilla cells in the passage range 1-3 grew hairs. In contrast none of the follicles that received late passage cells (range 6-15) produced hairs; and spontaneous regeneration of hair occurred in only 3% of the control follicles. These results demonstrate that cultured papilla cells of early passage numbers retain their ability to induce hair growth. Histological examination confirmed that the implanted papilla cells interacted with follicular epidermis to organize the development of new, hair-producing bulbs, each containing a discrete dermal papilla. An important observation was that aggregative behaviour leading to papilla formation was only manifested by early passage papilla cell implants. This persisting embryonic characteristic appears to be an essential functional component of papilla cell activity which operates to regulate the profound morphogenetic changes that occur during the hair growth cycle.