Mesoderm differentiation in explants of carp embryos

An analysis of carp blastoderm development was carried out in culture after isolation from the yolk cell and its yolk syncytial layer (YSL). The blastoderms were separated from the YSL at four different stages of embryogenesis: the blastula, early epiboly, early gastrula and late gastrula stages. Absence of the YSL in explants was checked by scanning electron microscopy. From observations of living embryos and histological examination of tissues which were formed in explants from all stages studied it was observed that they contained notochordal, muscle and neural tissue as signs of dorsal types of differentiation. Only in explants from the early and late gastrula stages were histotypical tissues organized in an embryonic-like body pattern. The data indicate that mesoderm differentiation in fish embryos is independent from the YSL, contrary to normal pattern formation which needs the presence of the YSL before the onset of gastrulation.     

Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor)

Summary Recently the mesoderm-inducing effects of the transforming growth factor (TGF-) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF- family has mesoderm-inducing ability.     

Protein kinases in amphibian ectoderm induced for neural differentiation

Summary Ectoderm explants from early gastrula stages of Xenopus laevis were induced with a neutralizing factor. The factor was isolated from Xenopus gastrulae and partially purified by chromatography on DEAE cellulose. The ectoderm was cultured for different periods of time and then homogenized. Protein kinase activity was determined in the homogenates from induced and control explants with histone H 1 or C-terminal peptide derived from histone H 1 as substrates. The C-terminal peptide is a more specific substrate for protein kinase C, whereas histoneH 1 is a substrate for cAMP/cGMP-dependent protein kinases as well protein kinase C. With both substrates the enzyme activity increases after induction. With the C-terminal peptide as the substrate the protein kinase activity is lower, but its relative increase after induction higher. This suggests that besides cAMP/cGMP dependent protein kinases protein kinase C or related enzymes are involved in the neural induction and differentiation processes. This corresponds to previous experiments which have shown that treatment of ectoderm with phorbol myristate acetate, an activator of protein kinase C and protein kinase C related enzymes, initiates neural differentiation. Endogeneous substrates, which are more intensively phosphorylated after induction are proteins with apparent molecular weights 21 kDa and 31 kDa. Addition of protein kinase C to the induced and control homogenates abolishes the difference in the phosphorylation rate of these proteins.     

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with a quartet of micromeres

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with micromeres was examined. An animal cap consisting of mesomere-descendants was isolated from a 32-cell stage embryo, and recombined with a quartet of micromeres isolated from a 16-cell stage embryo. The recombined embryos were completely depleted of the progenitors of an archenteron, pigment cells, blastocoelar cells and muscle cells. Secondary mesenchyme-like cells (induced SMC) were released from the archenteron derived from the animal cap cells in the recombined embryos. Some induced SMC differentiated into pigment cells, confirming previous data for another echinoid species. Moreover, three different kinds of mesodermal cells-blastocoelar, muscle and coelomic pouch cells-were formed in the recombined larvae. Experiments using a fluorescent probe confirmed that the pigment, blastocoelar, muscle cells and cells in part of the coelomic pouches in the recombined larvae were derived from the animal cap mesomeres. These results indicated that the animal cap mesomere had the potential to differentiate through cell fate regulation into four mesodermal cell types-pigment, blastocoelar, muscle and coelomic pouch cells-.     

Mesoderm induction in the future tail region of Xenopus

Summary We have used interspecific grafts between Xenopus borealis and Xenopus laevis to study the signalling system that produces tail mesoderm. Early gastrula ectoderm grafted into the posterior neural plate region of neurulae responds to a mesodermal inducing signal in this region and forms mainly tail somites; this signal persists until at least the early tail bud stage. Ventral ectoderm grafted into the posterior neural plate loses its competence to respond to this signal after stage 10 1/2. We have established the specification of anterior and posterior neural plate ectoderm. In ectodermal sandwiches or when grafted into unusual positions, anterior regions gave rise to mainly nervous system and posterior regions to large amounts of muscle, together with some nervous system. Thus it was impossible to assess the competence of posterior neural plate ectoderm to form further mesoderm and hence to establish if mesodermal induction continues during neurulation in unmanipulated embryos.     

Monitoring early neuronal differentiation by ion channels in ascidian embryos

According to the evolutionary tree proposed by Garstang, the tunicate larva has a central role in directing the ancestral sessile animal derived from primitive echinoderms into the stem for vertebrates by evolution through neoteny. The close similarity of the tunicate larval body plan to those of vertebrates and the extraordinary simplicity indicated by an extremely small cell population make the ascidian embryo and larva an excellent model system for analysis of vertebrate embryonic development. Furthermore, isolated anterior animal blastomeres from the Halocynthia eight-cell cleavage-arrested embryo, which are known to include presumptive brain vesicle region, autonomously develop long-lasting Ca-dependent action potentials which are characteristic of epidermal differentiation. However, when blastometeres are cultured in contact with the anterior vegetal blastomere, which are known to include presumptive notochordal region, and raised in contacted two cell systems, the same anterior animal blastomeres now develop neuronal Na⁺ spikes characterized by expression of Na⁺ channels and triethylammonium sensitive delayed rectifier K⁺ channels. This unique two-cell system enables us to examine roles of cell contact in various aspects of inductive differentiation at the cellular level. In this review, we focus on this simple cellular preparation and in particular, attempt to show how to make the preparation. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 3–22, 1998     

Morphological differentiation of mitochondria in the early amphibian embryo

Three different cell types (epidermal cells, fibroblast-like Ruffini cells and undifferentiated cells) may be observed in cultures of cells isolated from the blastula of Ambystoma mexicanum. Electron micrographs show that the mitochondria in the two kinds of differentiated cells have the orthodox conformation; those in the undifferentiated cells the condensed conformation. Measurements of the area and the form factor demonstrate that the mitochondria in the differentiated cells are considerably larger than those in the undifferentiated cells, and that the three populations of mitochondria differ significantly from each other.     

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos

Summary Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

Lithium induces changes in the plasma membrane protein pattern of early amphibian embryos

Summary— The plasma membrane protein pattern of Rana ridibunda embryos subjected to lithium (Li) treatment at various stages of development was examined by two-dimensional gel electrophoresis. Differences were observed at the neurula stage not only as compared to controls but among lithium-treated embryos as well. Of particular interest was the presence of proteins, specific for the gastrula stage, in lithium-treated embryos. The results are discussed in relation to the well-known effect of lithium on amphibian morphogenesis.     

Developmental mutants showing abnormal organ differentiation in rice embryos

Summary Zygotes of rice (Oryza sativa L. cv Taichung 65) were treated with 1.0 mM solution of the chemical mutagen N-methyl-N-nitrosourea. Out of 1420 M₂ lines, 28 single-locus recessive mutants on embryogenesis were identified. Among them, we analyzed 11 mutants in the present study, which differentiated the shoot (plumule) and/or root (radicle) with abnormality. Of the 11 mutants, two showed no shoot differentiation with normal root. On the other hand, we could not detect any mutant which exhibited a normal shoot without a root. This suggests that shoot and root are genetically controlled by different loci and that the alleles associated with shoot formation mutate more frequently than do those of the root. Five mutants showed aberrant morphology of shoot when both the shoot and root developed. One of them, odm 5 (organ differententiation mutant 5) was germinable, but produced many fine and twisted leaves. This mutant was, however, lethal at the early post-germination stage under the usual cultural conditions. In another mutant (odm 4), shoot differentiation seemed to be initiated at an arbitrary position, resulting in a very abnormal morphology of the shoot when the position fronted the endosperm. The other two mutants showed abnormal morphology of both the shoot and root. One (odm 11) of the remaining two mutants showed a wide variation of abnormalities including no organ differentiation, either shoot or root differentiation and the development of both shoot and root with abnormalities. The last one (odm 16) was unique. It had an embryo with normal shoot and root but the embryo size was only one-third to one-half of normal embryos in length. Of course, the shoot and root are also small but viable. Therefore, odm 16 is considered to be a mutant in the size regulation of the embryo. Although an allelism test has not yet been done, most of these mutants are probably non-allelic, as the phenotypic abnormality differs largely with each one. In rice, the shoot and root highly differentiate in contrast to dicotyledonous embryo. Accordingly, these developmental mutants are very useful materials for investigating the regulatory mechanism of gene expression in organ differentiation.     

Pattern and rate of ovary differentiation with reference to somatic development in anuran amphibians

The rate of somatic development of anuran amphibians is only roughly correlated with the rate of gonad differentiation and varies among species. The somatic stage of a tadpole often does not reflect its age, which seems to be crucial for gonad differentiation rate. We compared the morphology and differentiation of developing ovaries at the light and electron microscopy level, with reference to somatic growth and age of a female. Our observations were performed on 12 species of six families (Rana lessonae, R. ridibunda, R. temporaria, R. arvalis, R. pipiens, R. catesbeiana, Bombina bombina, Hyla arborea, Bufo bufo. B. viridis, Xenopus laevis, Pelobates fuscus) and compared with the results obtained by other authors. This allowed us to describe the unified pattern of anuran female gonad differentiation. Ovary differentiation was divided into 10 stages: I-III, undifferentiated gonad; IV, sexual differentiation; V, first nests of meiocytes; VI, first diplotene oocytes; VII-IX, increasing number of diplotene oocytes and decreasing number of oogonia and nests; X, fully developed ovary composed of diplotene oocytes with rudimental patches of oogonia. We distinguished three types of ovary differentiation rate: basic (most species), retarded (genus Bufo), and accelerated (green frogs of the subgenus Pelophylax genus Rana).     

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

The marginal sinus in the perifollicular region of the rat spleen

Summary The marginal sinus in the spleen of the Wistar rat surrounds the follicle and has more numerous PAS positive fibers on the inner wall than on the outer wall. India ink- and lead oxide-gelatin were injected into the abdominal aorta. It was found that much of the india ink-gelatin accumulated in the marginal sinus, the marginal zone, and part of the red pulp, while most of the lead oxide-gelatin collected in the marginal sinus.Ultrastructurally, the capillaries of the follicle were found to open into the marginal sinus. Regions not perforated by the marginal sinus lie between the follicle and the marginal zone. The wall of the marginal sinus is discontinuous and the discontinuities are wider on the marginal zone side than on the follicle side. The relationship of these findings is discussed.A part of this study was presented at the 64th Annual Meeting of the Japanese Pathological Society, Takatsuki, April, 1975     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

Localization of thymocyte stem cell precursors in the pharyngeal endoderm of early amphibian embryos.

The embryonic germ layer derivation of thymic lymphocytes in the leopard frog (Rana pipiens) was invesigated. The ectoderm and mesoderm—but not the endoderm—from the developing gill bud was reciprocally and orthotopically transplanted between diploid and triploid chromosomally marked 72-hr old embryos. The lymphocytes which subsequently developed in the thymus were of host ploidy. Thus, the head mesenchyme adjacent to the thymic anlagen does not appear to be the source of the stem cells from which thymocytes differentiate. Rather, the stem cells can he localized in the endoderm of the pharyngeal pouch which initially gives rise to the thymic epithelium. These findings suggest either the endodermal origin of thymocytes or the very early migration of stem cells into the thymus anlagen prior to any thymus histogenesis.     

Hepatocyte differentiation during early fetal development in the rat

Summary Rat hepatocyte differentiation between day 12 and 19 of fetal life was studied by electron microscopy. The cytoplasmic structures involved in synthetic and secretory function, i.e., rough endoplasmic reticulum and Golgi apparatus, appear to be the first to differentiate, and their development is probably related to the secretion of different kinds of plasma proteins. The cytoplasmic organelles involved in other hepatic functions develop later: lysosomes from day 15, peroxysomes, glycogen rosettes and smooth endoplasmic reticulum still later. However, the morphological differentiation of bile canaliculi begins from day 12.This work was supported in part by a grant from C.N.R. n. 78.02265.04