Ciliation and gene expression distinguish between node and posterior notochord in the mammalian embryo.

The mammalian node, the functional equivalent of the frog dorsal blastoporal lip (Spemann's organizer), was originally described by Viktor Hensen in 1876 in the rabbit embryo as a mass of cells at the anterior end of the primitive streak. Today, the term "node" is commonly used to describe a bilaminar epithelial groove presenting itself as an indentation or "pit" at the distal tip of the mouse egg cylinder, and cilia on its ventral side are held responsible for molecular laterality (left-right) determination. We find that Hensen's node in the rabbit is devoid of cilia, and that ciliated cells are restricted to the notochordal plate, which emerges from the node rostrally. In a comparative approach, we use the organizer marker gene Goosecoid (Gsc) to show that a region of densely packed epithelium-like cells at the anterior end of the primitive streak represents the node in mouse and rabbit and is covered ventrally by a hypoblast (termed "visceral endoderm" in the mouse). Expression of Nodal, a gene intricately involved in the determination of vertebrate laterality, delineates the wide plate-like posterior segment of the notochord in the rabbit and mouse, which in the latter is represented by the indentation frequently termed "the node." Similarly characteristic ciliation and nodal expression exists in Xenopus neurula embryos in the gastrocoel roof plate (GRP), i.e., at the posterior end of the notochord anterior to the blastoporal lip. Our data suggest that (1) a posterior segment of the notochord, here termed PNC (for posterior notochord), is characterized by features known to be involved in laterality determination, (2) the GRP in Xenopus is equivalent to the mammalian PNC, and (3) the mammalian node as defined by organizer gene expression is devoid of cilia and most likely not directly involved in laterality determination.

Formation of gut-like structures in vitro from mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into all cell types originating from the three germ layers; however, there are still few reports about the formation of functional organs from embryonic stem cells. Recently, we reported that by hanging drops of mouse ES cells, embryoid bodies (EBs) formed gut-like structures in vitro composed of three layers corresponding to the epithelium, lamina propria, and musculature. The morphological features and the process of formation are similar to gut and its organogenesis in vivo. Thus, this is a good model for development of the gut and a useful tool for analysis of the factors required for gut organogenesis. The protocol basically involves a method of hanging drops to make EBs, which are then plated on coated dishes for outgrowth. EBs develop to form gut-like structures when induced to spontaneously enter a program of differentiation in vitro without addition of any extrinsic factors.

Gastrulation in the sea anemone Nematostella vectensis occurs by invagination and immigration: an ultrastructural study.

The sea anemone Nematostella vectensis has recently been established as a new model system for the understanding of the evolution of developmental processes. In particular, the evolutionary origin of gastrulation and its molecular regulation are the subject of intense investigation. However, while molecular data are rapidly accumulating, no detailed morphological data exist describing the process of gastrulation. Here, we carried out an ultrastructural study of different stages of gastrulation in Nematostella using transmission electron microscope and scanning electron microscopy techniques. We show that presumptive endodermal cells undergo a change in cell shape, reminiscent of the bottle cells known from vertebrates and several invertebrates. Presumptive endodermal cells organize into a field, the pre-endodermal plate, which undergoes invagination. In parallel, the endodermal cells decrease their apical cell contacts but remain loosely attached to each other. Hence, during early gastrulation they display an incomplete epithelial-mesenchymal transition (EMT). At a late stage of gastrulation, the cells eventually detach and fill the interior of the blastocoel as mesenchymal cells. This shows that gastrulation in Nematostella occurs by a combination of invagination and late immigration involving EMT. The comparison with molecular expression studies suggests that cells expressing snailA undergo EMT and become endodermal, whereas forkhead/brachyury expressing cells at the ectodermal margin of the blastopore retain their epithelial integrity throughout gastrulation.

Kidney development and disease in the zebrafish.

Unraveling the molecular pathogenesis of human disease presents many experimental challenges, not the least of which is that experiments on humans are generally frowned upon. Model organisms, including the zebrafish, allow for experimental analysis of gene function and the detailed characterization of disease processes. Zebrafish have matured as a vertebrate model organism now that genetic tools for targeted "knockdowns" and unbiased mutagenesis approaches are in hand. The fish larval pronephros is a relevant kidney in which to pursue many aspects of human kidney development and disease. This short review outlines recent progress in applying the zebrafish pronephros to issues of human health and development.

Relación útero-embrionaria y su variación morfológica durante el periodo implantacional en conejo / Uterus-embryonnic relatioship and its morphologic variation during implantational period in the rabbit

La implantación embrionaria en úteros de mamíferos es iniciada por la formación de un contacto directo célula a célula entre el trofoblasto del blastocisto y el epitelio uterino. El conejo ha demostrado ser un excelente modelo para los estudios de implantación y se presenta como uno de los mamíferos con mayor eficiencia reproductiva. Nuestro objetivo fue reconstruir la secuencia de los eventos, tanto morfológicos como morfométricos que ocurren durante la implantación en el conejo, entre los 7 a 10 días post coito. Se utilizaron 16 conejas neozelandesas blancas adultas (Oryctolagus cuniculus), mantenidas en cautiverio y obtenidas del Bioterio de la Facultad de Medicina de la Universidad de La Frontera, Temuco, Chile. Una vez sacrificadas, el útero de cada coneja gestante fue fijado en formalina al 10 por ciento y postfijado en Dubosq brasil. Se utilizaron las técnicas histológicas: H. E. y Tricrómico de Masson e histoquímicas: PAS, PAS diastasa, Azul de Alcián pH 2.5 y pH 1.0, y Picrosirius de Junqueira. Otras vesículas se fijaron en methacarn para su estudio inmunocitoquímico con el anticuerpo monoclonal (CK) AE1, con la finalidad de evidenciar cambios en los filamentos intermedios de las células epiteliales. Se consignó el diámetro de la vesícula en mm y glándulas uterinas en µm. Desde el día 7 al 10 post coito, el diámetro de la vesícula embrionaria aumentó 2 mm por día. Las glándulas uterinas experimentaron un significativo y distinto crecimiento, dependiendo si éstas se encontraban en la pared mesometrial o antimesometrial. En conejo, el sinciciotrofoblasto del hemisferio abembriónico del blastocisto, se adhiere y fusiona con el epitelio uterino, luego la cámara implantacional, en coneja, se forma como resultado de la expansión del blastocisto que mantiene contacto con varios puntos de la pared uterina, ya sea en la región antimesometrial como mesometrial. La primera adhesión, ocurre entre el sinciciotrofoblasto y las células epiteliales de la pared antimesometrial antes de que ocurra una modificación epitelial general y del tejido conjuntivo subyacente, con aumento de glicógeno y glicosaminoglicanos.

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

Perturbation of extracellular matrix prevents association of the otic primordium with the posterior rhombencephalon and inhibits subsequent invagination.

In the avian embryo, the otic primordia become visible by Hamburger and Hamilton stage 10 as a pair of thickened regions of head ectoderm. In contrast to other epithelial primordia, invagination occurs by means of formation of a series of folds in distinct areas of the primordium, giving the otic vesicle a box-like appearance. Because previous work has shown that otic invagination is ATP and calcium independent, it is unlikely that cytoskeletal changes are the primary mechanism responsible for invagination as in other epithelial primordia. Interaction of the primordium with surrounding tissues may provide the force for otic invagination. These extracellular forces may be transduced through extracellular matrix macromolecules and their cell surface receptors. This investigation tests the hypothesis that fusion of the otic and hindbrain basal laminae between stages 11 and 13 is necessary for normal invagination. Perturbation of binding of the otic primordium to the neural tube was accomplished by means of microinjection of antibodies to various extracellular matrix components and integrin subunits into the head mesenchyme in the otic region at stage 10. Only antibodies to laminin and integrins caused detachment of the otic primordium from the hindbrain. These experiments suggest that fusion of the otic and hindbrain basal laminae is required for subsequent invagination and, furthermore, that this event is mediated by components of the extracellular matrix.

Ultrastructural aspects of the final stage of hemoglobin biosynthesis

Electron microscopic observations on rabbit embryo, adult rabbit, guinea pig, and human immature erythroid cells showed characteristic hemoglobinized organelles distinguishable from mitochondria by their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. The presence of hemoglobin (Hb) within these organelles was also demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed and osmotically lysec organelle fraction. The term hemosome has been suggested for these organelles because of their Hb content. We propose that they are the sites of heme integration into the four polypeptide globin chains. The frequency of hemosomes is higher in the peripheral blood erythorid cells of embryos than in the liver erythroid cells, coinciding with the higher Hb synthesis rate in peripheral blood than in the liver. Peripheral blood reticulocytes of rabbits with anemia induced by bleeding presented a lower hemosome frequency than normal reticulocytes. The decrease paralleled the decay of Hb biosynthesis activity. Moreover, Hb biosynthesis induced in HeLa cell and epithelial cell tissue cultures was always associated with the formation of hemosomes. Hemosomegenesis was studied in epithelial tissue culture cells experimentally induced to synthesize Hb, allowing the identification of several stages of hemosome formation in erythroid cells. The morphological data suggest that mitochondria are successively modified to lamellated...

Ultrastructural Studies on Mitochondria of Preimplantaion Rabbit Embryos

The ultrastructural changes of mitochondria in the ovarian oocytes from Graafian follicles, the ovulated tubal ova, and the various stages of preimplantation rabbit embryos have been observed with an electron microscope. From the ovarian oocytes to the 4-cell stage, mitochondria showed oval and round forms with a few cristae arranged concentrically and peripherally at the inner membrane. In 8-cell and 16-cell stages, mitochondria tended to change their forms to be elongated, and their sizes, and the outer membrane of the mitochondria had a tendency to become rough and irregular although there were few changes in the inner structure. In morula, some mitochondria began to show several transverse cristae proceeding into the matrix. Mitochondria rapidly increased in number at the late blastocyst stage. Matrix of mitochondria with transverse cristae found in the morula and in blastocyst stages was less dense than that of the earlier stages. The authors believe that the morphological changes of mitochondria during early embryonal development indicate the level of enzymatic activity at which this organelle is engaged in energy metabolism.