Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.
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Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis. (+info)
Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo.
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Fragments of the germ layer tissues isolated from the early-primitive-streak (early-streak) stage mouse embryos were tested for axis induction activity by transplantation to late-gastrula (late-streak to early-bud) stage host embryos. The posterior epiblast fragment that contains the early gastrula organizer was able to recruit the host tissues to form an ectopic axis. However, the most anterior neural gene that was expressed in the ectopic axis was Krox20 that marks parts of the hindbrain, but markers of the mid- and forebrain (Otx2 and En1) were not expressed. Anterior visceral endoderm or the anterior epiblast alone did not induce any ectopic neural tissue. However, when these two anterior germ layer tissues were transplanted together, they can induce the formation of ectopic host-derived neural tissues but these tissues rarely expressed anterior neural genes and did not show any organization of an ectopic axis. Therefore, although the anterior endoderm and epiblast together may display some inductive activity, they do not act like a classical organizer. Induction of the anterior neural genes in the ectopic axis was achieved only when a combination of the posterior epiblast fragment, anterior visceral endoderm and the anterior epiblast was transplanted to the host embryo. The formation of anterior neural structures therefore requires the synergistic interaction of the early gastrula organizer and anterior germ layer tissues. (+info)
Germ line development in fishes.
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Classical work on germ cells in fishes has dealt with three main issues; their embryonic origin, the proliferation, and migration pathway during embryonic and larval development. Until recently, primordial germ cells (PGCs) have been studied in a number of fishes using morphological criteria only. The identification of the Drosophila vasa homolog gene of zebrafish now allows comparison of these morphological data with vasa RNA expression patterns in zebrafish. Teleost PGCs can be distinguished from somatic cells by their distinct morphology, at the earliest during gastrulation, and in most fishes their number varies between 10 and 30 during pregonial development. Mitosis is generally not observed in PGCs at extragonadal locations, whereas they are mitotically active once at the gonadal ridges. During gastrulation, PGCs appear to translocate from the epiblast to the hypoblast and during somitogenesis they are found associated with the most peripheral yolk syncitial layer (YSL). From the peripheral YSL they migrate through the median mesoderm into the dorsal mesoderm and then to the dorsal mesentery, where they establish the gonad primordia with mesenchymal cells. Vasa RNA positive cells, the PGCs of the zebrafish conform to these general observations. Interestingly, classical descriptive and experimental data can now be reevaluated using vasa as a molecular marker of the fish germ line. The power of zebrafish genetics together with possibilities of experimental embryology should accelerate research on aspects of vertebrate germ line development such as PGC migration, division and apoptosis, as well as (in) fertility. The present review summarizes some of the classical data on germ line development in fishes in relation to recent data on vasa expression in zebrafish and compares these findings, where appropriate, with those in other model organisms. Special emphasis is placed on vasa gene expression as a potential universal germ line marker and suggestions are made for novel, zebrafish specific approaches to investigate the vertebrate germ line. (+info)
Fate mapping the mouse embryo.
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The use of clonal analysis to obtain a fate map of the epiblast of the mouse embryo and to investigate cell distribution during gastrulation and early neurulation is described in a personal reminiscence. A revised fate map of the epiblast at 6.5 days gestation is provided, and the development of 3-dimensional, quantitative image analysis techniques outlined. (+info)
Repression by suppressor of hairless and activation by Notch are required to define a single row of single-minded expressing cells in the Drosophila embryo.
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Notch signal transduction appears to involve the ligand-induced intracellular processing of Notch, and the formation of a processed Notch-Suppressor of Hairless complex that binds DNA and activates the transcription of Notch target genes. This suggests that loss of either Notch or Su(H) activities should lead to similar cell fate changes. However, previous data indicate that, in the Drosophila blastoderm embryo, mesectoderm specification requires Notch but not Su(H) activity. The determination of the mesectodermal fate is specified by Single-minded (Sim), a transcription factor expressed in a single row of cells abutting the mesoderm. The molecular mechanisms by which the dorsoventral gradient of nuclear Dorsal establishes the single-cell wide territory of sim expression are not fully understood. We have found that Notch activity is required for sim expression in cellularizing embryos. In contrast, at this stage, Su(H) has a dual function. Su(H) activity was required to up-regulate sim expression in the mesectoderm, and to prevent the ectopic expression of sim dorsally in the neuroectoderm. We have shown that repression of sim transcription by Su(H) is direct and independent of Notch activity. Conversely, activation of sim transcription by Notch requires the Su(H)-binding sites. Thus, Notch signalling appears to relieve the repression exerted by Su(H) and to up-regulate sim transcription in the mesectoderm. We propose a model in which repression by Su(H) and derepression by Notch are essential to allow for the definition of a single row of mesectodermal cells in the blastoderm embryo. (+info)
Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.
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BACKGROUND: Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized. The recent isolation of human ES cells calls for exploring their pluripotential nature. MATERIALS AND METHODS: Human ES cells were grown in suspension to induce their differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression pattern of several lineage-specific molecular markers using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: Here we report the induction in vitro of cystic embryoid bodies from human ES cells. Our findings demonstrate induction of expression of cell-specific genes during differentiation of the human ES cells into EBs. In the human EBs, we could show a characteristic regional expression of embryonic markers specific to different cellular lineages, namely, zeta-globin (mesoderm), neurofilament 68Kd (ectoderm), and alpha-fetoprotein (endoderm). Moreover, we present a synchronously pulsing embryoid body that expresses the myocardium marker alpha-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologies, among them cells with neuronal appearance that express neurofilament 68Kd chain. CONCLUSION: Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development as well as in transplantation medicine. (+info)
Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.
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Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells. (+info)
Tbr1 regulates differentiation of the preplate and layer 6.
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During corticogenesis, early-born neurons of the preplate and layer 6 are important for guiding subsequent neuronal migrations and axonal projections. Tbr1 is a putative transcription factor that is highly expressed in glutamatergic early-born cortical neurons. In Tbr1-deficient mice, these early-born neurons had molecular and functional defects. Cajal-Retzius cells expressed decreased levels of Reelin, resulting in a reeler-like cortical migration disorder. Impaired subplate differentiation was associated with ectopic projection of thalamocortical fibers into the basal telencephalon. Layer 6 defects contributed to errors in the thalamocortical, corticothalamic, and callosal projections. These results show that Tbr1 is a common genetic determinant for the differentiation of early-born glutamatergic neocortical neurons and provide insights into the functions of these neurons as regulators of cortical development. (+info)