Cell fusion-independent differentiation of neural stem cells to the endothelial lineage.

Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential (referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures. Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation. In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells, with human endothelial cells, which form the lining of blood vessels. We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.

Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus.

Adult neurogenesis in the dentate gyrus may contribute to hippocampus-dependent functions, yet little is known about when and how newborn neurons are functional because of limited information about the time course of their connectivity. By using retrovirus-mediated gene transduction, we followed the dendritic and axonal growth of adult-born neurons in the mouse dentate gyrus and identified distinct morphological stages that may indicate different levels of connectivity. Axonal projections of newborn neurons reach the CA3 area 10-11 d after viral infection, 5-6 d before the first spines are formed. Quantitative analyses show that the peak of spine growth occurs during the first 3-4 weeks, but further structural modifications of newborn neurons take place for months. Moreover, the morphological maturation is differentially affected by age and experience, as shown by comparisons between adult and postnatal brains and between housing conditions. Our study reveals the key morphological transitions of newborn granule neurons during their course of maturation.

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG.

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.

The Role of Extracellular Ca2+ in the Activation of Pectinaria Oocytes.

Several events are associated with fertilization in oocytes. Two such events are an increase in cytoplasmic Ca2+ concentration and the resumption of meiosis. Oocytes of the marine annelid, Pectinaria gouldii, are in metaphase I arrest when they are spawned. In this report we investigate the relationship between Ca2+ and resumption of meiosis in this species. Meiosis in unfertilized oocytes could be re-initiated with the divalent cation ionophore, A23187. Oocytes in Ca2+ free sea water, however, did not resume meiosis in the presence of the ionophore. Furthermore, it was observed that Ca2+ must be present for at least 15 min following ionophore treatment for meiosis to resume. These results suggest that extracellular Ca2+ is required for the re-initiation of meiosis in this species.

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos: (ubiquitin/embryogenesis/proteolysis).

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin (ca. 8 × 108 molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus. The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.

A Stereometric Analysis of Karyokinesis, Cytokinesis and Cell Arrangements during and following Fourth Cleavage Period in the Sea Urchin, Lytechinus variegatus: (sea urchin embryo/cell division patterns/stereo imaging/3-D reconstruction).

Fourth cleavage of the sea urchin embryo produces 16 blastomeres that are the starting point for analyses of cell lineages and bilateral symmetry. We used optical sectioning, scanning electron microscopy and analytical 3-D reconstructions to obtain stereo images of patterns of karyokinesis and cell arrangements between 4th and 6th cleavage. At 4th cleavage, 8 mesomeres result from a variant, oblique cleavage of the animal quartet with the mesomeres arranged in a staggered, offset pattern and not a planar ring. This oblique, non-radial cleavage pattern and polygonal packing of cells persists in the animal hemisphere throughout the cleavage period. Contrarily, at 4th cleavage, the 4 vegetal quartet nuclei migrate toward the vegetal pole during interphase; mitosis and cytokinesis are latitudinal and subequatorial. The 4 macromeres and 4 micromeres form before the animal quartet divides to produce a 12-cell stage. Subsequently, macromeres and their derivatives divide synchronously and radially through 8th cleavage according to the Sachs-Hertwig rule. At 5th cleavage, mesomeres and macromeres divide first; then the micromeres divide latitudinally and unequally to form the small and large micromeres. This temporal sequence produces 28-and 32-cell stages. At 6th cleavage, macromere and mesomere descendants divide synchronously before the 4 large micromeres divide parasynchronously to produce 56- and 60-cell stages.

A role for bone marrow-derived cells in the vasculature of noninjured CNS.

The contribution of hematopoietic cells to the formation of blood vessels is currently the focus of intense scrutiny. Bone marrow-derived endothelial progenitor cells are thought to generate endothelial cells in many tissues, including myocardium, muscle, and certain tumors. In the central nervous system (CNS), however, the possible role of bone marrow-derived angiocompetent cells remains unclear. Here we have investigated the long-term involvement of bone marrow-derived cells in the maintenance of endothelial structures in the brain, spinal cord, and retina. Using hematopoietic chimeras stably expressing green fluorescent protein (GFP) in bone marrow-derived tissues, we found large numbers of hematopoietic cells closely associated with vessels in the CNS. None of these cells, however, showed an endothelial phenotype. They were positive for monocytic and microglial surface markers and demonstrated active phagocytosis of neighboring endothelial elements. Bone marrow-derived, vasculature-associated cells in the noninjured adult CNS are distinct from endothelial cells, but play an active role in vascular structures.

An ultrastructural investigation of the striated subumbrellar musculature of the anthomedusan, Pennaria tiarella.

The presence of striated subumbrellar musculature in hydromedusae can be related to the development in hydrozoans of a free-swimming life stage. The detailed ultrastructure of the striated subumbrellar musculature of the anthomedusan, Pennaria tiarella is presented. The striated musculature of Pennaria resembles vertebrate striated muscle in filament arrangement (L2 lattice pattern) and M line structure. The striation pattern, out-of-register myofilaments, filament structure as determined by rotational symmetry, Z line structure, types of intercellular junctions, and sarcoplasmic reticulum are more similar to structures found in other invertebrate striated muscle.

A morphological analysis of the first cleavage mitotic cytoskeleton isolated from sea urchin embryos (Strongylocentrotus droebachiensis).

Morphological changes in the mitotic cytoskeleton (MC) that occurred through the course of the first cleavage of Strongylocentrotus droebachiensis, cultured at 8°C or at 0°C, temperatures within the natural range for this species, have been investigated. Electron microscopy of MCs isolated from zygotes has revealed that they consisted largely of microtubules (mts). Thus, the morphology of these MCs is derived from the arrangement of the mts which form them. During anaphase, astral rays elongated while kinetochore fibers shortened. Asters enlarged during anaphase as a result of two events: astral ray lengthening and centrosphere enlargement. At the end of anaphase, asters of 8°C MCs filled the entire cell volume. The pattern of changes that occurred in 8°C mitotic apparatuses (MAs) also occurred in 0°C MCs. The observation of asters in 0°C MCs is contradictory to that of Stephens ('72b), who reported that 0°C MCs in this species were anastral. However, in 0°C metaphase MCs, the astral rays and spindle fibers were not as long as those in 8°C MCs. Also in 0°C MCs, the centrosphere was largely filled with dense material, whereas the centrosphere in 8°C MCs was larger and contained little dense material. Asters of 0°C MCs did not attain a large enough size to fill the egg volume completely, as did asters of 8°C MCs.

Mice lacking methyl-CpG binding protein 1 have deficits in adult neurogenesis and hippocampal function.

DNA methylation-mediated epigenetic regulation plays critical roles in regulating mammalian gene expression, but its role in normal brain function is not clear. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, has been shown to bind methylated gene promoters and facilitate transcriptional repression in vitro. Here we report the generation and analysis of MBD1-/- mice. MBD1-/- mice had no detectable developmental defects and appeared healthy throughout life. However, we found that MBD1-/- neural stem cells exhibited reduced neuronal differentiation and increased genomic instability. Furthermore, adult MBD1-/- mice had decreased neurogenesis, impaired spatial learning, and a significant reduction in long-term potentiation in the dentate gyrus of the hippocampus. Our findings indicate that DNA methylation is important in maintaining cellular genomic stability and is crucial for normal neural stem cell and brain functions.