The BMP antagonists cerberus-like and noggin do not interact during mouse forebrain development

Mouse cerberus-like encodes for a secreted factor of the Cerberus/Dan family. This molecule has neural inducing capabilities and can bind to BMP-4 and nodal molecules in the extracellular space. When cerberus-like is inactivated, its function may be compensated for another molecule, since no abnormalities can be observed in the mouse mutant. Compensation mechanisms have been shown to occur between the BMP antagonists chordin and noggin. Here we report the generation of cerberus-like-/-; noggin-/- double mutants to uncover a possible compensation by noggin in cer-l-/- mutant. Double mutants were obtained and failed to show any further detectable defects beside the ones presented by the noggin-/- single mutant. Contrarily to chordin and noggin, mouse cerberus-like and noggin cannot compensate for each other during mouse embryogenesis.     

Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes

A human autoantiserum (5051) directed against pericentriolar material (PCM) was used to study the distribution of microtubule-organizing centers (MTOCs) in the oocyte and during the first cell cycle of mouse development. In oocytes, the PCM was found not only at the poles of the barrel-shaped metaphase II spindle but also at many discrete loci around the cytoplasm near the cell cortex. The spindle poles were also composed of several PCM foci. In metaphase-arrested eggs only the PCM foci located near the chromosomes acted as MTOCs. However, after reduction of the critical concentration for tubulin polymerization by taxol, the cytoplasmic PCM foci were also found to be associated with nucleation of microtubules. After fertilization the cortical PCM foci remained in a peripheral position until the end of the S phase, when they appeared to migrate centrally towards the pronuclei. At prometaphase of the first mitotic division, numerous MTOCs were found around the two sets of chromosomes; these MTOCs then aligned to form two bands on either side of the metaphase plate of the first mitosis.     

Microtubule organizing centers regulate spindle positioning in mouse oocytes

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Cytoplasmic transfer of microtubule organizing centers in mouse tissue culture cells

A chloramphenicol-resistant, aminopterin-sensitive cell line (AMT) derived from a mouse mammary tumor MT-29240 was enucleated, and the cytoplasts were fused with nucleated chloramphenicol-sensitive, HAT-resistant SV40 3T3 mouse cells. The resulting cytoplasmic hybrids (cybrids) were selected for their resistance to chloramphenicol and the chromosome complement of the SV40 3T3 cells. In addition to transfer of chloramphenicol resistance, these cybrid clones, as studied in the electron microscope, contained the intracisternal A particle phenotype characteristic of only the AMT cells. The cytoplasmic microtubule complex (CMTC) in these cybrids was also studied and appears to resemble the elaborate CMTC of the AMT cells more closely than the more reduced CMTC of the SV40 3T3. When treated with a colcemid block and then a brief reverse, the microtubule organizing centers (MTOC) appear as bright fluorescent foci when tubulin antibody and indirect immunofluorescence techniques are used. When AMT or SV40 3T3 cells are treated in this manner, only one MTOC is present in interphase cells. One clone of these cybrids, however, contained two MTOCs in interphase cells. This CMTC and MTOC phenotype has persisted in this cybrid clone for over 3 months of continuous culture.     

Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.     

End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers

During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent “end-on” fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein’s interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.     

Cooperative activity of noggin and gremlin 1 in axial skeleton development

Inductive signals from adjacent tissues initiate differentiation within the somite. In this study, we used mouse embryos mutant for the BMP antagonists noggin (Nog) and gremlin 1 (Grem1) to characterize the effects of BMP signaling on the specification of the sclerotome. We confirmed reduction of Pax1 and Pax9 expression in Nog mutants, but found that Nog;Grem1 double mutants completely fail to initiate sclerotome development. Furthermore, Nog mutants that also lack one allele of Grem1 exhibit a dramatic reduction in axial skeleton relative to animals mutant for Nog alone. By contrast, Pax3, Myf5 and Lbx1 expression indicates that dermomyotome induction occurs in Nog;Grem1 double mutants. Neither conditional Bmpr1a mutation nor treatment with the BMP type I receptor inhibitor dorsomorphin expands sclerotome marker expression, suggesting that BMP antagonists do not have an instructive function in sclerotome specification. Instead, we hypothesize that Nog- and Grem1-mediated inhibition of BMP is permissive for hedgehog (Hh) signal-mediated sclerotome specification. In support of this model, we found that culturing Nog;Grem1 double-mutant embryos with dorsomorphin restores sclerotome, whereas Pax1 expression in smoothened (Smo) mutants is not rescued, suggesting that inhibition of BMP is insufficient to induce sclerotome in the absence of Hh signaling. Confirming the dominant inhibitory effect of BMP signaling, Pax1 expression cannot be rescued in Nog;Grem1 double mutants by forced activation of Smo. We conclude that Nog and Grem1 cooperate to maintain a BMP signaling-free zone that is a crucial prerequisite for Hh-mediated sclerotome induction.     

End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers

During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent “end-on” fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein’s interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.     

Structural and immunocytochemical characterization of microtubule organizing centers in pteridophyte spermatogenous cells

Summary During the development of the spermatogenous cells, the pteridophyteCeratopteris richardii produces three structurally well-defined microtubule organizing centers (MTOCs). The blepharoplast, a spherical body that occurs during the last two spermatogenous divisions, organizes two microtubule (MT) arrays, one associated with a nuclear indentation and the other that organizes the spindle apparatus for the final divisions. After the last spermatogenous division, the blepharoplast reorganizes to produce two new putative MTOCs: the lamellar strip (LS) of the multilayered structure (MLS), which apparently organizes the spline microtubule array, and an amorphous zone (AM), that connects the basal bodies. Thin and semi-thin sections of this tissue were probed with antisera which recognize MTOCs in lower eukaryotes and animals to determine if any of these structures contain MTOC-associated proteins or epitopes recognized by monoclonal antisera. Gamma tubulin antibodies, which recognizeonly the minus ends of MTs in mammalian cells, label along the MT in all arrays found in the pteridophyte spermatogenous cells. Kinetochore MTs are unlabelled near the kinetochore, however. The monoclonal antibodies MPM-2 and C-9, that recognize centrosomal and nuclear epitopes in mammalian cells, label the interphase nucleus, the cytoplasm of mitotic cells, and the blepharoplast during both nuclear indentation and spindle formation. Double labelling of the blepharoplast-containing cells with anti-tubulin and either MPM-2 or C-9 reveals that the blepharoplast-associated fluorescence is the focus of the tubulin arrays. Centrin labels the reorganizing blepharoplast, the MLS, the AM, and a stellate pattern in the transition region of the flagella. These data indicate the usefulness of the structurally well-recognized MTOCs in pteridophyte spermatogenous cells in investigation of land plant MTOCs.     

Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos

Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2^−/− or Chd^−/− mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2^−/− mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2^−/− embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2^−/−vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.     

Distribution of aromatase-immunoreactive cells in the mouse forebrain

Summary The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens.     

Distribution of microtubule organizing centers in migrating sheets of endothelial cells

This study was designed to investigate the relationship between the position of the microtubule organizing center (MTOC) and the direction of migration of a sheet of endothelial cells (EC). Using immunofluorescence and phase microscopy the MTOC's of migrating EC were visualized as the cells moved into an in vitro experimental wound produced by mechanical denudation of part of a confluent monolayer culture. Although the MTOC's in nonmigrating EC were randomly positioned in relation to the nucleus, in migrating cells the position of the MTOC's changed so that 80% of the cells had the MTOC positioned in front of the nucleus toward the direction of movement of the endothelial sheet. This repositioning of the MTOC occurred within the first 4 h after wounding and was associated with the beginning of migration of EC's into the wounded area as seen by time-lapse cinemicrophotography. These studies focus attention on the MTOC as a cytoskeletal structure that may play a role in determining the direction of cell movement.     

Noggin基因与中枢神经系统发育的研究进展

Noggin作为一种重要的胚胎蛋白,在胚胎背腹轴模式形成、神经管发育及神经诱导方面有重要功能。干细胞研究的新进展提示,中枢神经系统发育将持续至生后及成年,包括胚胎及成体干细胞的增殖与分化,而noggin通过拮抗骨形成蛋白（BMPs）参与胚胎及成体干细胞的增殖与分化。本文就noggin基因在中枢神经系统发育中的重要功能予以阐述。     

Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the Vldlr knockout mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.     

Major events in the development of the forebrain

The development of the central nervous system can be divided into a number of phases, each of which is characterized by particular developmental disorders. In recent years, much progress has been made in elucidating the mechanisms by which the forebrain develops and in our understanding of major developmental disorders such as holoprosencephaly and neuronal migration disorders. In this general introduction to this symposium the major stages in the development of the forebrain, its regionalization and the genes involved, and some of the developmental disorders derailing cortical development with subsequent damage to the main cortical fiber connections (pyramidal tract and corpus callosum) are discussed.     

Early steps in the development of the forebrain

The tremendous complexity of the adult forebrain makes it a challenging task to elucidate how this structure forms during embryonic development. Nevertheless, we are beginning to understand how a simple epithelial sheet of ectoderm gives rise to the labyrinthine network of cells that constitutes the functional forebrain. Here, we discuss early events in forebrain development--those that lead to the establishment of the anterior neural plate and the regional subdivision of this territory into the different domains of the prospective forebrain.