Uninjected Blastomere Research Articles

Loss of Neurofilaments Alters Axonal Growth Dynamics

The highly regulated expression of neurofilament (NF) proteins during axon outgrowth suggests that NFs are important for axon development, but their contribution to axon growth is unclear. Previous experiments in Xenopus laevis embryos demonstrated that antibody-induced disruption of NFs stunts axonal growth but left unresolved how the loss of NFs affects the dynamics of axon growth. In the current study, dissociated cultures were made from the spinal cords of embryos injected at the two-cell stage with an antibody to the middle molecular mass NF protein (NF-M), and time-lapse videomicroscopy was used to study early neurite outgrowth in descendants of both the injected and uninjected blastomeres. The injected antibody altered the growth dynamics primarily in long neurites (>85 microm). These neurites were initiated just as early and terminated growth no sooner than did normal ones. Rather, they spent relatively smaller fractions of time actively extending than normal. When growth occurred, it did so at the same velocity. In very young neurites, which have NFs made exclusively of peripherin, NFs were unaffected, but in the shaft of older neurites, which have NFs that contain NF-M, NFs were disrupted. Thus growth was affected only after NFs were disrupted. In contrast, the distributions of alpha-tubulin and mitochondria were unaffected; thus organelles were still transported into neurites. However, mitochondrial staining was brighter in descendants of injected blastomeres, suggesting a greater demand for energy. Together, these results suggest a model in which intra-axonal NFs facilitate elongation of long axons by making it more efficient.

Gap junctional communication in the early Xenopus embryo.

In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16-128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral and dorsal aspects of the embryo, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8 did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral patterning.

B24 protein stored in lampbrush spheres is involved in early cleavage in urodele amphibians

A cDNA encoding a protein (B24) belonging to the Mcm/P1 family was isolated from the newt Triturus carnifex. In eukaryotes, the members of the Mcm/P1 family are essential factors in the DNA replication process. B24 protein (TcMcm3) is present in salamandrid ovarian oocytes and early embryos; its role was tested by injecting specific anti-B24 monoclonal antibodies into the cytoplasm of one blastomere of two-cell stage embryos. The injected blastomere encountered cleavage arrest either soon after the injection or following one or two divisions; later, it degenerated. Instead, the uninjected blastomere went on developing and organizing a hemi-embryo, which does not grow beyond the tailbud stage. These results are consistent with the hypothesis that the B24 protein is involved in DNA replication at cleavage. The B24 protein studied here appears to play a specific role in early development; other variants of the Mcm3 group seem to be employed by different adult tissues. J. Exp. Zool. 280:142–151, 1998. © 1998 Wiley-Liss, Inc.

B24 protein stored in lampbrush spheres is involved in early cleavage in urodele amphibians

A cDNA encoding a protein (B24) belonging to the Mcm/P1 family was isolated from the newt Triturus carnifex. In eukaryotes, the members of the Mcm/P1 family are essential factors in the DNA replication process. B24 protein (TcMcm3) is present in salamandrid ovarian oocytes and early embryos; its role was tested by injecting specific anti-B24 monoclonal antibodies into the cytoplasm of one blastomere of two-cell stage embryos. The injected blastomere encountered cleavage arrest either soon after the injection or following one or two divisions; later, it degenerated. Instead, the uninjected blastomere went on developing and organizing a hemi-embryo, which does not grow beyond the tailbud stage. These results are consistent with the hypothesis that the B24 protein is involved in DNA replication at cleavage. The B24 protein studied here appears to play a specific role in early development; other variants of the Mcm3 group seem to be employed by different adult tissues.

Cleavage Arrest of Early Frog Embryos by the G Protein-activated Protein Kinase PAK I

PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.

Inhibition of axonal development after injection of neurofilament antibodies into a Xenopus laevis embryo

The ability to target specific cytoskeletal components in axons for disruption within intact developing embryos would provide a valuable tool for studying neuronal development. Neurofilaments are an attractive target for such an approach, because they are neuron specific and are expressed late in embryogenesis principally beginning during axon outgrowth. No pharmacological agents are currently available that disrupt neurofilaments without also affecting general development. One approach that has been used successfully to affect proteins in vivo is to inject specific antibodies into living cells. We employed this approach in Xenopus laevis embryos by injecting two antibodies directed against the middle molecular weight neurofilament protein (NF-M) into a single blastomere of a two-cell stage embryo. Injected antibodies could be detected for as long as 3.5 days in cells descended from the injected blastomere. Only cell bodies of neurons descended from anti-NF-M-injected blastomeres contained abnormal accumulations of intermediate filament proteins, and peripheral nerve development was unilaterally retarded in these neurofilament antibody-injected tadpoles. Such accumulations and peripheral nerve defects were not seen in neurons derived from uninjected blastomeres or from blastomeres injected with control antibodies. These data demonstrate the usefulness of specific antibodies to perturb neuronal development in intact frog embryos and, in addition, suggest a role for neurofilaments in axon elongation.

Fibronectin-rich fibrillar extracellular matrix controls cell migration during amphibian gastrulation.

We have reviewed the evidence supporting the notion that the fibrillar extracellular matrix on the basal surface of the blastocoel roof in amphibian embryos directs and guides mesodermal cell migration during gastrulation. Based on extensive experimental evidence in several different systems, we conclude the following: (i) the fibrillar extracellular matrix contains fibronectin (FN) and laminin. (ii) The fibrils are oriented in such a way as to promote directional migration of mesodermal cells during migration. (iii) We have used several different probes to disrupt the interaction between migrating mesodermal cells and the fibrillar extracellular matrix. These probes include: (a) nucleocytoplasmic and interspecific hybridization. Such embryos have defects in FN synthesis and gastrulation. (b) Fab' fragments of anti-FN and anti-integrin VLA-5 IgGs prohibit mesodermal cell adhesion both in vitro and in vivo and gastrulation is arrested. (c) Peptides containing the RGDS sequence specifically inhibit interactions between migrating mesodermal cells and the FN-fibrillar matrix. (d) Tenascin blocks cell adhesion to FN in vitro and gastrulation in vivo. (e) Antibodies against the cytoplasmic domain of beta 1 integrin, when injected into blastomeres, prevent FN-fibrillogenesis in progeny of injected blastomeres and delay mesodermal cell migration selectively in the progeny of injected blastomeres but not in the uninjected blastomere progeny.

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca 2+ channels

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Iontophoretic injection of lucifer yellow CH into zygotes and blastomeres of the hydroidHydractinia echinata

The dye Lucifer yellow CH was iontophoresed into recently fertilized eggs and early blastomeres ofHydractinia echinata. Iontophoresis was carried out on the stage of an inverted microscope in order to follow filling of the injected cells by short pulses of epifluorescent illumination. Lucifer yellow proved to be nontoxic and development in embryos with injected blastomeres proceeded normally. When zygotes were injected all the cells of the forming embryo contained dye. When one of the first two blastomeres was injected all the progeny of the injected cell also contained dye. Dye-coupling between injected and uninjected blastomeres did not occur in two-cell embryos nor between descendants of either line. Development of Lucifer-yellow-filled blastomeres or zygotes could be stopped by blue light irradiation. In a number of injected cells, the dye tended to accumulate forming brightly shining spots. The dye did not penetrate the nuclear envelope of injected cells.

Microinjection of Lucifer yellow CH into sea urchin eggs and embryos.

Eggs and embryos of Arbacia punctulata were microinjected with the fluorescent dye, Lucifer yellow CH, using a simple pressure injection system. When injected into eggs that were subsequently fertilized, the dye was distributed throughout all cells of the developing embryo. If one cell of a two-cell embryo was injected, dye did not diffuse into the uninjected blastomere. During subsequent development, all progeny of the injected cell contained dye resulting in an embryo that was half-fluorescent. Blue light irradiation of a two-cell embryo, one cell of which had been injected with Lucifer yellow, caused the injected blastomere to stop further divisions while the uninjected blastomere developed normally and was free of dye. These results indicate that the first two blastomeres of Arbacia embryos are not electrically coupled, nor up to the time of hatching, is there any coupling between cells in one half of the first cleavage plane and cells in the other half.