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L Solnica-Krezel

Publications and source records attributed to L Solnica-Krezel.

47 records · Page 3Linked to original sources
PubMed1994

Implications for dorsoventral axis determination from the zebrafish mutation janus.

THE mechanisms underlying the formation of dorsoventral polarity in the zebrafish Danio rerio are unknown. Here we describe the zebrafish recessive maternal-effect mutation janusm55. The mutant phenotype is a division of the blastoderm along the first cleavage plane into two detached half-sized blastoderms. Partial-axis bifurcation occurs in a subset of mutants. Analysis of goosecoid expression in the mutant embryos indicates that only one organizer region is present in each embryo. Furthermore, the position of this organizer region is random with respect to the first cleavage plane bisecting the two blastoderms. Finally, cell tracing in wild-type embryos demonstrates that there is no strict correlation of the dorsoventral axis with early cleavage planes in zebrafish. These findings support the notion that the establishment of the dorsoventral axis and the first cleavage planes are determined by separate mechanisms in the zebrafish embryo.

Animals↗
PubMed1994

Zebrafish: genetic tools for studying vertebrate development.

Zebrafish have entered the arena of vertebrate biology as a mainstream model system, and the use of genetic tools in this tropical fish should enhance our understanding of vertebrate development. The zebrafish system allows genetic experiments that are not possible in other vertebrates, and the mutations isolated thus far attest to its usefulness, complementing knowledge obtained from other model organisms.

Animals↗
PubMed1994

Efficient recovery of ENU-induced mutations from the zebrafish germline.

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.

Alleles↗
PubMed1994

Microtubule arrays of the zebrafish yolk cell: organization and function during epiboly.

In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer. At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter. We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endocytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to a delay in all epibolic movements. We propose that the yolk cell microtubules contribute either directly or indirectly to all epibolic movements. However, the epibolic movements of the yolk syncytial layer nuclei and of the blastoderm are not coupled, and only movements of the yolk syncytial nuclei are absolutely dependent on microtubules. We hypothesize that the microtubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the mechanisms by which the two microtubule arrays might function during epiboly are discussed.

Animals↗
PubMed1992

Cellular and molecular analysis of plasmodium development in Physarum.

The development of an amoeba into a plasmodium involves extensive changes in cellular organisation and gene expression. The genetic basis of a number of recessive mutations that block plasmodium development has been elucidated. The stage at which development becomes abnormal has been determined for all the mutants, as has the terminal phenotype. In order to investigate the changes in gene expression that accompany plasmodium development, a cDNA library has been made using RNA isolated from cell populations in which development was occurring.

Animals↗
PubMed1992

A developmental mutation (npfL1) resulting in cell death in Physarum polycephalum.

In Physarum, microscopic uninucleate amoebae develop into macroscopic multinucleate plasmodia. In the mutant strain, RA614, plasmodium development is blocked. RA614 carries a recessive mutation (npfL1) in a gene that functions in sexual as well as apogamic development. In npfL+ apogamic development, binucleate cells arise from uninucleate cells by mitosis without cytokinesis at the end of an extended cell cycle. In npfL1 cultures, apogamic development became abnormal at the end of the extended cell cycle. The cells developed a characteristic rounded, vacuolated appearance, nuclear fusion and vigorous cytoplasmic motion occurred, and the cells eventually died. Nuclei were not visible by phase-contrast microscopy in most of the abnormally developing cells, but fluorescence microscopy after DAPI staining revealed intensely staining, condensed nuclei without nucleoli. Studies of tubulin organization during npfL1 development indicated a high frequency of abnormal mitotic spindles and, in some interphase cells, abnormally thick microtubules. Some of these features were observed at low frequency in the parental npfL+ strain and may represent a pathway of cell death, resembling apoptosis, that may be triggered in more than one way. Nuclear fusion occurred during interphase and mitosis in npfL1 cells, and multipolar spindles were also observed. None of these features were observed in npfL+ cells, suggesting that a specific effect of the npfL1 mutation may be an incomplete alteration of nuclear structure from the amoebal to the plasmodial state.

Animals↗
PubMed1991

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum.

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.

Cell Division↗
PubMed1990

Variable pathways for developmental changes in composition and organization of microtubules in Physarum polycephalum.

The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated alpha 3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: alpha 3-tubulin is present in the majority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of beta 2-tubulin and disappearance of alpha 3-tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.

Cytoskeleton↗
PubMed1989

The localization of the divergent beta 2-tubulin isotype in the microtubular arrays of Physarum polycephalum.

The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.

Fluorescent Antibody Technique↗
PubMed1988

Activation of a beta-tubulin gene during early development of the plasmodium in Physarum polycephalum.

Uninucleate amoebae of Physarum polycephalum strain CL undergo apogamic development to form multinucleate plasmodia via an intermediate stage of large, uninucleate cells irreversibly committed to plasmodial development. This amoebal-plasmodial transition involves major changes in tubulin gene expression and the organization of microtubular structures. We analysed the expression of the betC locus, which encodes the plasmodial-specific beta 2-tubulin, during plasmodial development. A key question addressed was the timing of expression of betC in relation to the last open mitosis of the amoeba and the first closed mitosis of the plasmodium during the transition. Culture conditions were improved to yield partly synchronous differentiating cultures containing 50-60% committed cells, in order to facilitate biochemical analysis of development. Northern blotting indicated that betC RNA was virtually absent from amoebae and from early differentiating cultures. However, betC transcripts could already be detected in differentiating cultures containing only 0.1% of committed cells; the relative amount of betC transcripts increased as the percentage of committed cells in differentiating cultures increased. In fully developed plasmodia, there was at least a 330-fold increase in the betC transcript level compared to that in amoebae. We conclude that betC is activated during the amoebal-plasmodial transition immediately before or during the commitment event. Small amounts of beta 2-tubulin polypeptide could first be detected by Western blotting around the stage of the first closed mitosis. Thus beta 2-tubulin may participate in the first closed mitosis that committed cells undergo during their development into plasmodia.

Cell Differentiation↗