Pedro Ripoll

Minutes: Mutants of Drosophila autonomously affecting cell division rate☆

Abstract “Minute” (M) mutants of Drosophila show a characteristic prolongation of developmental time. We chose three different Minute mutants to examine the proposition that this slower rate of development is also reflected in a decreased mitotic rate in imaginal disc cells. The division rate of normal cells (M+) dividing in Minute individuals and of Minute cells dividing in normal individuals has been analyzed by inducing mitotic recombination in the growing wing disc and the abdominal histoblasts. In both kinds of experiments, clones of non-Minute cells are always larger than simultaneously arising Minute cell clones implying a higher mitotic rate for M+, as compared to M, cells. In addition, Minute cells seem to be unable to compete with normal cells in the wing disc; however, their viability in the abdominal histoblasts is nearly normal. The possibility of inducing, at any developmental age, cells with different cell-cycle lengths and clones that are larger or smaller than normal provides a powerful new technique with which to approach developmental problems.

Developmental compartmentalization in the dorsal mesothoracic disc of Drosophila

Abstract The clonal analysis of the development of the dorsal mesothoracic (wing) disc shows that clones initiated after a given time do not cross over certain demarcation lines in the adult cuticle. The property of M + M + (non-Minute) recombinant cells to overgrow the M M + background cells was used to demonstrate the establishment of clonal restrictions during development. It has been shown that M + M + clones initiated at a given time of development share common demarcation lines that delimit what we call “a developmental compartment.” As development time and cell proliferation of the anlage proceed, large compartments become split into pairs of smaller ones. A study of the number of cells in a given compartment at the time of its splitting into subcompartments indicates that the “developmental segregation” takes place in groups of neighboring cells and suggests that the number of segregated cells is different and characteristic for each compartment. Within a given compartment, a single clone of M + M + cells gives rise to 60–90% of the total number of adult cells. This phenomenon is reminiscent of the regulative properties of the morphogenetic fields, and the relation of these to developmental compartments is discussed. Since homoeotic mutants transform entire developmental compartments into one another, the hypothesis is advanced that homoeotic genes control compartment development.

A cell division mutant of drosophila with a functionally abnormal spindle

Normal distribution of chromosomes to daughter cells is insured by the proper functioning of the spindle. Homozygosity for a semi-lethal mutation of Drosophila melanogaster (abnormal spindle) altering this structure has the following effects: the mitotic cycle is arrested in metaphase, leading to a high frequency of polyploid cells; sex chromosome disjunction during male meiosis is severely affected, as revealed by the resulting exceptional (diplo and nullo) gametes (microscopic examination of spermiogenesis confirms this aberrant segregation); meiotic spindles of living cells are morphologically abnormal; and tubulins extracted from mutant larvae are normal in amount, electrophoretic mobility, and ability to form microtubules in vitro. The results suggest that the mutant phenotype is due to an altered structural component of the spindle other than tubulins.

Cell lineage and differentiation in Drosophila.

Embryonic differentiation is usually monitored by the appearance of cell, or tissue-specific enzymes or their products or structural proteins. These phenotypic traits correspond to the activities of specific genes, and therefore, in the long run, differentiation has to be explained in terms of selective gene activities. The mechanisms by which particular genes are expressed in certain cell types and not in others remain one of most appealing problems of modern biology.

A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation

Abstract.Dicentric chromosomes are rarely found, because they interfere with normal cell division causing chromosome instability. By in situ hybridization of region-specific heterochromatic yeast artificial chromosomes we have found that the artificially generated C(1)A chromosome of Drosophila melanogaster has two potential centromeres: one carries all the sequences of the centromere of the Y chromosome and the other carries only a part of the Y centromeric region that is rich in telomere-related sequences. Immunostaining with anti-Bub1 (a kinetochore-specific marker) shows that, in spite of the differences in sequence, both centromeres can be active although as a rule only one at a time. In a small fraction of the chromosomes centromere inactivation is incomplete, giving rise to true dicentric chromosomes. The centromere inactivation is clonally inherited, providing a new example of epigenetic chromosome imprinting and the possibility of genetically dissecting this process. The involvement of telomere-related sequences in centromere function is discussed.

The spindle is required for the process of sister chromatid separation in Drosophila neuroblasts

We have studied two aspects of the process of sister chromatid separation in the Drosophila melanogaster neuroblasts. First, we analyzed the requirement of a functional spindle for sister chromatid separation to take place using microtubule depolymerizing drugs such as colchicine or a reversible analogue (MTC). Incubation of this tissue in colchicine causes the cells to block irreversibly at metaphase and no significant levels of sister chromatid separation were observed even after long periods of incubation. Exposure of neuroblasts to MTC also causes cells to block at metaphase, but after reversion most of the cells enter anaphase and are thus able to complete sister chromatid separation. These results imply that a functional spindle is required for sister chromatid separation. Second, we studied the role of heterochromatin during chromatid pairing and subsequent separation in chromosomes which carry either one or two extra pieces of heterochromatin. The results indicate that sister chromatids establish strong pairing along the translocated heterochromatin. During the early stages of anaphase, these chromosomes separate first the centromeric region and later the regions bearing extra heterochromatin. These results indicate that constitutive heterochromatin plays an important role for sister chromatid pairing and might be involved in the process of separation.

Relationship between chromosome content and nuclear diameter in early spermatids of Drosophila melanogaster

We have studied, using light microscopy, the relationship between chromosome content and nuclear diameter in early spermatids of males carrying different combinations of wild-type and compound chromosomes in Drosophila melanogaster. By using these genotypes we have been able to observe spermatid nuclei bearing various numbers of chromosomes ranging from only one sex chromosome and no major autosomes to almost twice the normal chromosome complement. We have found that variations in the chromosome content are accompanied by increasing the variance in early spermatid nuclear diameter; the more gametic classes produced, the higher the variance of nuclear diameters. These results indicate that measuring nuclear diameters in early spermatids represents a useful way to estimate the levels of meiotic non-disjunction and thereby to improve the characterization of lethal or male sterile mutants in which analysis of meiotic chromosome non-disjunction cannot be achieved by conventional genetic methods.

Genetic analysis of cell division in Drosophila

Publisher Summary This chapter discusses the genetics of cell division in Drosophila and the effect of different alterations in cell division on Drosophila development. Drosophila developmental genetics accommodate many old-fashioned techniques that should be routinely used to complement the knowledge of mitotic genes and their function. For many reasons, Drosophila has occupied a privileged position as a model system for genetics in the reduced historical time scale. It also occupies a privileged position in the evolutionary time scale. Many of the future relevant findings on the genetic control of cell division will come from the laboratories working with fruit flies, and these findings will be extrapolated with ease to other eukaryotes, including humans. The chapter elaborates on the effect of mitotic mutations on embryonic development, mitosis during postembryonic development, and tubulin and kinesin gene families.

Gonadal Mosaicism Induced by Chemical Treatment of Sperm in Drosophila melanogaster

Accurate interpretation of forward genetic screens of chromosomes exposed in mature spermatozoa to a mutagenic chemical requires understanding—incomplete to date—of how exposed chromosomes and their replicas proceed through early development stages from the fertilized ovum to establishment of the germline of the treated male’s offspring. We describe a model for early embryonic development and establishment of the germline of Drosophila melanogaster and a model-validating experiment. Our model proposes that, barring repair, DNA strands modified by treatment with alkylating agents are stable and mutagenic. Each replication of an alkylated strand can result in misreplication and a mutant-bearing daughter nucleus. Daughter nuclei thenceforth replicate faithfully and their descendants comprise the embryonic syncytium. Of the 256 nuclei present after the eighth division, several migrate into the polar plasm at the posterior end of the embryo to found the germline. Based upon distribution of descendants of the alkylated strands, the misreplication rate, and the number of nuclei selected as germline progenitors, the frequency of gonadal mosaicism is predictable. Experimentally, we tracked chromosomes 2 and 3 from EMS-treated sperm through a number of generations, to characterize autosomal recessive lethal mutations and infer gonadal genetic content of the sons of treated males. Over 50% of 106 sons bore germlines that were singly, doubly, or triply mosaic for chromosome 2 or chromosome 3. These findings were consistent with our model, assuming a rate of misreplication between 0.65 and 0.80 at each replication of an alkylated strand. Crossing treated males to mismatch-repair-deficient females had no apparent effect on mutation rate.