Dose-dependent regulation of pair-rule stripes by gap proteins and the initiation of segment polarity

A key step in Drosophila segmentation is the establishment of periodic patterns of pair-rule gene expression in response to gap gene products. From an examination of the distribution of gap and pair-rule proteins in various mutants, we conclude that the on/off periodicity of pair-rule stripes depends on both the exact concentrations and combinations of gap proteins expressed in different embryonic cells. It has been suggested that the distribution of gap gene products depends on cross-regulatory interactions among these genes. Here we provide evidence that autoregulation also plays an important role in this process since there is a reduction in the levels of Kruppel (Kr) RNA and protein in a Kr null mutant. Once initiated by the gap genes each pair-rule stripe is bell shaped and has ill-defined margins. By the end of the fourteenth nuclear division cycle, the stripes of the pair-rule gene even-skipped (eve) sharpen and polarize, a process that is essential for the precisely localized expression of segment polarity genes. This sharpening process appears to depend on a threshold response of the eve promoter to the combinatorial action of eve and a second pair-rule gene hairy. The eve and hairy expression patterns overlap but are out of register and the cells of maximal overlap form the anterior margin of the polarized eve stripe. We propose that the relative placement of the eve and hairy stripes may be an important factor in the initiation of segment polarity.     

The zygotic control of Drosophila pair-rule gene expression. I. A search for new pair-rule regulatory loci

The examination of pair-rule gene expression in wild-type and segmentation mutant embryos has identified many, but not necessarily all, of the elements of the regulatory system that establish their periodic patterns. Here we have conducted a new type of search for previously unknown regulators of these genes by examining pair-rule gene expression in blastoderm embryos lacking parts of or entire chromosomes. This method has the advantage of direct inspection of abnormal pair-rule gene patterns without relying upon mutagenesis or interpretation of larval phenotypes for the identification of segmentation genes. From these experiments we conclude that: (i) most zygotically required regulators of the fushi tarazu (ftz), even-skipped (eve) and hairy (h) pair-rule genes have been identified, except for one or more loci we have uncovered on chromosome arm 2L; (ii) the repression of the ftz and eve genes in the anterior third of the embryo is under maternal, not zygotic control; and (iii) there are no general zygotically required activators of pair-rule gene expression. The results suggest that the molecular basis of pair-rule gene regulation can be pursued with greater confidence now that most key trans-acting factors are already in hand.     

Cytochalasin induces spindle fusion in the syncytial blastoderm of the early Drosophila embryo.

Microfilament integrity is needed to maintain the regular arrangement of the spindle microtubules and to guarantee the normal progression of the last syncytial mitoses in Drosophila embryo. To investigate when and how microfilaments participate in this process, we incubated permeabilized embryos with the inhibitor of actin polymerization, cytochalasin B, at different times during the nuclear cycle. Our results suggest that the correct microfilament distribution is only required for the appropriate segregation of nuclei during the 11th, 12th and 13th syncytial mitoses rather than during the 10th mitosis when the spindles are too far apart to interact. When cytochalasin B treatment was performed during the last syncytial mitoses many spindles fuse among them and the regular mitotic progression is perturbed.     

Distribution of F-actin during cleavage of the Drosophila syncytial blastoderm

The process of cleavage during the syncytial blastoderm stage of the Drosophila embryo was studied in fixed whole-mounts using a triple- staining technique. Plasmalemma was stained with Concanavalin A conjugated to tetramethylrhodamine isothiocyanate, the underlying cortical F-actin with a fluorescein derivative of phalloidin, and nuclei with 4',-6 diamidine-2-phenylindole dihydrochloride. The surface caps, which overlie the superficial nuclei at this stage, were found to be rich in F-actin as compared with the rest of the cortex. After the caps formed, they extended over the surface and flattened. Whilst this was occurring the F-actin network within the caps became more diffuse. By the end of the expansion process F-actin had become concentrated at both poles of the caps. The caps then split in two. The cleavage was not accompanied by the formation of any apparent contractile ring of microfilaments across the cap, rather the break region was depleted in F-actin. The cortical actin associated with each half of the old cap then became reorganized around a nucleus to form a new daughter cap, and the cycle began again.     

Ultrastructural differentiations during formation of the blastoderm in the Drosophila melanogaster embryo

The ultrastructure of the Drosophila melanogaster embryo is described during the transition from the syncytial to the cellular blastoderm. The centriole is unusually short, only 150–175 mμ in length, and has a central cylinder and radial “spokes.” Although two centrioles have not been found together, a structure resembling a procentriole is frequently found with the centriole. Differentiations of cisternae of ER with the characteristics of annulate lamellae are found in the blastoderm adjacent to the yolk. Some evidence suggests that these differentiations first form adjacent to yolk nuclei during the blastema and early blastoderm stages and that subsequently they are incorporated into the blastoderm cells.A new supranuclear cytoplasmic region is formed in blastoderm cells after the nuclei have moved from the surface. Within this area a cytoplasmic complex forms consisting of Golgi, agranular tubules, and granular tubules and cisternae. The possible origins of the new ER are discussed. Definite differences in fine structure, both in the number of mitochondria and in the type of differentiation of the supranuclear complex, were found between the mid-dorsal and mid-ventral regions of the embryo. The differentiation of the supranuclear complex is discussed in relation to the establishment of the determinative nature of the embryo and to cellular differentiation.     

Clonal analysis of the blastoderm anlage of the Malpighian tubules in Drosophila melanogaster

Summary Genetically marked maroon-like (mal) clones were induced by mitotic recombination with X-rays at the blastoderm stage in mal/mal ⁺ heterozygotes and were analysed in differentiated Malpighian tubules (MT). Marked cells were not confined to single anterior (MA) or posterior (MP) tubules, but were distributed among the four tubules. About 70% of the clones with two or more cells were fragmented, i.e. mal cells were separated by wild-type cells. Since the clones contain, on average, 6 cells and the differentiated MT consist of 484 cells (2 × 136 MA cells, 2 × 106 MP cells), we estimate that there are about 80 cells in the blastoderm anlage which on average pass through two to three mitoses. With increasing radiation doses (254 R, 635 R, 1270 R) a linear increase in clone frequency is observed. The mean sizes and size distributions of clones, however, remain unchanged. Since the increasing radiation dose also results in fewer differentiated Malpighi cells, we assume that regeneration does not occur. Therefore, size distributions of marked clones presumably represent real mitotic patterns in normogenesis. We suggest that essentially three successive mitoses take place, with a decreasing fraction of cells showing mitotic activity. Only a small fraction of cells goes through a fourth or even a fifth mitosis. Marked non-Minute clones induced in Minute heterozygotes are more frequent, but are not larger than non-Minute clones in wild-type background. Therefore, compartment boundaries cannot be recognized by this method. However, frequencies of marked cells found simultaneously in MA and MP pairs or in several single tubules of the same individuals are significantly higher than frequencies of multiple recombination events predicted by the Poisson distribution. From this, we conclude that neither the MA pair nor the MP pair nor single tubules represent compartments of the MT anlage.On the occasion of his 60th birthday, this work is dedicated to Prof. Dr. H.J. Becker, who initiated cell lineage studies in Drosophila     

Localized defects of blastoderm formation in maternal effect mutants of Drosophila

In the three maternal effect lethal mutant strains of D. melanogaster described in this report, the homozygous mutant females produce defective eggs that cannot support normal embryonic development. The embryos from these eggs begin to develop for the first 2 hr after fertilization in an apparently normal way, forming a blastula containing a cluster of pole cells at the posterior end and a layer of syncytial blastoderm nuclei. During the subsequent transition from a syncytial to a cellular blastoderm, cell formation in the blastoderm is either partially or totally blocked. In mutant mat(3)1 no blastoderm cells are formed, indicating that there are separate genetic controls for pole cells and blastoderm cells. The other two mutants form an incomplete cellular blastoderm in which certain regions of the blastoderm remain noncellular. The noncellular region in mutant mat(3)3 is on the posterior-dorsal surface, covering about 30% of the total blastoderm. In mutant mat(3)6 blastoderm cells are formed only at the anterior and posterior ends, separated by a noncellular region that covers about 70% of the total blastoderm. The selective effects on blastoderm cell formation in the three mutants emphasize the importance of components present in the egg before fertilization for the transition from a syncytial to a cellular blastoderm.The genes defective in the three mutants are essential only for oogenesis and not for any other period of development, as indicated by a strict dependence of the lethal phenotypes on the maternal genotypes. Heterozygous embryos from the eggs of homozygous mutant females die, whereas homozygous mutant embryos from the eggs of heterozygous females develop into viable adults.One of the mutants, mat(3)3, has a temperature-sensitive phenotype. Homozygous mat(3)3 females maintained at a restrictive temperature of 29°C show the lethal maternal effect. However, at a permissive temperature of 20°C the females produce viable adult progeny. The temperature-sensitive period in mat(3)3 females occurs during the last 12 hr of oogenesis, consistent with the maternal effect phenotype of the mutant.     

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm

The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three- dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. We propose that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form.     

How one becomes many: blastoderm cellularization in Drosophila melanogaster

Embryonic development in Drosophila melanogaster begins with a rapid series of mitotic nuclear divisions, unaccompanied by cytokinesis, to produce a multi-nucleated single cell embryo, the syncytial blastoderm. The syncytium then undergoes a process of cell formation, in which the individual nuclei become enclosed in individual cells. This process of cellularization involves integrating mechanisms of cell polarity, cell-cell adhesion and a specialized form of cytokinesis. The detailed molecular mechanism, however, is highly complex and, despite extensive analysis, remains poorly understood. Nevertheless, new insights are emerging from recent studies on aspects of membrane polarization and insertion, which show that membrane components from intracellular organelles are involved. In addition, actin and actin-associated proteins have been heavily implicated while new evidence shows that microtubule cytoskeletal elements are mechanistically involved in all aspects of cellularization. This review will draw on both the traditional models and the new data to provide a current perspective on the nature of cellular blastoderm formation in Drosophila melanogaster.