Cellular Blastoderm Stage Research Articles

Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo

The organization of microtubules within the surface caps of Drosophila embryos is described for the mitotic cycles of the syncytial blastoderm stage (particularly cycle 10), and for the subsequent cellularization process. Tubulin was labelled with the well characterized monoclonal antibody YL 1 2 (Kilmartin et al., J cell biol 93 (1982) 576) [1]. Each surface cap was found to contain an array of microtubules running around the nucleus. The microtubules originated at prominent centrosomes located close to the apical surface of each cap nucleus. During mitosis the spindle microtubules stained strongly for tubulin. A novel finding was that the spindle microtubules of the interzone region appeared to reduce their connections with the centrosomes at the end of anaphase. The spindle remnant remained in position during telophase but then became smaller in size, disappearing by interphase. At this phase of the cell cycle duplication of the aster centrosomes occurred. The cellular blastoderm stage was marked by a change in the main axis of microtubule orientation. The centrosomes of each cap separated somewhat and formed initiation centres for the development of a well developed basket of microtubules around each nucleus, but now perpendicular to the surface. The microtubule baskets were seen to extend in parallel with nuclear elongation, but not in concert with growth of the cell membranes, which extended some way beneath the bases of the nuclei.

Studies on embryonic diapause in the pnd mutant of the silkworm, Bombyx mori: Genetic control of embryogenesis

The pigmented and non-diapausing egg mutant ( pnd) of the silkworm, Bombyx mori, has a defect as a result of which embryos never enter into diapause, even though the eggs have been exposed to the diapause factor. In the mutant homozygous embryos, two distinct peaks of DNA synthesis which correspond to the stages of cellular blastoderm and organogenesis were detected until blastokinesis. On the other hand, in the heterozygous embryos derived from moths of homozygous pnd females, a paternal pnd + gene caused the embryos never to develop beyond late gastrulation, at which stage the embryos enter into diapause; the heterozygous embryos exhibit only one peak of DNA synthesis corresponding to the cellular blastoderm stage. We conclude that the paternal pnd + gene might participate in the process of suppressing DNA synthesis at the early-middle gastrula stage.

Isolation of caudal , a Drosophila homeo box-containing gene with maternal expression, whose transcripts form a concentration gradient at the pre-blastoderm stage

We report the isolation and characterization of caudal (cad), a previously unknown Drosophila homeo box-containing gene from the 38E region on the left arm of the second chromosome. This homeo box has diverged from the prototype sequence in Antennapedia, but contains subregions which are highly homologous. By Northern analysis and in situ hybridization experiments two transcripts of 2.4 kb were found to accumulate in nurse cells and in the oocyte during oogenesis. These transcripts generate a transient concentration gradient along the antero-posterior axis at the syncytial blastoderm stage. At the cellular blastoderm stage transcripts accumulate in a single band from 13-19% egg length at the posterior end. One zygotic transcript of 2.6 kb is detected. At later stages this transcript is localized in ectodermally and endodermally derived tissues such as the proctodeum, the Malpighian tubules and the posterior midgut. The 2.6-kb transcript is detectable until the onset of metamorphosis.

Developmental analysis of Drosophila position-specific antigens

The distributions of three position-specific (PS) antigens have been examined in different Drosophila tissues and at various developmental times, using both immunofluorescence and affinity purification procedures. In the imaginal discs the PS antigens show nonuniform and nonhomologous distributions, and the expression of the antigens in a particular disc region can vary during development. In general, PS antigen expression appears to correlate with morphogenetic events in the disc epithelia, suggesting that the antigens are involved in cell-cell recognition and/or adhesion processes. PS antigens are also found in many other tissues, and in embryos as early as the cellular blastoderm stage. Affinity-purified PS antigens from different tissues or stages appear to be similar, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results are discussed in relation to Drosophila developmental events, with particular regard to the dorsoventral cell lineage restriction in the wing disc.

Spatial distribution of transcripts from the segmentation gene fushi tarazu during Drosophila embryonic development

The locus fushi tarazu appears to be involved in the establishment of the segmentation pattern of the Drosophila embryo. The cuticle of ftz mutant embryos is missing structures in alternating segments such that only half the normal number of segments are present. We have localized ftz + transcripts in tissue sections of wild-type Drosophila embryos by in situ hybridization. Transcripts from the ftz + gene were first detected during nuclear cleavage prior to cell formation. During the last two nuclear divisions ftz + transcription becomes gradually restricted such that at the cellular blastoderm stage the ftz + transcripts are localized in seven evenly spaced bands of cells. The size of each band is similar to the size of the segment primordia at the blastoderm. By the time segmentation becomes morphologically distinct ftz + transcripts are no longer detected. These results suggest that the ftz + gene plays a key role in the determination of the segmentation pattern in the embryo.

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : III. Zygotic loci on the X-chromosome and fourth chromosome.

In order to identify X-chromosomal genes required inDrosophila for early patterning and morphogenesis, we examined embryos hemizygous for EMS-induced lethal mutations to determine which of those mutations cause gross morphological defects. Embryos from 2711 lethal lines, corresponding to 3255 lethal point mutations were studied. Only 21% caused death during embryogenesis and of these, only one-sixth, or 3% of the total lethals, were associated with defects visible in the final cuticle pattern. Of the 114 point mutants causing visible cuticle defects, 76 could be assigned to 14 complementation groups. An additional 25 mutations mapping to regions of the X-chromosome not covered by male fertile duplications were assigned to six complementation groups based on similarities of map position and phenotype. Thirteen mutations could not be assigned to complementation groups. All mutations allowed normal development through the cellular blastoderm stage, the first defects associated with the earliest acting loci being observed shortly after the onset of gastrulation. The phenotypes of the various loci range from alterations in segment pattern or early morphogenetic movements to defects in final pigmentation and denticle morphology.Cuticle preparations were also examined for 63 deletions spanning in total 74% of the X-chromosome, as well as for 8 deletions and point mutations derived in saturation mutagenesis screens of the fourth chromosome (Hochman 1976). With the exception of defects in head morphology and defects in cuticle differentiation, none of the hemizygous deletions showed phenotypes other than those predicted by point mutations known to lie in those regions. No deletion caused new or unknown alterations in gastrulation, segmentation or cuticle pattern.These results suggest that the number of genes required zygotically for normal embryonic patterning is small and that most, if not all such loci, are represented by point mutations in our collection.

Changes of RNA precursor pools (ATP, UTP) during embryogenesis ofDrosophila melanogaster (Diptera) andBruchidius obtectus (coleoptera)

The total amount of ATP and UTP was measured in embryos ofDrosophila melanogaster andBruchidius obtectus at different development stages. The pool-sizes were measured by their ability to enhance poly(AU) synthesis in an in vitro test, using RNA polymerase ofE. coli, poly-d(AT), ATP, and3H-UTP. In embryos ofD. melanogaster, the amount of UTP decreased from 4.5 pmoles/ embryo during cleavage and early blastoderm to 2 pmoles at later stages. The ATP pool decreased from 14 pmoles/ embryo to 6 pmoles at the same developmental stages. In embryos ofB. obtectus, the UTP pool expanded from about 1 pmole/embryo during cleavage and early blastoderm to about 3 pmoles during gastrulation and organogenesis. Much more ATP was found inB. obtectus embryos: during fertilization 23 pmoles/embryo were found. At late syncytial blastoderm stages the amount of ATP rose to about 50 pmoles/embryo. At cellular blastoderm stages and during gastrulation only 26 pmoles/embryo of ATP were found. During organogenesis the ATP pool decreased to about 14 pmoles/embryo.

A fate map for the larval epidermis of Drosophila melanogaster: localized cuticle defects following irradiation of the blastoderm with an ultraviolet laser microbeam

Drosophila melanogaster embryos at the cellular blastoderm stage were irradiated with a uv laser microbeam (257 nm, 7 erg) of 20-μm diameter. Depending on the site of irradiation, up to 90% of the resulting first-instar larvae showed defects in the integument. The location of the defects within the cuticular pattern corresponded closely to the site of irradiation. The data indicate that the anlagen for the left and right parts of the larval epidermis are separated ventrally and each occupies an area extending from 20 to 60% of the egg length (EL) in the anteroposterior direction (0% EL = posterior pole). Within this area, the anlagen for the three thoracic and eight abdominal segments are equally spaced, each occupying a transverse stripe of a width of about 3.9 EL, corresponding to three to four cell diameters. The region of the blastoderm from which the epidermis of these segments develops comprises about 2200 cells which is one-third of the cells of the blastoderm. The paper includes a detailed description of the cuticular pattern of a Drosophila melanogaster first-instar larva.

RNA synthesised during oogenesis and early embryogenesis in an insect egg (Euscelis plebejus).

RNA labelled during oogenesis or early embryogenesis was isolated from eggs of the leaf hopperEuscelis plebejus. The polyadenylated RNA fraction deposited during early oogenesis accounted for approximately 2.7% of the total RNA content of the newly laid egg. This fraction differed significantly in molecular weight (15-32 S) from poly(A)-containing RNA synthesised between early cleavage and early germ anlage stages (4-20S). Locally injected3H-uridine spread through the egg within approximately 3 h. A considerable fraction (25-35%) of label injected as3H-uridine during early cleavage was recovered in DNA at subsequent stages (10-20 h later); labelled RNA was not found prior to the cellular blastoderm stage. When the yolk-endoplasm was separated from the blastoderm cells, only the latter contained demonstrable amounts of RNA synthesised by the embryo. Of the precursor incorporated into embryonic RNA, approximately 10% was found in the polyadenylated fraction at the early blastoderm stage, but only 3% at the early germ anlage stage. No differences in size distribution of polyadenylated RNA were evident between anterior and posterior halves of the early germ anlage stage.

Determination During Early Embryogenesis inDrosophila melanogaster

Ligation of developing embryos of Drosophila melanogaster was performed at three different stages of nuclear multiplication and at the cellular blastoderm stage. Egg fragments of variable sizes are able to continue development up to the hatching stage. Partial embryos differentiate larval structures, anterior fragments forming larval head and posterior fragments larval abdominal structures. These fragments differentiate a variable number of the twelve larval cuticular bands formed by intact embryos. We found that ligation at cellular blastoderm can lead to anterior and posterior fragments which differentiate together all the twelve bands, indicating that at this stage the embryo develops these patterns in a mosaic fashion. Ligation of younger embryos prevents the differentiation of some intermediate larval cuticular bands, while the terminal ones are consistently differentiated. The number and position of the deleted bands is correlated with the time and position of ligation. This indicates that the mosaic pattern present in the egg at blastoderm is not fully formed at earlier stages in development.

Regionalisation of segment-forming capacities during early embryogenesis inDrosophila melanogaster.

Transverse fragmentation of the egg ofDrosophila melanogaster results in the formation of partial larvae. Anterior and posterior egg fragments develop the respective partial larval patterns. The partial patterns do not add up to the complete pattern.Fragmentation near the middle of the egg during early cleavage causes a gap of 3-4 segments on average in the larva. This gap is reduced to 2 segments on average if operations are performed at the early syncytial blastoderm stage. Fragmentation near the pole regions from early cleavage stages onwards causes a gap of only 2 larval segments on average. When the egg is fragmented at the columnar cellular blastoderm stage or later, the gap at all positions amounts to the size of one segment or less. A gap is also found after incomplete fragmentation, when the ooplasmic bridge between both egg parts was constricted beyond a certain limit.A specific shift of the segment-forming capacities along the egg axis is observed from syncytial blastoderm stages onwards.After partial longitudinal fragmentation no additional structures are observed. In general, the partial transverse patterns add up to the complete pattern, but minor structures like single denticles are missing near the fragmentation site.The results are discussed with respect to current concepts of segment pattern formation during early embryogenesis in dipterans.

Defective development after puncturing the periplasm of nuclear multiplication stage Drosophila embryos

Nuclear multiplication stage embryos were punctured in either the anterior, midlateral, or posterior regions. Both embryos and adults were examined for defects, and the defects were correlated with whether there had been any leakage of cytoplasmic material from the egg at the time of puncturing. Embryonic defects were found, correlated to the site of damage, in all three regions. A number of embryos was followed through development and it was found that 15.1% of the embryos which leaked cytoplasm hatched into larvae, compared to 82.3% of those which did not leak any cytoplasm. Morphological defects arising as a result of lateral puncture only were observed in adults. Many sterile adults were obtained from eggs in which the posterior region had been punctured. The results show that nuclear multiplication embryos are well able to tolerate the disturbance of the cortical cytoplasm created by puncture, but only rarely are they able to compensate for the actual loss of material by regulation. The results were similar to those observed after puncturing Drosophila embryos at the cellular blastoderm stage.

Ultrastructural patterns of RNA synthesis during early embryogenesis of Drosophila melanogaster

Chromatin was obtained from Drosophila melanogaster during early embryogenesis and examined by transmission electron microscopy. Nuclear contents spread at progressive stages of syncytial development show a low level of only non-nucleolar template activity, and very few RNP fibril gradients extending over 2 μm in length are observed. At the cellular blastoderm stage, newly activated nucleolar genes appear during the early portion of the first true cell cycle. Variation in the lengths of incomplete rRNP gradients indicates that the activation of each rRNA gene is independently controlled. All rRNA loci, whether having complete or incomplete gradients, exhibit high densities of nascent transcripts per unit length, suggesting that the rate of chromatin transcription, rather than the RNA polymerase I pool size, limits rRNA synthesis on individual genes. No more than half the rRNA genes are derepressed at this stage, indicating that total rRNA synthesis is regulated by the number of genes activated. Non-nucleolar RNP fibril gradients covering up to 8 μm of genome are also first observed at the cellular blastoderm stage. Most of these gradients are differentiated from the short gradients first seen during syncytial growth by a lower density of transcribing RNA polymerases.

Adult differentiation from partial Drosophila embryos after egg ligation during stages of nuclear multiplication and cellular blastoderm

When embryos are ligated during different stages of nuclear multiplication and cellular blastoderm they develop into partial larvae which never hatch. The partial larvae were injected into adult females for further development. During this in vivo culture the imaginal disk cells divide and achieve competence to differentiate into adult structures. We find that independent of the fragment size anterior embryonic tissues give rise to cranial adult structures and posterior fragments to adult caudal structures. This indicates that during the first nuclear divisions cranial versus caudal development is already determined. The two complementary fragments do not add up to a total embryo when separated very early; however, if separation of the two parts occurs at cellular blastoderm stage all adult structures of the fly can be found.

Larval and adult abdominal defects resulting from microcautery of blastoderm staged Drosophila embryos.

Drosophila embryos were damaged by microcautery at the cellular blastoderm stage at the sites of presumptive histoblasts, identified from fate maps. The resulting adults were analyzed for abnormal abdominal structures in one series, and in two further series the pupal cases of the defective adults which hatched were also checked for irregularities in segmentation of the larva, both dorsally and ventrally. The relationships between the larval segmentation and adult pattern are described. A sample of pupal cases of morphologically normal flies hatching from microcautery were checked and showed that regulation only rarely occurred, i.e., abnormal larvae sometimes produced normal adults. Both tergite and sternite defects occurred, and duplications of parts of these structures were observed in both cases. In general, abnormal fusions, missing hemi-segments, and partial deletions were associated with larval defects and were therefore probably the result of damage to larval cells, or both larval cells and histoblasts. Duplications and partial segment deficiencies also resulted from apparantly normal pupal cases and were therefore probably the result of directly damaging the presumptive histoblast cells of the blastoderm. It is suggested that the various nests of histoblasts in each segment act as one morphogenetic field, with larval cells within the field.

Adult deficiencies and duplications of head and thoracic structures resulting from microcautery of blastoderm stage Drosophila embryos

Drosophila embryos were damaged by microcautery at the cellular blastoderm stage at the sites of presumptive imaginal cells, identified from fate maps. The resulting adults were analyzed for abnormal structures. Cautery of any of the presumptive imaginal regions can lead to defects in the adult cuticle, though the majority of adults which hatch are morphologically normal. The abnormal adults had one or more discs either deleted, incompletely formed, or misarranged. Several of the structures which were incomplete had duplicated regions. The results suggest that, from the time fo their initial formation in the cellular blastoderm, a group of cells determined to be an 'adult' structure possesses a gradient of developmental capacity which is expressed by certain regions duplicating and other cells regenerating. The types of duplications found were similar to those resulting from other experimental treatments of imaginal discs at later stages in development, indicating that the presumptive imaginal disc cells, when they are first established in the cellular blastoderm, have a similar organization to mature imaginal discs.

The mitochondrial derivative of the spermatozoon of Drosophila before and after fertilization

The ultrastructure of the paracrystalline mitochondrial derivative of the Drosophila spermatozoon has been investigated in sperms stored for different intervals after mating within the genital apparatus of inseminated females and in sperms which penetrated the egg. The paracrystalline lattice remains structurally intact even after the most prolonged storage. Only after fertilization does the derivative exhibit structural modifications which following a characteristic pattern lead to the formation of lamellar bodies present up to the onset of the cellular blastoderm stage of the egg development. The behaviour of the mitochondrial derivative during the sperm storage, its fate at fertilization, and the absence of cytochrome c oxidase activity from its crystalline texture, as demonstrated by cytochemical tests, suggest that the typical biochemical activity of mitochondria is not a major function of this highly differentiated sperm component. The functional significance of the derivative is discussed in relation to a possible role at fertilization and to sperm metabolism.

Studies on Embryonic Determination of the Harlequin-fly, Chironomus dorsalis : I. Effects of centrifugation and of its Combination with Constriction and Puncturing

ABSTRACT By centrifuging, alone or in combination with constricting and puncturing of the egg, the embryonic determination in the harlequin-fly C. dorsalis has been studied. In eggs centrifuged moderately, three zones were distinguished: a centrifugal yolk zone, a centripetal oil zone, and an intermediate zone consisting of cytoplasm with nuclei. The mode of redistribution of the constituents of the centrifuged eggs differs according to the developmental stage at the time of centrifugation and to the centrifugal force. The strata of weakly centrifuged eggs begin to mix with each other immediately after centrifugation. Redistribution of the constituents of strongly centrifuged eggs always occurs in a definite way, even though the redistribution after centrifuging at the nuclear division stage does not take place until later, in the nuclear migration stage, while the cytoplasm of eggs centrifuged during and after nuclear migration begins to migrate as soon as the centrifuge is stopped. By centrifuging the eggs at the nuclear division stage, various types of malformation related to the direction of centrifugation are caused. These malformations are characterized by having duplicated parts. The frequency of occurrence of the malformations increases with increase in the centrifugal force. The structure of the double monsters is further modified by separating a part of the yolk or oil in the centrifuged eggs by strong centrifuging or by constricting, and by removing a part of the cytoplasm from the centrifuged eggs by puncturing. Modification always occurs in the mid-part of the double monster in the form of loss of segments. The number of omitted segments depends on the volume of the separated or the removed part. By applying two successive centrifugations in reverse directions it was found that a causal relationship between the direction of centrifugation before nuclear migration and the resulting malformation type is established at about the nuclear migration stage. From late centrifugation, two other groups of malformations resulted which are different from those obtained by early treatment. Those of the first group, which were caused by centrifuging at the syncytial blastoderm stage, are characterized by having a defect on the centrifugal side and no duplicated parts. Those of the second group were obtained from eggs centrifuged at the cellular blastoderm stage. In the extreme degree of this malformation the cells which reach the final level of differentiation are not organized into an embryo, forming instead small tissue masses. The specific features of the embryonic development of C. dorsalis, particularly thorax development, and the more general problem of the relation of the mode of development of Chironomus to that of other Dipterans, are discussed.