Calpain A controls mitotic synchrony in the Drosophila blastoderm embryo.

The beautiful mitotic waves that characterize nuclear divisions in the early Drosophila embryo have been the subject of intense research to identify the elements that control mitosis. Calcium waves in phase with mitotic waves suggest that calcium signals control this synchronized pattern of nuclear divisions. However, protein targets that would translate these signals into mitotic control have not been described. Here we investigate the role of the calcium-dependent protease Calpain A in mitosis. We show that impaired Calpain A function results in loss of mitotic synchrony and ultimately halted embryonic development. The presence of defective microtubules and chromosomal architecture at the mitotic spindle during metaphase and anaphase and perturbed levels of Cyclin B indicate that Calpain A is required for the metaphase-to-anaphase transition. Our results suggest that Calpain A functions as part of a timing module in mitosis, at the interface between calcium signals and mitotic cycles of the Drosophila embryo.

Invasion by matrix metalloproteinase-expressing cells is important for primitive streak formation in early chick blastoderm.

Epiblast cells in the early chick embryo differentiate to form all three germ layers through ingression of cells at the primitive streak across the basement membrane that underlies the epiblast. We tested the idea that degradation of the extracellular matrix components by matrix metalloproteinase(s) (MMPs) is involved in this process. Epiblast cells and primitive streak cells were dissociated into single cells and seeded onto a reconstituted basement membrane gel in vitro. Following overnight culture, approximately half the cells made holes in the substratum by dissolving the gel matrix. This invasive phenomenon was reproduced in vitro even when the cells were cultured upside down using a hanging culture system. We detected gelatinase activity in the culture supernatants from both prestreak epiblast cells and primitive streak cells. Pro-MMP-2 was detected in the culture media of the prestreak/streak cells as a 72-kDa band by gelatin zymography. In RT-PCR experiments, mRNAs for MMP-2, membrane-type (MT)3-MMP and MMP-11(stromelysin-3) were expressed in the epiblast cells before and during primitive streak formation. Injection of GM 6001 or other MMP inhibitors into the subgerminal cavity of the embryo inhibited the formation of the primitive streak and/or the primitive groove in more than 82% of the injected embryos. On the other hand, injection of a negative control compound instead of GM 6001 did not cause substantial inhibition. These results suggest that MMPs are involved in the enzymatic degradation of the basement membrane underlying the epiblast and are thus important for the ingression of mesendodermal cells along the primitive streak.

[Dynamics of the annulate lamellae in Drosophila syncytial embryos].

In eukaryotic cells, mitotic events are controlled by evolutionarily conserved cyclin-dependent kinases (cdk): these kinases phosphorylate cell proteins, which causes structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that cdk1 activity is a key factor that controls nuclear pore complex assembly/disassembly and affects the structure of cytoplasmic pores in the annulate. In this paper, we report a comparative analysis of these cytoplasmic organelles throughout the cell-cycle and throughout the development of Drosophila syncytial embryos. Based on the results obtained, it was presupposed that distribution of annulate lamellae containing cytoplasmic pores could reflect the inactivation of the mitotic kinase cdk1 in Drosophila syncytial embryos.

Effect of egg turning and incubation time on carbonic anhydrase gene expression in the blastoderm of the Japanese quail (Coturnix c. japonica).

1. The gene expression of carbonic anhydrase, a key enzyme for the production of sub-embryonic fluid (SEF), was assessed in turned and unturned eggs of the Japanese quail. The plasma membrane-associated isoforms CA IV, CA IX, CA XII, CA XIV, and the cytoplasmic isoform CA II, were investigated in the extra-embryonic tissue of the blastoderm and in embryonic blood. 2. Eggs were incubated at 37.6 degrees C, c.60% RH, and turned hourly (90 degrees ) or left unturned. From 48 to 96 h of incubation mRNA was extracted from blastoderm tissue, reverse-transcribed to cDNA and quantified by real-time qPCR using gene-specific primers. Blood collected at 96 h was processed identically. 3. Blastoderm CA IV gene expression increased with the period of incubation only in turned eggs, with maxima at 84 and 96 h of incubation. Only very low levels were found in blood. 4. Blastoderm CA II gene expression was greatest at 48 and 54 h of incubation, subsequently declining to much lower levels and unaffected by turning. Blood CA II gene expression was about 25-fold greater than in the blastoderm. 5. The expression of CA IX in the blastoderm was the highest of all isoforms, yet unaffected by turning. CA XII did not amplify and CA XIV was present at unquantifiable low levels. 6. It is concluded that only gene expression for CA IV is sensitive to egg turning, and that increased CA IV gene expression could account for the additional SEF mass found at 84 to 96 h of incubation in embryos of turned eggs.

Differential requirements of a mitotic acetyltransferase in somatic and germ line cells.

During mitosis different types of cells can have differential requirements for chromosome segregation. We isolated two new alleles of the separation anxiety gene (san). san was previously described in both Drosophila and in humans to be required for centromeric sister chromatid cohesion (Hou et al., 2007; Williams et al., 2003). Our work confirms and expands the observation that san is required in vivo for normal mitosis of different types of somatic cells. In addition, we suggest that san is also important for the correct resolution of chromosomes, implying a more general function of this acetyltransferase. Surprisingly, during oogenesis we cannot detect mitotic defects in germ line cells mutant for san. We hypothesize the female germ line stem cells have differential requirements for mitotic sister chromatid cohesion.

Expression of protein tyrosine kinases and stem cell factor in chicken blastodermal cells.

Chicken blastodermal cells (CBC) from Stage X embryos, which were isolated from newly laid, fertile, unincubated eggs, are pluripotent cells and can produce somatic and germline chimeras when injected into recipient stage X embryos. The CBC retain their pluripotential ability for up to 7 d in vitro. The molecular mechanisms that control proliferation and differentiation of CBC are largely unknown, although protein tyrosine kinases (PTK) are known to play important roles in these processes in similar cells. To understand better the molecular mechanisms of proliferation and differentiation in CBC, expression profiles of PTK and stem cell factor (SCF) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific and degenerate oligonucleotide primers. Seventeen distinct PTK, including 14 receptor-type and 3 nonreceptor-type PTK and SCF were identified by RT-PCR. Expression of all of the genes was confirmed by northern blot analysis. The northern blot analysis showed that all probes hybridized with one or more transcripts at various expression levels. The expression of the 17 PTK and SCF genes in CBC suggests that they might play a role in signal transduction pathways that control the proliferation or differentiation in CBC.

Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis.

We have developed a simple fluorescent assay for detection of phospholipase A2 (PLA2) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA2 inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca2+-dependent cytosolic PLA2 (cPLA2) subtype. Embryonic cPLA2 activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA2 homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA2, lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.

The Drosophila melanogaster homologue of the Xeroderma pigmentosum D gene product is located in euchromatic regions and has a dynamic response to UV light-induced lesions in polytene chromosomes.

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy. XPD has a 5'- to 3'-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages.

A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion was observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved. The mRNA is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole amount in situ hybridization, revealed that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation.

Target sequences for hunchback in a control region conferring Ultrabithorax expression boundaries.

Boundaries of Ultrabithorax expression are mediated by long-range repression acting through the PBX or ABX control region. We show here that either of these control regions confers an early band of beta-galactosidase expression which is restricted along the anteroposterior axis of the blastoderm embryo. This band is succeeded by a stripe pattern with very similar anteroposterior limits. Dissection of the PBX control region demonstrates that the two patterns are conferred by distinct cis-regulatory sequences contained within separate PBX subfragments. We find several binding sites for hunchback protein within both PBX subfragments. Zygotic hunchback function is required to prevent ectopic PBX expression. Moreover, the PBX pattern is completely suppressed in embryos containing uniformly distributed maternal hunchback protein. Our results strongly suggest that hunchback protein directly binds to the PBX control region and acts as a repressor to specify the boundary positions of the PBX pattern.

Microinjection of glycosaminoglycan-degrading enzymes in the chicken blastoderm. An ultrastructural study.

The relationship between the presence of glycosaminoglycans (GAGs) and the morphology of the middle layer or mesoblast was examined by performing transmission electron microscopy of chicken blastoderms microinjected with GAG-degrading enzymes. The controls included microinjections with saline or trypsin, as well as solid-phase assays for proteolytic activity in commercially available GAG-degrading preparations. The results indicate that, in normal as well as in saline-injected blastoderms, middle-layer cells are rounded or cuboidal in shape, and are linked to each other by small intercellular junctions in the primitive-streak region. As they migrate laterally along the basal lamina, they appear as typical mesenchymal cells, being separated by large intercellular spaces and covered by cell processes. The removal of hyaluronate (by the microinjection of hyaluronidases) led to compaction of the middle-layer cells in the area lateral to the primitive streak. These cells lost their mesenchymal aspect and retracted their processes, and intercellular junctions were observed. The presence of proteolytic activity in the enzyme preparations did not interfere with the results. On the basis of the results obtained using this microinjection technique, we were able to confirm at the ultrastructural level that hyaluronate, due to its space-creating properties, promotes the detachment of ingressed primitive-streak cells and preserves the mesenchymal aspect of the middle layer during the lateral migration of single cells along the basal lamina. Whether the presence of hyaluronate is necessary to allow positioning of the mesoblast could not be inferred using our experimental procedure. We present evidence that this molecule, as well as having physicochemical properties, is also involved in the modulation of tissue interactions during gastrulation.

[Acetylcholinesterase distribution in the blastoderm of chick embryos]. / Raspredelenie atsetilkholinésterazy v blastoderme kruinykh zarodyshei.

The distribution of acetylcholine esterase (AChE; EC 3.1.1.7) was studied cytochemically in the blastoderm of chick embryos (stages 2-5 by Hamburger and Hamilton). The onset of primitive streak formation was accompanied by a sharp increase in the AChE activity in the anterior part of hypoblast. Changes in the AChE distribution during the early embryogenesis correlated fairly well with the movement of the primary hypoblast cells.

Nonuniform distribution of enzymes in fish eggs.

The distribution of enzymes in the blastoderm and the yolk was investigated in the eggs of the bony fish, Misgurnus fossilis. The total activities of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PD), and aldolase (ALD) did not change during early development. However, the enzymes were redistributed in the egg: at the blastula stage the blastoderm contains about 60% of the total ALD activity and 80% of the total LDH and G-6-PD activities. The "free cytoplasm" volume, in which the enzymic proteins can be solubilized, is greater in the yolk than in the blastoderm. Low levels of homologous enzymes from other species injected into the egg are distributed between the blastoderm and the yolk in the same way as are the native enzymes; they are primarily localized in the blastoderm. Injected exogenous enzyme concentrates preferentially in the yolk, apparently being distributed proportionately with respect to the "free cytoplasm" volumes. The ratio of these "free cytoplasmic" volumes, determined by distribution of the injected enzyme (LDH), in the yolk and in the blastoderm is 6.8. On the basis of our results we propose that many egg enzymes are noncovalently bound to blastoderm structures, and that the "nonbound" fraction of the enzymes in the cytosol is distributed proportionally to the "free cytoplasm" volumes in the blastoderm and in the yolk. Fractionation of the subcellular constituents of blastoderm extracts by ultracentrifugation or by gel filtration on Sepharose in the presence of an excess of LDH reveals the high-molecular weight LDH-containing complexes. These complexes are absent in the yolk and in the unfertilized eggs. These results are consistent with the hypothesis that the binding of the enzyme to subcellular structures in the blastoderm is an important mechanism in ooplasmic segregation.

[Distribution of acid phosphatase in the chicken blastoderm]. / Raspredelenie kisloi fosfatazy v blastoderme kuritsy.

The distribution of acid phosphatase in the chick blastoderm (stages 2--4 by HH) has been studied using cytochemistry. A marked increase of enzymatic activity all over the blastoderm was shown to coincide with the beginning of primitive streak formation. A part of the cells after their immigration are characterized by the fall of acid phosphatase activity. The percentage of such cells increases in the cranio-caudal direction of the definitive primitive streak. The patterns of yolk utilization upon the separation of individual embryonic rudiments are discussed.

Acetylcholinesterase differentiation during myogenesis in early chick embryonic cells caused by an inducer RNA.

In the stage 4 chick blastoderm, an area located 0.6 mm posterior to Hensen's node, the post-nodal piece (PNP), consists of an undifferentiated population of cells, since the explants when cultivated in vitro in a variety of media do not develop into any histologically identifiable structures. However, addition of a specific low molecular weight RNA isolated from the 16-day-old chick embryonic heart promotes the appearance of a distinct mode of morphological and biochemical changes that is similar to that of embryonic cardiogenic process. The RNA-induced changes in the PNP also include a marked increase in acetylcholinesterase activity. The increase in enzymatic activity can be measured biochemically, as well as visualized histochemically.

Distribution of acid phosphatase in chick Hensen's node.

Acid phosphatase distribution and yolk drop ultrastructure in Hensen's node of chick blastoderm at Hamburger-Hamilton stages 3 and 4 are described. At stage 3 large deposits of the reaction product are localised in type-A yolk drops, where signs of intensive degradation are seen. It seems that during this degradation lipid droplets are being formed in the degrading yolk drops. Partly digested yolk drops with a vesicular appearance are extruded from the cells, especially in deeper layers of the node. The phosphatase reaction product is also distributed into intercellular spaces. At stage 4 the cells of the node are more vacuolated and contain acid phosphatase and electron-dense yolk drops in lesser amounts. The possible physiological role of acid phosphatase in Hensen's node during gastrulation is discussed.

[The specificity of intracellular changes in chick embryo blastoderm during the latent period of embryogenesis]. / Spetsifika vnutrikle tochnykh izmenenii v blastoderme kurinogo zarodysha v techenie latentnogo periodo émbriogeneza

Yolk inclusions, lipids and polysaccharides found in the chicken embryo blastoderm cells are utilized during the latent period of embryogenesis. The yolk outside the blastoderm is not utilized. A delay in the development of the embryo of first days of incubation is related to a switching over the metabolism from utilizaiton of intracellular nutrient material to assimilation of the extracellular yolk. In the course of morphogenetical movements of the embryo, in the process of gastrulation, took place an increased biosynthesis in the blastoderm cell membranes.