Oriented cell divisions in the extending germband of Drosophila

Tissue elongation is a general feature of morphogenesis. One example is the extension of the germband, which occurs during early embryogenesis in Drosophila. In the anterior part of the embryo, elongation follows from a process of cell intercalation. In this study, we follow cell behaviour at the posterior of the extending germband. We find that, in this region, cell divisions are mostly oriented longitudinally during the fast phase of elongation. Inhibiting cell divisions prevents longitudinal deformation of the posterior region and leads to an overall reduction in the rate and extent of elongation. Thus, as in zebrafish embryos, cell intercalation and oriented cell division together contribute to tissue elongation. We also show that the proportion of longitudinal divisions is reduced when segmental patterning is compromised, as, for example, in even skipped (eve) mutants. Because polarised cell intercalation at the anterior germband also requires segmental patterning, a common polarising cue might be used for both processes. Even though, in fish embryos, both mechanisms require the classical planar cell polarity (PCP) pathway, germband extension and oriented cell divisions proceed normally in embryos lacking dishevelled (dsh), a key component of the PCP pathway. An alternative means of planar polarisation must therefore be at work in the embryonic epidermis.     

Armadillo/beta-catenin-dependent Wnt signalling is required for the polarisation of epidermal cells during dorsal closure in Drosophila

At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarisation of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarisation defects and fail to close properly. We have investigated the role of the Wingless signalling pathway in the polarisation of the DME cells and DC. We find that the beta-catenin-dependent Wingless signalling pathway is required for polarisation of the DME cells. We further show that although the DME cells are polarised in the plane of the epithelium and present polarised localisation of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g. Flamingo, PCP Wingless signalling is not involved in DC.     

Distribution of the wingless gene product in Drosophila embryos: a protein involved in cell-cell communication

wingless, a segment polarity gene required in every segment for the normal development of the Drosophila embryo, encodes a cysteine-rich protein with a signal peptide. A polyclonal antiserum localizes the wingless protein in approximately the same region of the embryo as the wingless mRNA. The pattern of antigen localization changes rapidly during development. In the extended germband stage, stripes of wingless staining are present in the trunk region just anterior to the parasegment boundary; wingless-expressing cells abut engrailed-expressing cells across that boundary. wingless antigen is seen both inside and outside the cell by electron microscopy: inside the cell, in small membrane-bound vesicles and in multivesicular bodies; outside the cell, close to or on the plasma membrane and associated with material in the intercellular space. The multivesicular bodies containing the wingless protein are occasionally found in engrailed-positive cells, suggesting that the wingless protein behaves as a paracrine signal.     

Nuclear Prospero allows one-division potential to neural precursors and post-mitotic status to neurons via opposite regulation of Cyclin E

In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. Here we show that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization.     

Cellular changes during heat shock induction and embryo development of cultured microspores ofBrassica napus cv. Topas

Summary Brassica napus cv. Topas microspores, isolated and cultured near the time of the first pollen mitosis and subjected to a heat treatment of 24 h, can be induced to develop into haploid embryos. This is a study of microspore structure during induction and embryo determination. Early during the 32.5 °C incubation period the nucleus moved away from the edge of the cell, and granules, 30 to 60 nm in diameter, appeared in the mitochondria and as a cluster in the cytoplasm. Cells divided symmetrically and at the end of the heat treatment, acquired the features of induced bicellular structures described previously. The features persisted as the cells divided randomly within the exine for 4–7 days following heat induction. Multicellular structures released from the exine underwent periclinal divisions resulting in protoderm differentiation of the globular embryo, thus determining embryo development. The cytoplasm of early heart-stage embryos contains abundant polyribosomes. Non-embryogenic development was indicated by large accumulations of starch and/or lipid and thickened cell walls or an unorganized pattern of cell division following release of the multicellular structures from the exine. Embryogenesis is discussed in terms of induction, embryo determination and development.     

short gastrulation, a mutation causing delays in stage-specific cell shape changes during gastrulation in Drosophila melanogaster

Mutations at the short gastrulation locus affect the timing of certain early morphogenetic events occurring during gastrulation in Drosophila melanogaster. Specifically, the invagination and subsequent closing of the posterior midgut and the anterior midgut appear to be delayed in these embryos. In addition, their germbands do not extent the full distance anteriorly on the dorsal side of the embryo. The dorsal cells are abnormally thick and fall into extremely deep dorsal folds as the germband extends. sog embryos continue development, but form disorganized first instar larvae. Normal sog expression is required in the zygote, but not in the mother for normal embryonic development and viability. Analysis of adult and larval gynandromorphs indicates that sog expression is required only in the ventral and/or anterior and posterior ends of the embryo, arguing that the dorsal abnormalities caused by the mutation are secondary consequences of defects elsewhere in mutant embryos.     

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.     

Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.     

Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea Urchin

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.     

Sister chromatids fail to separate during an induced endoreplication cycle in Drosophila embryos

When mitosis is bypassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes. Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication. Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles. Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16(th) embryonic mitotic cycle to an endocycle; however, we show that Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated. Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G2(14)), Cyclins A, B, and B3 disappeared and cells reentered S phase. This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis. Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1. The observed cyclin destruction suggests that cell cycle resetting by Rux was associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis was not sufficient to trigger sister disjunction.     

The torpedo (DER) receptor tyrosine kinase is required at multiple times during Drosophila embryogenesis.

The torpedo (DER) gene of Drosophila, which encodes a receptor tyrosine kinase of the EGF receptor subfamily, is essential for oogenesis, embryogenesis and imaginal disc development. To gain insight into the nature of the signals transduced by the torpedo product, we have characterized the gene's loss-of-function phenotype in the embryo. Through the induction of germline clones, we provide a genetic demonstration that maternal torpedo product does not contribute to zygotic development. Thus, the embryonic lethal phenotypes examined accurately reflect the consequences of eliminating all gene activity from the zygote. Temperature-shift experiments with the conditional allele topIF26 show that torpedo is required at two distinct times during embryonic development: the gene is first needed for germband retraction and for the production of anterior, posterior and ventral cuticle, then later for the secretion of ventral denticles. Since denticle formation can be severely disrupted in topIF26 animals without affecting cuticle production, the early and late requirements for torpedo appear to be functionally unrelated. torpedo, therefore, is required at multiple times in the development of the ventral epidermis, and may transduce qualitatively different signals. Since the early requirement for torpedo correlates with the first visible defect in embryonic development, increased cell death in the amnioserosa, cephalic ectoderm and ventral epidermis, the abnormalities in cuticle production and germband shortening seen in the mutant may be secondary consequences of a primary defect in cell viability. Given that the onset of cell death in torpedo embryos is not preceded by any obvious defects in mitogenesis, the establishment of cell identities or the maintenance of gene expression, it is possible that torpedo transduces a signal necessary for cell survival per se during early embryogenesis. During late embryogenesis, torpedo may mediate the reception of a second signal which regulates ventral epidermal cell differentiation.     

多胚发育寄生蜂腰带长体茧蜂在寄主亚洲玉米螟幼虫体内的发育

多胚发育的幼虫内寄生蜂腰带长体茧蜂Macrocentrus cingulum的卵、胚胎和幼虫在寄主亚洲玉米螟Ostrinia furnacalis幼虫血腔内发育,通常1枚卵可以分裂增殖为数百只胚胎。本文通过定时解剖寄生的寄主幼虫,初步了解了腰带长体茧蜂多胚的形成过程及其在寄主体内的发育情况。结果表明：以4龄末期亚洲玉米螟幼虫为寄主时,寄生蜂卵产入寄主体内10 min左右开始卵裂,1天左右,初级胚胎从卵壳中被释放出来。之后胚胎在胚外膜内持续分裂产生大量二级胚胎形成桑葚胚。寄生后3天左右,二级胚胎从胚外膜中被释放出来,进入胚胎发育阶段。寄生后6天左右,胚胎进入胚带形成阶段。寄生后8天左右,胚带伸长,头尾形成。寄生后9天左右,身体分节完成,部分幼虫孵化,蜕去胚外膜。寄生后13天左右,蜂幼虫从寄主体内啮出。胚胎在发育初期体积变化不大,但从胚带形成开始,体积则迅速增大。腰带长体茧蜂与另一多胚发育寄生蜂佛州点缘跳小蜂Copidosoma floridanum在胚胎发育进程上明显不同,体现了它们对各自寄主的适应。     

Terminal mitoses require negative regulation of Fzr/Cdh1 by Cyclin A, preventing premature degradation of mitotic cyclins and String/Cdc25

Cyclin A expression is only required for particular cell divisions during Drosophila embryogenesis. In the epidermis, Cyclin A is strictly required for progression through mitosis 16 in cells that become post-mitotic after this division. By contrast, Cyclin A is not absolutely required in epidermal cells that are developmentally programmed for continuation of cell cycle progression after mitosis 16. Our analyses suggest the following explanation for the special Cyclin A requirement during terminal division cycles. Cyclin E is known to be downregulated during terminal division cycles to allow a timely cell cycle exit after the final mitosis. Cyclin E is therefore no longer available before terminal mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence, Cyclin A, which can also function as a negative regulator of Fizzy-related/Cdh1, becomes essential to provide this inhibition before terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1 activity results in the premature degradation of the Cdk1 activators Cyclin B and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without these activators, entry into terminal mitoses is not possible. However, entry into terminal mitoses can be restored by the simultaneous expression of versions of Cyclin B and Cyclin B3 without destruction boxes, along with a Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover, terminal mitoses are also restored in Cyclin A mutants by either the elimination of Fizzy-related/Cdh1 function or Cyclin E overexpression.     

Acquisition of embryogenic competency does not require cell division in carrot somatic cell

Totipotency is the ability of a cell to regenerate the entire organism, even after previous differentiation as a specific cell. When totipotency is coupled with active cell division, it was presumed that cell division is essential for this expression. Here, using the stress-induction system of somatic embryos in carrots, we show that cell division is not essential for the expression of totipotency in somatic/embryonic conversion. Morphological and histochemical analyses showed that the cell did not divide during embryo induction. Inhibitors of cell division did not affect the rate of somatic embryo formation. Our results indicate that the newly acquired trait of differentiation appears without cell division, but does not arise with cell division as a newborn cell.     

Upregulation of Mitimere and Nubbin acts through cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Here, we report that maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. Our results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to our understanding of maintenance and loss of pluripotential stem cell identity.     

Requirements for zygotic gene activity during gastrulation in Drosophila melanogaster

Mutations at the folded gastrulation (fog) and twisted gastrulation (tsg) loci interfere with early morphogenetic movements in Drosophila melanogaster. fog embryos do not form a normal posterior midgut and although their germbands do elongate, they do not extend dorsally. As a result, when normal embryos have fully extended germbands, the germbands in mutant embryos are folded into the interior on the ventral side of the embryo. tsg embryos have abnormally deep dorsal folds during early gastrulation, associated with the failure of dorsal cells to slip laterally to make way for the expanding germband. Both fog and tsg embryos continue to develop, but form disorganized first instar larvae. fog and tsg are zygotically active genes expressed at least by 10 and 20 min after the onset of gastrulation. Both mutations are viable in homozygous germ cells and the wild-type genes need not be expressed during oogenesis for survival of heterozygous progeny. Elimination of fog+ gene product from maternal germ cells does, however, affect the extent of folding observed during gastrulation in viable heterozygotes. Analysis of fog adult and larval gynandromorphs indicates that normal folded gastrulation gene function is only required at the posterior region of the embryo, most probably in the cells giving rise to the posterior midgut or proctodeum. The relative survival of fog mosaics suggests that embryos with mosaic "lethal foci" also die during embryogenesis, although the typical fog phenotype is only produced when the entire focus is mutant. In contrast to the fog focus, no particular cell must be wild type in tsg mosaics for survival. Wild-type cells on the dorsal side of the embryo, however, are most effective in rescuing the embryo. This indicates that normal tsg gene product may be required only on the dorsal side of the embryo, potentially in the region which gives rise to the amnion serosa.     

Patterns of expression of cyclins A, B1, D, E and cdk 2 in preimplantation mouse embryos

Cell cycle regulatory proteins have been characterized in somatic cells and exhibit phase-specific expression patterns. Changes in expression of these regulatory proteins have not been clearly characterized in early preimplantation mouse embryos. This study utilized indirect immunofluorescence to determine the expression pattern of G1/S phase cyclins D and E; S, G2/M phase cyclins A and B1, and cdk 2 during the first three cell cycles of mouse embryo development. Cyclin D demonstrated low expression throughout the first cell cycle but had a somatic-like pattern of expression in cycles 2 and 3 with peak expression at G1 declining through S phase to a low during G2. Cyclin E was present at peak levels in G1 declining through S to a low in G2 during both the first and third cell cycles, but remained at moderate levels during the entire second cell cycle. Cyclin A was maintained at moderate levels throughout the first two cell cycles but showed a somatic-like pattern with a low level in G1 increasing during S phase with peak levels during G2 of the third cell cycle. Cyclin B consistently demonstrated a pattern opposite to a somatic G2/M cyclin, with peak levels in G1 declining through S phase to a low in G2 during each of the three cell cycles examined. Cdk 2 was present at consistent levels during G1 and S phases of all three cell cycles declining slightly in G2.