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.     

Crumbs Affects Protein Dynamics In Anterior Regions Of The Developing Drosophila Embryo

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.     

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.     

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。     

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.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Significance of the polar lobe for the determination of dorsoventral polarity in Dentalium vulgare (da Costa)

The development of dorsoventral polarity in Dentalium dentale has been analyzed after inhibiting first polar lobe formation with cytochalasin B and bisecting the egg into two equal parts at an early trefoil stage. Cleavage pattern and morphogenesis have been studied in both in vivo and permanent cytological preparations. After bisecting the egg, each blastomere may fuse with its adhering polar lobe half and subsequently behave as a CD blastomere. The polar lobe substance may induce both halves to develop an apical tuft and probably also a posttrochal region. Cytochalasin B embryos which pass through an equal first cleavage form a four-cell stage in which the two D blastomeres are situated opposite or adjacent to each other (CDCD or CCDD embryos, respectively). During further development the larvae show a duplication of lobe-dependent structures. It is concluded that dorsoventral polarity originates epigenetically by fusion of the polar lobe with one of the first two blastomeres and is not preformed in the uncleaved egg.     

Analysis of pattern formation during embryonic development of Hydractinia echinata

Summary Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.     

Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera,Tephritidae)

In a sampling of untreated embryos of the economically important fruit pest species, Anastrepha ludens, the cumulative hatch percentage in the lab was noted to be ∼85%. Approximately 70% of the larvae had eclosed through the posterior pole of the egg. This process is effected by the act of Pole Reversal (PR) of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. Investigation of the effects of cryopreservation and various pretreatments prior to cryostorage on the PR behavior was prompted by the observation of significantly lower proportion of cryopreserved embryos exhibiting the PR behavior. Pretreatments (dechorionation and permeabilization) followed by vitrification resulted in delayed hatching, reflecting a slower embryonic development rate of ∼10 h. A smaller proportion of the treated embryos either eclosed from the anterior end of the egg or did not eclose at all despite complete development and prehatch gnawing activity. In the untreated controls, 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation, 83% and 55% (adjusted hatch) of the embryos were noted to hatch this way, respectively. An analysis of the hatch count after the treatments shows that factors contributing to the embryos' inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pre-treatment contributed the highest to this phenomenon lending support to the view that chemical toxicity rather than physical effects of cryopreservation play a major role in post-cryopreservation effects.     

The genital disc of Drosophila melanogaster

 The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development. Received: 4 February 1997 / Accepted: 22 May 1997     

Multiple functions of segment polarity genes in Drosophila

l(1)dishevelled (l(1)dsh) is a late zygotic lethal mutation that exhibits a rescuable maternal effect lethal phenotype. l(1)dsh/Y embryos, derived from females possessing a homozygous l(1)dsh germline clone, exhibit a segment polarity embryonic phenotype. Analysis of the development of these embryos indicates: (1) that segmental boundaries do not form although the correct number of tracheal pits is formed; (2) that pockets of cell death occur between the tracheal pits; and (3) that engrailed expression becomes abnormal during germ band shortening. We propose that, in the absence of both maternal and zygotic expression of l(1)dsh+, cells from each posterior compartment die. Subsequently, cells from the anterior compartment must rearrange their positional values to generate the segment polarity phenotype. We have compared the phenotype of five other segment polarity loci: four embryonic lethals [l(1)armadillo, l(2)gooseberry, l(2)wingless, and l(3)hedgehog]; and the late zygotic lethal, l(1)fused. Only l(2)wingless embryos exhibit early segmentation defects similar to those found in l(1)dsh/Y embryos derived from homozygous germline clones. In contrast, segmentation is essentially normal in l(1)armadillo, l(2)gooseberry, l(3)hedgehog, and l(1)fused embryos. The respective maternal and zygotic contribution and the roles of the segment polarity loci for the patterning of the embryo and the adult are discussed.     

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.     

Egg development in parthenogenetic nematodes: variations in meiosis and axis formation

In the well studied model nematode Caenorhabditis elegans entrance of the sperm induces an anterior-posterior polarity in the egg and determines the orientation of the primary embryonic axis. Subsequently, fusion of two haploid gamete nuclei results in a diploid zygote as a prerequisite for normal embryogenesis. Here we analyze the establishment of embryonic polarity and diploidy in the absence of sperm in three parthenogenetic nematode species from three different families, Diploscapter coronatus (Diploscapteridae), Acrobeloides nanus (Cephalobidae) and Plectus sp. (Plectidae). We find that they not only differ from C. elegans in these two aspects but also from each other, indicating variant solutions for the same developmental challenges and supporting the view that the parthenogenetic mode of reproduction has been acquired multiple times independently.     

Parameters influencing reversal of segment sequence in posterior egg fragments ofCallosobruchus (Coleoptera)

Summary The double abdomen type of embryonic segment pattern can develop in posterior fragments ofCallosobruchus eggs. In this type of pattern, a series of posterior segments is joined in reversed polarity to an equal set from the original pattern persisting in normal polarity. Reversed and non-reversed sets are fused in a plane of mirror symmetry, which shows in the larval cuticle as a symmetry line. This line may be located anywhere in the posterior thorax or the anterior abdomen. The reversed abdomen may be incomplete caudally due to secondary causes. Polarity reversal and concomitant double abdomen formation occurred only when temporary constriction was terminated before cellularization of the blastoderm, and only when the anterior fragment was degenerating. Maximum reversal frequency was 94% of analyzable posterior partial larvae when the constriction was applied slightly anterior to the middle of the egg when the egg contained 4–32 nuclei. Reversal was often restricted to longitudinal strips of the larval cuticle. The longitudinal borderlines between the reversed and the non-reversed strips ran predominantly along the larval midlines. Such borderlines probably existed in the blastoderm anywhere around its circumference, but borderlines in the future mesoderm and serosa would be internalized during gastrulation and dorsal closure, respectively, and the embryonic midlines would then become secondary borderlines visible in the larval cuticle. If a morphogen is involved in segment pattern formation, its transport in the egg must be polarized longitudinally in order to account for reversals restricted to longitudinal cuticular strips.     

Intracellular sperm/egg interactions in Drosophila: a three-dimensional structural analysis of a paternal product in the developing egg

During fertilization in Drosophila, a single 1.75 mm long sperm enters the egg through the anterior end. Using a sperm-specific monoclonal antibody and indirect immunofluorescence of whole fixed eggs and embryos, intracellular interactions between the sperm and egg are examined as they occur inside the fertilized egg. The sperm nucleus remains attached to the axoneme throughout the entire process of fertilization including the stages of pronuclear maturation, pronuclear fusion and karyogamy indicating an intracellular function for the sperm during these stages. Optical sections and three-dimensional reconstructions of whole mount specimens reveal that a stereotypically folded structure forms during fertilization strongly suggesting that this structure positions the male pronucleus in the proper region of the egg in anticipation of pronuclear fusion. This, and the appearance of regional structural changes in the sperm upon entry suggests that sperm are localized via specific interactions with the maternal cytoplasm. Following fertilization and during the ensuing cleavage divisions, the sperm remains intact and localized at the anterior end of the egg. During cellular blastoderm formation the sperm tail is sequestered into the anterior yolk area where it continues to persist well into embryonic development. This structural analysis identifies intracellular sperm/egg interactions as an important aspect of fertilization, and provides a unique model system for the study of sperm/egg interactions not presently available in other systems.     

The influence of ultrasound and constant magnetic field on gametes, zygotes, and embryos of the sea urchin

The influence of constant magnetic field, power 7 T, and ultrasound, frequency 2, 4 and 8 MHz, on gametes, fertization, embryos and larvae of the sea urchin was studied. It was shown that magnetic field breaks the process of the gamete fusion but does not influence gametes, embryos, and larvae. Ultrasound impairs the motility of spermatozoa and larvae, prevents the fertilization, and breaks the embryonic development. It is assumed that the effect of the magnetic field is connected with the response of the cortical cytoskeleton, which consists of bundles of actin microfilaments. The rearrangement of the cortical cytoskeleton occurs during the first 20 minutes after the contact of sperm with the egg. Also there is effect of magnetic fields on calcium ions, which are liberated during the first seconds after gamete contact. The effect of the ultrasound is explained by a small increase in water temperature and cavitation process, which break celluar structures.     

Allocation and specification of the genital disc precursor cells in Drosophila

The adult structures of Drosophila melanogaster are derived from larval imaginal discs, which originate as clusters of cells within the embryonic ectoderm. The genital imaginal disc is composed of three primordia (female genital, male genital, and anal primordia) that originate from the embryonic tail segments A8, A9, and A10, respectively, and produce the sexually dimorphic genitalia and analia. We show that the genital disc precursor cells (GDPCs) are first detectable during mid-embryogenesis as a 22-cell cluster in the ventral epidermis. Analysis of mutant and double mutant phenotypes of embryonic patterning genes in the GDPCs, together with their expression patterns in these cells, revealed the following with respect to the origins and specification of the GDPCs. The allocation of the GDPCs from the ventral epidermis requires the function of ventral patterning genes, including the EGF receptor and the spitz group of genes. The ventral localization of the GDPCs is further restricted by the action of dorsal patterning genes. Along the anterior-posterior axis, several segment polarity genes (wingless, engrailed, hedgehog, and patched) are required for the proper allocation of the GDPCs. These segment polarity genes are expressed in some, but not all of the GDPCs, indicating that anterior and posterior compartments are not fully established in the GDPCs. In addition, we found that the three primordia of the larval genital disc have already been specified in the GDPCs by the coordinated actions of the homeotic (Hox) genes, abdominal-A, Abdominal-B, and caudal. By identifying how these different patterning networks regulate the allocation and primordial organization of the 22 embryonic precursors of the compound genital disc, we demonstrate that at least some of the organization of the larval disc originates as positional information in the embryo, thus providing a context for further studies on the development of the genital disc.     

Dicephalic — ADrosophila mutant affecting polarity in follicle organization and embryonic patterning

Summary The mutationdicephalic (dic) affects follicle development and thereby alters the antero-posterior polarity of embryonic patterning. It maps at a single locus (3–46.0±1.0) and can be characterized as a semi-dominant maternal effect mutation with low penetrance. Indic follicles, the 15 nurse cells form two clusters located at opposite poles of the oocyte; the numerical distribution of the nurse cells among the clusters varies from 7:8 to 1:14. Thedic egg shell carries a micropyle (anterior marker) at either pole, but the misshapen respiratory appendages are restricted to one of the two poles in most eggs. The malformed eggs rarely yield larvae and these are always abnormal anteriorly and/or posteriorly. The segment pattern expressed in their cuticle may represent two anterior parts of opposite polarities (double head type), two posterior parts of opposite polarities (double abdomen type, rare) or show uniform polarity. Lability of organization at the cystocyte stage appears as the primary developmental defect of the mutant.