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We investigated the hypothesis that several transcriptional repressors are necessary to set the boundaries of anterior pair-rule stripes in Drosophila. Specifically, we tested whether Tailless (Tll) is part of a repression mechanism that correctly sets the anterior boundaries of hairy 1 (h 1) and even-skipped 1 (eve 1) stripes. Single mutant tll embryos displayed subtle deviations from the normal positions of h 1 and eve 1 stripes. Moreover, we observed stronger stripe deviations in embryos lacking both Tll and Sloppy-paired 1 (Slp 1), a common repressor for anterior pair-rule stripes. Using h 1 and eve 1 reporter constructs in the genetic assays, we provided further evidence that interference with normal mechanisms of stripe expression is mediated by Tll repression. Indeed, Tll represses both h 1 and eve 1 reporter stripes when misexpressed. Investigating the expression of other anterior gap genes in different genetic backgrounds and in the misexpression assays strengthened Tll direct repression in the regulation of h 1 and eve 1. Our results are consistent with tll being a newly-identified component of a combinatorial network of repressor genes that control pair-rule stripe formation in the anterior blastoderm of Drosophila.

During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. Here, we show that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. We found that mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. We propose that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding.

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.

In mouse Otx2 plays essential roles in anterior-posterior axis formation and head development in anterior visceral endoderm and anterior mesendoderm. The Otx2 expression in these sites is regulated by VE and CM enhancers at the 5' proximal to the translation start site, and we proposed that these enhancers would have been established in ancestral sarcoptergians after divergence from actinopterigians for the use of Otx2 as the head organizer gene (Kurokawa et al., 2010). This would make doubtful an earlier proposal of ours that a 1.1 kb fragment located at +14.4 to +15.5 kb 3' (3'En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (Kimura-Yoshida et al., 2007). In the present study, we demonstrate that fugu Otx2a is not expressed in the dorsal margin of blastoderm, shield and early anterior mesendoderm, and that the fugu Otx2a 3'En do not exhibit activities at these sites of fugu embryos. We conclude that the fugu Otx2a 3'En does not harbor an organizer enhancer, but encodes an enhancer for the expression in later anterior mesendodermal tissues. Instead, in fugu embryos Otx2b is expressed in the dorsal margin of blastoderm at blastula stage and shield at 50% epiboly, and this expression is directed by an enhancer, 5'En, located at -1000 to -800 bp, which is uniquely conserved among teleost Otx2b orthologues.

Medaka is an ideal model system for developmental studies as it combines the advantages of powerful genetics and classical embryology. Due to the accessibility, transparency and fast development, embryogenesis and morphogenesis can be followed in vivo. Microscopic time-lapse imaging, however, requires the immobilization of the object to be observed. In medaka rhythmical contractile movements of the blastoderm during early development hampered time-lapse studies, as they cause the embryo to rotate vividly. Here we show that the contractile movements can be reduced by continuous treatment with the gap-junction uncoupling agent n-heptanol up to the 12-somite stage (stage 23) without interfering with development. This allows for the first time to perform high-resolution time-lapse studies in medaka.

Unfertilized eggs of Oryzias latipes were artificially inseminated and incubated at 26+/-1 degrees C. Careful observation of the process of embryonic development by light microscopy allowed division of the process into 39 stages based on diagnostic features of the developing embryos. The principal diagnostic features are the number and size of blastomeres, form of the blastoderm, extent of epiboly, development of the central nervous system, number and form of somites, optic and otic development, development of the notochord, heart development, blood circulation, the size and movement of the body, development of the tail, membranous fin (fin fold) development, and development of such viscera as the liver, gallbladder, gut tube, spleen and swim (air) bladder. After hatching, development of the larvae (fry) and young can be divided into six stages based on such diagnostic features as the fins, scales and secondary sexual characteristics.

In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. We identified a novel maternal-effect recessive mutation, tokkaebi (tkk), that affects formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5-100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos displayed defects in the nuclear accumulation of beta-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly affected. Injection of RNAs for activated beta-catenin, dominant-negative forms of Axin1 and GSK3beta, and wild-type Dvl3, into the tkk embryos suppressed the ventralized phenotypes and/or dorsalized the embryos, and restored or induced an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs was affected in the tkk embryos. Inhibition of cytoplasmic calcium release elicited an ectopic and expanded expression of chordin in the wild-type, but did not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the beta-catenin-degradation machinery to control the stability of beta-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of beta-catenin is involved in dorsal axis formation in zebrafish.

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.

The generation of polarity and patterning in multicellular organisms depends in part on the asymmetric localization of molecules to specific subdomains within a cell. Localized transcripts for several molecules are known to be required for patterning oocytes and embryos in Drosophila as well as Caenorhabditis elegans. Here, we describe the localization of transcripts encoding the nodal-related morphogen, Squint (sqt), in zebrafish oocytes and early embryos, and the mechanisms by which sqt RNA is localized. sqt transcripts are uniformly distributed in oocytes through all stages of oogenesis. Upon egg activation, sqt RNA is localized to the blastoderm, and excluded from the yolk cell. The mechanism of sqt RNA transport was examined using cytoskeletal inhibitors. Disruption of actin microfilaments by treatment with latrunculin A does not alter the localization of sqt RNA to the blastoderm. However, disruption of the microtubule cytoskeleton by treatment with nocodazole affects sqt RNA localization. These results indicate that sqt transcripts are translocated by an RNA localization pathway which is initiated upon egg activation, and that sqt RNA localization through this pathway is mediated via the microtubule cytoskeleton.

TNF-receptor associated factors (TRAFs) comprise a family of adaptor proteins that act as downstream signal transducers of the TNF receptor superfamily and the Toll/interleukin-1 receptor family. The mammalian TRAFs 2, 5 and 6 are known to activate JNK- and NF-kappaB signaling pathways, whereas the function of the other three mammalian family members, TRAF 1, 3 and 4 is less well characterized. Vertebrate TRAFs have a very similar structure with the exception of TRAF1: aside the characteristic C-terminal TRAF domain, they share a N-terminal RING finger followed by five or, in the case of TRAF4, seven regularly spaced zinc fingers. Two TRAF homologues are present in the genome of Drosophila melanogaster, DTRAF1 and DTRAF2 (also known as DTRAF6) and both have been implicated in the Toll-receptor pathways leading to the activation of NF-kappa B and JNK. DTRAF1 is most closely related to mammalian TRAF4 which is predominantly expressed during nervous system development and in ephitelial progenitor cells. In order to gain insight into possible roles of DTRAF1 during development, we have performed a detailed transcriptional analysis of the gene at various embryonic and larval stages.

The homeobrain (hbn) gene is a new paired-like homeobox gene which is expressed in the embryonic brain and the ventral nerve cord. Expression of homeobrain initiates during the blastoderm stage in the anterior dorsal head primordia and the gene is persistently expressed in these cells which form parts of the brain during later embryonic stages. An additional weaker expression pattern is detected in cells of the ventral nerve cord from stage 11 on. The homeodomain in the Homeobrain protein is most similar to the Drosophila proteins DRx, Aristaless and Munster. In addition, the localized brain expression patterns of homeobrain and DRx resemble each other. Two other homeobox genes, orthopedia and DRx are clustered in the 57B region along with homeobrain. The current evidence indicates that homeobrain, DRx and orthopedia form a homeobox gene cluster in which all the members are expressed in specific embryonic brain subregions.

The larval gut of Drosophila is coated with visceral muscles of mesodermal origin. In the midgut region this musculature comprises circular and longitudinal fibres. The complete visceral musculature is described to be removed during metamorphosis and to be replaced by a newly differentiated imaginal tissue resembling the morphology of the larval musculature. However, progenitors of this imaginal visceral musculature have never been detected prior to differentiation. Here I present results indicating that the longitudinal visceral musculature of the midgut completely persists through metamorphosis. Single cells expressing green fluorescent protein (GFP) as a marker were transplanted at the blastoderm stage. All clones contributing to the longitudinal visceral musculature detected in third instar larvae were recovered after metamorphosis in adult flies. Further evidence for the persistence of the larval visceral musculature was obtained from the P[Gal4] insertion line 5053A. It expresses GAL4 specifically in the longitudinal visceral muscles of the midgut of all developmental stages to the adult fly beginning at the end of embryogenesis. By using GFP as a reporter, it was possible to follow these cells through the entire metamorphosis. Although the muscles undergo dramatic morphological changes including the loss of their contractile system, no evidence for a replacement of the larval visceral musculature by imaginal precursor cells was detected.

Small GTPases of the rho family function as signal transducer for extra-cellular stimuli to control cytoskeletal re-organization and a variety of other cellular processes including adhesion, proliferation and transcriptional regulation (Hall, A., 1998. RhoGTPases and the actin cytoskeleton. Science 279, 509–514). Usually widely expressed, their activities are tightly controlled by conformational changes induced by hydrolysis of the GTP bound molecule (Bourne H.R., Sanders D.A., 1990. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132). Conversion of GTP to GDP relies on a rho intrinsic GTPase domain that requires GTPase activating proteins (GAPs) for potent activity (Lamarche, N., Hall. A., 1994. GAPs for rho-related GTPases. Trends Genet. 10, 436–440). Here we report on the identification of a novel Drosophila GAP gene, d-CdGAPr, encoding a protein related to mammalian CdGAPs. The gene is expressed throughout development as well as in adults. Spatio-temporal transcription pattern of d-CdGAPr during embryogenesis is highly dynamic. Abundant in the pre-blastoderm embryo prior to the onset of zygotic transcription, messengers accumulate at the blastoderm posterior pole after cellularisation. During gastrulation and subsequent development, all cells accumulate low levels of d-CdGAPr RNA, while a few territories transiently display stronger expression. Sites of preferential expression include the posterior pole of the early cellular blastoderm, the neuro-ectoderm prior to neuroblast delamination, rows of epidermal cells in the most posterior part of thoracic and first abdominal segments and a ring of epidermal cells at the posterior end of the embryo.

Asymmetric distribution of maternal mRNAs has not been well documented in zebrafish. Recently, we have shown that dazl mRNA is localized at the vegetal pole. Here we report a novel zebrafish gene, bruno-like (brul), which provides another example of vegetal mRNA localization. brul encodes an Elav-type RNA-binding protein that belongs to the Bruno-like family that includes mammalian CUG-BP, Xenopus EDEN-BP, and Drosophila Bruno. At 24 hpf, brul mRNA was abundant in lens fiber cells. At the onset of embryogenesis, maternal brul mRNA was detected at the vegetal pole, and it then migrated rapidly toward the blastoderm through yolk cytoplasmic streams. During oogenesis, brul mRNA became localized at the vegetal cortex at stage II, later than dazl mRNA. We found that anchoring of brul mRNA was dependent on microfilaments.

The Drosophila gap gene Krüppel (Kr) encodes a zinc finger-type transcription factor required for controlling the spatial expression of other segmentation genes during early blastoderm stage. Here we show that two independent and transferable repressor domains of Krüppel act to control expression of the pair-rule gene hairy, and that the minimal cis-acting element of hairy stripe7 (h7) mediates either Krüppel-dependent activation or repression in different regions of the blastoderm embryo. The C-terminal region of Krüppel which encompasses the predominant repressor domain is not essential for activation, but is required to fully suppress h7-mediated transcription in response to high levels of Krüppel activity. This domain contains an interaction motif for dCtBP, a homologue of the human co-repressor CtBP. dCtBP activity is, however, dispensable for Krüppel-mediated repression in the embryo since Krüppel-mediated repression functions in the absence of dCtBP. Possible modes of h7-mediated gene regulation in response to the different domains and levels of Krüppel are discussed.

We report the expression pattern of a new adenomatous polyposis coli (APC) homolog called E-APC during Drosophila development. E-APC protein is expressed in all embryonic and larval cells we have examined. In the early blastoderm embryo, we see a striking concentration of E-APC in the cortical actin caps. Microtubules are closely associated with these caps. Since human APC has been reported to bind to microtubules, we investigated whether the cortical E-APC co-localizes with tubulin. However, this was not the case, implying that the putative tubulin-binding property of human APC is not well conserved.

The vertebrate Six genes are homologues of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Here we describe two new Six genes in Drosophila, D-Six3 and D-Six4, which encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, we report the partial sequences of 12 Six gene homologues from several lower vertebrates and show that the class of Six proteins can be subdivided into three major families, each including one Drosophila member. Similar to so, both D-Six3 and D-Six4 are initially expressed at the blastoderm stage in narrow regions of the prospective head and during later stages in specific groups of head midline neurectodermal cells. D-Six3 may also be essential for development of the clypeolabrum and several head sensory organs. Thus, the major function of the ancestral Six gene probably involved specification of neural structures in the cephalic region.

Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the anterior–posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In the anterior region, such elements mediate transcriptional activation in response to the maternal concentration gradient of the anterior determinant BICOID and repression by spatially distinct activities of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on the activity of the gap gene knirps , suggesting that posterior stripe expression is exclusively controlled by zygotic regulators. Here we show that the zygotic activation of hairy stripe 6 expression is preceded by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior stripes. The results suggest that activation and the expression level mediated by the hairy stripe 6-element depend on the number of activator binding sites, likely to involve additive rather than synergistic interactions. We found an identical transacting factor requirement for hairy stripe 6 and 7 expression. The arrangement of the corresponding binding sites for the common factors involved in the control of the two stripes share a high degree of similarity, but some of the factors exert opposite regulatory functions within the two enhancer elements.

The region-specific homeotic gene spalt is involved in the specification of terminal versus trunk structures during early Drosophila embryogenesis. Later in development spalt activity participates in specific processes during organogenesis and larval imaginal disc development. The multiple functions of spalt are reflected in distinct spatio-temporal expression patterns throughout development. Here we show that spalt cis-regulatory sequences for region-specific and organ-specific expression are clustered. Their organization may provide the structural basis for the diversification of expression pattern within the spalt/spalt related/spalt adjacent gene complex. We also examined the transacting factor requirement for the blastodermal spalt expression domains. They are under the genetic control of maternal and gap gene products and we show that these products are able to bind to corresponding spalt cis-acting sequences in vitro. The results suggest that the transacting factors, as defined by genetic studies, functionally interact with the spalt regulatory region. In addition, we provide evidence that a zygotic gene product of the terminal system, Tailless, cooperates with the maternal gene product Caudal and thereby activates gene expression in the terminal region of the embryo.

We have analysed the cell lineage relationships between larval and imaginal mesodermal primordia at the blastoderm stage by homotopic single cell transplantations. The primordia of adepithelial cells, the precursors of adult thoracic muscles, are restricted to the region from 50 to 65% egg length within the ventrally located mesodermal anlage. Clones of adepithelial cells always show a common cell lineage with larval muscles and in some cases additionally with larval fat body. This proves that at the blastoderm stage the determination of larval vs. imaginal mesodermal primordia has not yet taken place. Larval somatic muscle clones, in contrast to clones in the ectoderm, can overlap several segments, whereas clones of adepithelial cells are always restricted to imaginal discs of one segment.

The Drosophila gene buttonhead ( btd ) is a gap-like head segmentation gene which encodes a triple zinc finger protein structurally and functionally related to the human transcription factor Spl. Here we report the pattern of btd expression during embryogenesis. btd is not only expressed and required in the blastoderm anlagen of the antennal, intercalary and mandibular segments as reported previously, but both expression and requirement extend into the anlage of the maxillary segment. From gastrulation onwards, btd is expressed in distinct spatial and temporal patterns, suggesting that btd might be required for a number of developmental processes beyond head segmentation. In fact, analysis of btd mutant embryos revealed that btd participates in the formation of the peripheral nervous system. However, no other morphologically apparent phenotype was observed. We identified a btd -related gene, termed D-Spl , which is expressed in temporal and spatial patterns similar to btd during postblastodermal development. No localized expression domains of D-Spl , which is located in the same X-chromosomal band as btd , were seen during the blastoderm state. The results suggest that D-Spl and btd represent a novel gene pair with partially redundant edundant functions after the blastoderm stage.

Many of the mechanisms that govern the patterning of the Drosophila neuroectoderm and mesoderm are still unknown. Here we report the sequence, expression, and regulation of the homeoɡ gene msh, which is likely to play an important role in the early patterning events of these two tissue primordia.msh expression is first observed in late blastoderm embryos and occurs in longitudinal bands of cells that are fated to become lateral neuroectoderm. This expression is under the control of dorsoventral axis-determination genes and depends on dpp-mediated repression in the dorsal half of the embryo and on flb-(EGF-) mediated repression ventrally. The bands of msh expression define the cells that will form the lateral columns of proneural gene expression and give rise to the lateral row of SI neuroblasts. This suggests that msh may be one of the upstream regulators of the achaete-scute (AS-C) genes and may play a role that is analogous to that of the homeoɡ gene vnd/NK2 in the medial sector of the neuroectoderm. During neuroblast segregation, msh expression is maintained in a subset of neuroblasts, indicating that msh, like vnd/NK2, could function in both dorsoventral patterning of the neuroectoderm and neuroblast specification. The later phase of msh expression that occurs after the first wave of neuroblast segregation in defined ectodermal and mesodermal clusters of cells points to similar roles of msh in patterning and cell fate specification of the peripheral nervous system, dorsal musculature, and the fat body. A comparison of the expression patterns of the vertebrate homologs of msh, vndNK2, and AS-C genes reveals striking similarities in dorsoventral patterning of the Drosophila and vertebrate neuroectoderm and indicates that genetic circuitries in neural patterning are evolutionarily conserved.

In wildtype males, binding of the MSL-1 gene product to the X chromosome is first seen at the cellular blastoderm stage (stage 5). MSL-2 is associated with the X chromosome in male embryos at a later stage, but the difference in apparent binding time between these two proteins is probably due to a difference in the sensitivity of their respective antisera. Early binding of MSL-1 is never seen in wildtype female embryos, and we have determined that this inhibition is mediated by the SXL product made by the activation of the early Sxl promoter. Once it is allowed to occur, the early X chromosome association of the MSLs is relatively stable, persisting in some cases through the first larval instar in spite of the presence of SXL levels concordant with normal female development. The results of these experiments are discussed in light of their relevance to the established observations that (1) the SXL made by the early promoter inhibits the hypertranscription of run at the blastoderm stage, and (2) severe disturbances in SXL function (loss in XX individuals and gain in haplo-X individuals) result in lethality during embryogenesis while loss of msl function kills males much later.

By marking cells of early gastrula stage embryos, we showed that in embryos mutant for a strong tll allele the fate map is shifted posteriorly and the hindgut anlage is deleted. We therefore used aspects of hindgut development to characterize the phenotype of new and previously described tll alleles. In embryos mutant for the various alleles, relative levels of blastoderm expression of Trg (T-related gene, required to establish the hindgut) and of mature hindgut size were determined; the results of these assays correlated with each other. Of the alleles that map to the sequence encoding the Tailless nuclear receptor protein, all (four) affect the zinc fingers of the DNA binding domain; surprisingly, substitutions of highly conserved residues allow a range of activities as detected by our bioassays.

We have analyzed the expression pattern and localization of MLE, MLS-1, MSL-2, and histone H4Ac16 during embryogenesis to determine when msl-dependent dosage compensation begins. Maternal MSL-1 and MLE are present in both sexes at fertilization. MSL-2 lacks a maternal component, and male-specific zygotic expression is detectable at the end of blastoderm. During germ band extension, MSL-1, MSL-2, MLE, and histone H4Ac16 display coincident sub-nuclear localization in male embryos. In embryos lacking one of the MSL proteins, the sub-nuclear localization of the other MSLs and of histone H4Ac16 is not detected. We conclude that the MSL proteins associate with the X chromosome and are interdependent since early embryogenesis.

The Drosophila melanogaster trithorax gene encodes several large RNAs which are expressed in complex patterns in the embryo. The D. virilis trithorax gene was isolated and sequenced. It produces a similar to D. melanogaster set of transcripts, and it encodes a protein that shows sequence similarity in several domains which are also conserved in the human homologue, ALL-1 HRX . Previous experiments have suggested that a distinct expression domain of trithorax in the posterior region of the embryo is required to maintain expression of the BX-C genes (Sedkov et al., 1994, Development 120, 1907–1917). At cellular blastoderm, trithorax RNA expression in D. virilis embryos is also confined to the posterior portion of the presumptive mesoderm. This finding supports the idea that the specific BX-C-related expression domain is an essential feature of the trithorax gene.

Cytoplasmic incompatibility (CI) in Drosophila is related to the presence of Wolbachia, an intracelluar microorganism found in many species of insects. In order to study the intracellular localization of Wolbachia in eggs and embryos, we have purified the bacteria from fly embryos and subsequently generated a monoclonal antibody (Mab Wol-1) specific for Wolbachia. Indirect immunofluorescence staining using Wol-1 reveals that during mitosis, Wolbachia are localized near spindle poles and centrosomes. Double label immunofluorescence experiments using anti-tubulin and anti-Wolbachia antibodies show that Wolbachia co-localize with centrosomal microtubules throughout the cell cycle. Direct interactions between the bacteria and centrosome-organized microtubules are implied from seven observations: (1) throughout the mitotic cycle, the position and movement of Wolbachia precisely mimic the behavior of the centrosome and apparently associates with centrosome-organized microtubules; (2) Wolbachia segregate equally to each spindle pole during mitosis; (3) Wolbachia do not associate with spindle microtubules during mitosis; (4) Wolbachia located in the egg cortex localize to the domains of cytoplasm organized by microtubules during blastoderm formation; (5) polar body nuclei that lack centrosomes but contain associated microtubules do not contain Wolbachia; (6) Wolbachia no longer associate with yolk nuclei, following differentiation and loss of centrosomes; (7) during pole cell formation, Wolbachia co-localize with the centrosome on the apical side of the nucleus as pole cells form. Quantitative data indicates that no Wolbachia growth occurs during the preblastoderm period even though rapid nuclear, and subsequent cellular, proliferation takes place during this same period. This indicates that Wolbachia are under strict growth regulation by the host suggesting that host factors play a role in regulating growth of Wolbachia in the egg. Further cellular and molecular studies of the extensive, global interactions between host and symbiont observed in this egg should provide important new insights into the evolution of host/symbiosis and the cell biology of cytoplasmic incompatibility.

Like most organisms, Drosophila embryos develop in relation to two orthogonal axes, the anteroposterior and dorsoventral. These two axes are established by four independent systems of maternal information. Mutations in any system disrupt either the anteroposterior or the dorsoventral patterning of the embryo but never affect the orthogonal orientation of the axes relative to each other. Here we show by analyzing their embryonic fate map, that K10 embryos still possess a dorsoventral polarity. However, instead of forming a 90 degrees angle, the dorsoventral and the anteroposterior axes lie parallel to each other. This axis misorientation was partially corrected by decreasing the wild-type grk gene copy number such that the embryos issued from K10/K10; grk/+ females showed a variability in their fate map which could be interpreted as a progressive rotation of dorsoventral axis relative to the unmodified anteroposterior axis. This rotation is maximal in the K10 embryos where it reaches 90 degrees and results in the congruence of the two axes. The alteration of the embryonic fate map could be traced back to oogenesis where it was shown to correlate with the mislocalization of the grk transcripts.

Dorsoventral (DV) patterning in the trunk region of Drosophila embryo is established through intricate molecular interactions that regulate Dpp/Scw signaling during the early blastoderm stages. The hindgut of Drosophila, which derives from posterior region of the cellular blastoderm, also shows dorsoventral patterning, being subdivided into distinct dorsal and ventral domains. engrailed (en) is expressed in the dorsal domain, which determines dorsal fate of the hindgut. Here we show that a repressor Brk restricts en expression to the dorsal domain of the hindgut. Expression domain of brk during early blastdermal stages is defined through antagonistic interaction with dpp, and expression domains of dpp and brk in the early blastoderm include prospective hindgut domain. After stage 9, dpp expression in the dorsal domain of the hindgut primordium disappears, but, the brk expression in the ventral domain continues. It was found that Dorsocross (Doc), which is a targe gene of Dpp, is responsible for restricting brk expression to the ventral domain of the hindgut. On the other hand, activation of en is under the control of brachyenteron (byn) that is regulated independently of dpp, brk, and Doc. The cooperative interaction of common DV positional cues with byn during hindgut development represents another aspect of mechanisms of DV patterning in the Drosophila embryo.

The segmentation gene hunchback (hb) is involved in setting up the anterior-posterior axis of the Drosophila embryo. It is expressed maternally and zygotically and it plays a key role in integrating the effects of the anterior and posterior maternal systems. The hb gene from D. virilis has previously been cloned and was shown to be well conserved in its coding region, but less so in its upstream region which shows a more patchy pattern of conserved and diverged sequences. This work deals with the functional conservation of hb between the two species. We have mapped two additional regulatory elements for the expression of hb in the early embryo, namely the enhancer for the maternal expression and the enhancer region for the late blastoderm expression. Fragments containing these two elements, the previously identified bicoid dependent element for the early blastoderm expression of hb and the coding region were taken from D. virilis and tested in the D. melanogaster background. We find that all enhancer elements as well as the coding region are functionally conserved between the two species. Comparison of the upstream sequences that include the enhancer region for the late blastoderm expression reveal seven highly conserved blocks. Some of these contain consensus binding sites for transregulatory factors that are likely to control the respective expression domains.

We have studied the genetic requirement for the normal expression of the terminal gap genes huckebein (hkb) and tailless (tll) and their possible function in the posterior pole region of the Drosophila embryo. At the early blastoderm stage, both genes are expressed in largely coextensive expression domains. Our results show that in the posterior region of the embryo both the activation and the control of the spatial limits of tll and hkb expression are critically dependent on torso (tor) activity, which is thought to be a crucial component of a cellular signal transduction pathway provided by the terminal maternal system. Furthermore, the spatial control of hkb and tll expression does not require mutual interactions among each other, nor does it require regulatory input from other gap genes which are essential for the establishment of segmentation in the trunk region of the embryo ("central gap genes"). Therefore, the terminal gap genes have unique regulatory features which are distinct from the central gap genes. In the absence of terminal gap gene activities, as in hkb and tll mutant embryos, the expression domains of the central gap genes expand posteriorly, indicating that the terminal gap gene activities prevent central gap gene expression in the posterior pole region of the wildtype embryo. This, in turn, suggests that the terminal gap gene activities prevent metamerization by repression of central gap genes, thereby distinguishing the segmented trunk from the nonsegmented tail region of the embryo.

The germline cells of Drosophila are derived from pole cells, which form at the posterior pole of the blastoderm and become primordial germ cells (PGCs). To elucidate the signal transduction pathways for the development of embryonic PGCs, we examined the effects of various growth factors on the proliferation of PGCs. Up- and down-regulation of Wingless (Wg) in both of soma and PGCs caused an increase and a decrease in the number of PGCs, respectively. The Wg/β-catenin signaling pathway began to occur in PGCs at the same time as the PGCs began to divide during the embryonic stage in both sexes. In addition, PGCs were found to produce wg mRNA as they begin to divide. Thus, Wg functions as an autocrine factor to initiate mitosis in embryonic PGCs. Decapentaplegic affected the growth of PGCs from the end of the embryonic stage. The results indicate that these growth factors regulate the division of embryonic PGCs in a stage-specific manner.

Gap genes are among the first transcriptional targets of maternal morphogen gradients in the early Drosophila embryo. However, it remains unclear whether these gradients are indeed sufficient to establish the boundaries of localized gap gene expression patterns. In this study, we address this question using gap gene circuits, which are data-driven mathematical tools for extracting regulatory information from quantitative wild-type gene expression data. We present new, quantitative data on the mRNA expression patterns for the gap genes Kruppel (Kr), knirps (kni) and giant (gt) during the early blastoderm stage of Drosophila development. This data set shows significant differences in timing of gap gene expression compared to previous observations, and reveals that early gap gene expression is highly variable in both space and time. Gene circuit models fit to this data set were used for a detailed regulatory analysis of early gap gene expression. Our analysis shows that the proper balance of maternal repression and activation is essential for the correct positioning of gap domains, and that such balance can only be achieved for early expression of kni. In contrast, our results suggest that early expression of gt requires local neutralization of repressive input in the anterior region of the embryo, and that known maternal gradients are completely insufficient to position the boundaries of the early central Kr domain, or in fact any Kr-like domain in the central region of the blastoderm embryo. Based on this, we propose that unknown additional regulators must be involved in early gap gene regulation.

Specification of the terminal regions of the Drosophila embryo depends on the Torso RTK pathway, which triggers expression of the zygotic genes tailless and huckebein at the embryonic poles. However, it has been shown that the Torso signalling pathway does not directly activate expression of these zygotic genes; rather, it induces their expression by inactivating, at the embryonic poles, a uniformly distributed repressor activity. In particular, it has been shown that Torso signalling regulates accumulation of the Capicua transcriptional repressor: as a consequence of Torso signalling Capicua is downregulated specifically at the poles of blastoderm stage embryos. Extending the current model, we show that activation of the Torso pathway can trigger tailless expression without eliminating Capicua. In addition, analysis of gene activation by the Torso pathway and downregulation of Capicua unveil differences between the terminal and the central embryonic regions that are independent of Torso signalling, hitherto thought to be the only system responsible for confering terminal specificities. These data provide new insights into the mode of action of the Torso signalling pathway and on the events patterning the early Drosophila embryo.

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.

The transforming growth factor-beta (TGF-beta) superfamily of structurally related ligands regulates essential signaling pathways that control many aspects of cell behavior in organisms across the phylogenetic spectrum. Here we report the identification of maverick (mav), a gene that encodes a new member of the TGF-beta superfamily in Drosophila. Phylogenetic analysis and sequence comparison suggest that Mav cannot be easily assigned to any one sub-family, since it is equally related to BMP, activin and TGF-beta ligands. mav maps to the fourth chromosome and is expressed throughout development. In situ hybridization experiments reveal the presence of maternally derived mav transcript in precellular blastoderm embryos. Later in development, mav is expressed in a dynamic pattern in the developing gut, both in endodermal and visceral mesodermal cells. In adult females, high levels of mav mRNA are present in late stage egg chambers.

We describe a novel Drosophila transcription unit, located in chromosome region 60A. It encodes a zinc finger protein that is expressed in distinct spatial and temporal patterns during embryogenesis. Its initial expression occurs in a stripe at the anterior and the posterior trunk boundary, respectively. The two stripes are activated and spatially controlled by gap-gene activities. The P-element of the enhancer trap line l(2)02970 is inserted in the 5′-region of the transcript and causes a ken and barbie ( ken ) phenotype, associated with malformation of male genital structures.

The 14-3-3 protein family is known to interact with various proteins involved in signaling pathways. We report here the expression pattern of the Drosophila 14-3-3 (d14-3-3epsilon) protein during embryonic development. In syncytial blastoderm when the nuclei divided rapidly, d14-3-3epsilon localized in the nuclei. During cellularization d14-3-3epsilon gradually became membrane-bound. During gastrulation, an enhanced staining in the perinuclear region was observed in various tissues. Co-labeling with dp-ERK which recognized the activated form of MAPK suggested that d14-3-3epsilon was expressed prior to MAPK activation. During neuronal differentiation, the d14-3-3epsilon protein remained at a high level in the neuronal cytoplasm.

We have examined the cell lineage of larval and imaginal precursors of the mesodermal anlage between 10% and 60% egg length (EL) by homotopic single-cell transplantations at the blastoderm stage. Clones in the larval somatic muscles and in the fat body were derived from transplantations everywhere between 10% and 60% EL along the ventral side of the embryo. Clones frequently overlap these tissues and can extend over a maximum of four segments in the larval somatic muscles or over two morphologically-distinct parts in the fat body. Clones in the gonadal mesoderm overlap with other mesodermal derivatives and exhibit different mitotic behaviour in the two sexes. We present a blastoderm fate map for the fat body, the larval somatic muscles and the gonadal mesoderm. Clones in the imaginal muscle precursors of the abdomen, as well as of the thorax, always show a common cell lineage with larval somatic muscles and partly with other mesodermal tissues. These clones of imaginal derivatives are always found within a single segment, while the overlapping clone parts in the larval somatic muscles can label up to three segments.

Nuclear protein kinases are believed to play important roles in regulating gene expression. We report here the identification and developmental expression of Dmnk (Drosophila maternal nuclear kinase), a Drosophila gene encoding a putative nuclear protein serine/threonine kinase with no apparent homology to previously identified protein kinases and located at 38B on the second chromosome. Dmnk mRNAs are transcribed in nurse cells and are subsequently localized in the anterior of oocytes during oogenesis, in a manner similar to several maternal transcripts regulating oogenesis and early embryogenesis. At early cleavage-stages Dmnk transcripts are transiently present throughout the embryo, but become restricted to the posterior pole and then to the newly-formed primordial germ cells (pole cells) by the blastoderm stage. The transcripts are sustained in the pole cells during gastrulation until they pass through the midgut pocket wall into the body cavity. Immunostaining with specific antibodies revealed that Dmnk proteins are localized to the nuclei in a speckled pattern. Dmnk proteins become detectable in both somatic and germ line cell nuclei upon their arrival at the periplasm of the syncytial embryo, but then disappear from the somatic cell nuclei. Consistent with mRNA expression, Dmnk proteins in pole cell nuclei are sustained during gastrulation. Taken together, Dmnk represents a novel class of nuclear protein kinases and the dynamic expression of Dmnk suggests a role in germ line establishment. The results are discussed in the light of recent findings concerning germ line establishment in Caenorhabditis and Drosophila.

Basic-helix-loop-helix (bHLH) transcription factors are involved in the control of many developmental processes in vertebrates and invertebrates. The HLH domain mediates formation of homo- or heterodimers. We have taken advantage of these dimerisation properties to identify a novel Drosophila HLH protein using the yeast two-hybrid system. Expression of bHLH54F at the blastoderm stage is restricted to a small subpopulation of mesodermal cells near the posterior pole. During germ band retraction these cells spread along the future midgut region. Later bHLH54F-expressing cells make up the longitudinal portion of the visceral musculature. Characterisation of this expression pattern demonstrates that precursors of the outer, longitudinal muscles of the midgut are distinct in origin and morphology from precursors of the inner, circular muscles.

Co-evolution between developmental regulatory elements is an important mechanism of evolution. This work compares the hunchback-bicoid interaction in the housefly Musca domestica with Drosophila melanogaster. The Musca HUNCHBACK protein is 66% conserved and partially rescues a hunchback mutant, yet the BICOID-dependent promoter (P2) of Musca hunchback is unexpectedly diverged from D. melanogaster. Introduced into D. melanogaster, this promoter drives a normal P2 pattern during the syncytial blastoderm stage but is expressed ectopically at the anterior pole of the embryo at later stages. We also report differences in the early expression of hunchback in Musca. We suggest that conservation of the morphogenetic function of bicoid in different sized embryos of higher diptera requires co-evolution of bicoid and its target binding sites.

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene.