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Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

The neuroblast timer genes hunchback, Krüppel, nubbin and castor are expressed in temporal sequence in neural stem cells, and in corresponding spatial sequence along the Drosophila blastoderm. As canonical gap genes, hunchback and Krüppel play a crucial role in insect segmentation, but the roles of nubbin and castor in this process remain ambiguous. We have investigated the expression and functions of nubbin and castor during segmentation in the beetle Tribolium. We show that Tc-hunchback, Tc-Krüppel, Tc-nubbin and Tc-castor are expressed sequentially in the segment addition zone, and that Tc-nubbin regulates segment identity redundantly with two previously described gap/gap-like genes, Tc-giant and Tc-knirps. Simultaneous knockdown of Tc-nubbin, Tc-giant and Tc-knirps results in the formation of ectopic legs on abdominal segments. This homeotic transformation is caused by loss of abdominal Hox gene expression, likely due to expanded Tc-Krüppel expression. Our findings support the theory that the neuroblast timer series was co-opted for use in insect segment patterning, and contribute to our growing understanding of the evolution and function of the gap gene network outside of Drosophila., Summary:nubbin and the gap genes knirps and giant redundantly repress Krüppel expression during segmentation. Simultaneous knockdown of all three genes causes ectopic Krüppel expression and loss of abdominal segment identity.

Blastema formation, a hallmark of limb regeneration, requires proliferation and migration of progenitors to the amputation plane. Although blastema formation has been well described, the transcriptional programs that drive blastemal progenitors remain unknown. We transcriptionally profiled dividing and non-dividing cells in regenerating stump tissues, as well as the wound epidermis, during early axolotl limb regeneration. Our analysis revealed unique transcriptional signatures of early dividing cells and, unexpectedly, repression of several core developmental signaling pathways in early regenerating stump tissues. We further identify an immunomodulatory role for blastemal progenitors through interleukin 8 (IL-8), a highly expressed cytokine in subpopulations of early blastemal progenitors. Ectopic il-8 expression in non-regenerating limbs induced myeloid cell recruitment, while IL-8 knockdown resulted in defective myeloid cell retention during late wound healing, delaying regeneration. Furthermore, the il-8 receptor cxcr-1/2 was expressed in myeloid cells, and inhibition of CXCR-1/2 signaling during early stages of limb regeneration prevented regeneration. Altogether, our findings suggest that blastemal progenitors are active early mediators of immune support, and identify CXCR-1/2 signaling as an important immunomodulatory pathway during the initiation of regeneration.

The Drosophila blastoderm and the vertebrate neural tube are archetypal examples of morphogen-patterned tissues that create precise spatial patterns of different cell types. In both tissues, pattern formation is dependent on molecular gradients that emanate from opposite poles. Despite distinct evolutionary origins and differences in time scales, cell biology and molecular players, both tissues exhibit striking similarities in the regulatory systems that establish gene expression patterns that foreshadow the arrangement of cell types. First, signaling gradients establish initial conditions that polarize the tissue, but there is no strict correspondence between specific morphogen thresholds and boundary positions. Second, gradients initiate transcriptional networks that integrate broadly distributed activators and localized repressors to generate patterns of gene expression. Third, the correct positioning of boundaries depends on the temporal and spatial dynamics of the transcriptional networks. These similarities reveal design principles that are likely to be broadly applicable to morphogen-patterned tissues., Summary: This Review discusses similarities between the mechanisms patterning the Drosophila blastoderm and the vertebrate neural tube, suggesting a set of principles for morphogen-patterned tissues.

Underlying the plasma membrane of eukaryotic cells is an actin cortex that includes actin filaments and associated proteins. A special feature of all polarized and epithelial cells are cortical domains, each of which is characterized by specific sets of proteins. Typically, an epithelial cell contains apical, subapical, lateral and basal domains. The domain-specific protein sets contain evolutionarily conserved proteins, as well as cell-type-specific factors. Among the conserved proteins are, the Par proteins, Crumbs complex and the lateral proteins Scribbled and Discs large 1. Organization of the plasma membrane into cortical domains is dynamic and depends on cell type, differentiation and developmental stage. The dynamics of cortical organization is strikingly visible in early Drosophila embryos, which increase the number of distinct cortical domains from one, during the pre-blastoderm stage, to two in syncytial blastoderm embryos, before finally acquiring the four domains that are typical for epithelial cells during cellularization. In this Review, we will describe the dynamics of cortical organization in early Drosophila embryos and discuss the processes and mechanisms underlying cortical remodeling.

In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo. This is the first cellular resolution dataset of a genetically perturbed Drosophila embryo that captures all cells in 3D. We describe the technical developments required to build this atlas and how the method can be employed and extended by others. We also analyze this novel dataset to characterize the degree and timing of cell fate canalization in the segmentation network. We find that in two layers of this gene regulatory network, following depletion of bcd, individual cells rapidly canalize towards normal cell fates. This result supports the hypothesis that the segmentation network directly canalizes cell fate, rather than an alternative hypothesis whereby cells are initially mis-specified and later eliminated by apoptosis. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further computational modeling of canalization and gene regulation in this transcriptional network., Summary: Drosophila bicoid mutant embryos show severe patterning phenotypes, but individual cells retain wild-type fates - as judged by expression of key patterning genes at single-cell resolution.

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation., Summary: Early chick embryos share previously unappreciated features with anamniote embryos such as the timing of zygotic gene activation and yolk syncytial layer formation.

The transcription factor NF-κB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a Drosophila homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here, we use modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised dorsal levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, we suggest that it might be more prevalent than previously thought.

In order to gain insight into the impact of yolk increase on endoderm development, we have analyzed the mechanisms of endoderm formation in the catshark S. canicula, a species exhibiting telolecithal eggs and a distinct yolk sac. We show that in this species, endoderm markers are expressed in two distinct tissues, the deep mesenchyme, a mesenchymal population of deep blastomeres lying beneath the epithelial-like superficial layer, already specified at early blastula stages, and the involuting mesendoderm layer, which appears at the blastoderm posterior margin at the onset of gastrulation. Formation of the deep mesenchyme involves cell internalizations from the superficial layer prior to gastrulation, by a movement suggestive of ingressions. These cell movements were observed not only at the posterior margin, where massive internalizations take place prior to the start of involution, but also in the center of the blastoderm, where internalizations of single cells prevail. Like the adjacent involuting mesendoderm, the posterior deep mesenchyme expresses anterior mesendoderm markers under the control of Nodal/activin signaling. Comparisons across vertebrates support the conclusion that endoderm is specified in two distinct temporal phases in the catshark as in all major osteichthyan lineages, in line with an ancient origin of a biphasic mode of endoderm specification in gnathostomes. They also highlight unexpected similarities with amniotes, such as the occurrence of cell ingressions from the superficial layer prior to gastrulation. These similarities may correspond to homoplastic traits fixed separately in amniotes and chondrichthyans and related to the increase in egg yolk mass., This work was funded by Région Centre, Région Bretagne (EVOVERT grant number 049755; PEPTISAN project), National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d'Orléans and Université Pierre et Marie Curie. BGG benefited from a Région Bretagne fellowship.

Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved.

Transcription factors and microRNAs (miRNAs) are two important classes of trans-regulators in differential gene expression. Transcription factors occupy cis-regulatory motifs in DNA to activate or repress gene transcription, whereas miRNAs specifically pair with seed sites in target mRNAs to trigger mRNA decay or inhibit translation. Dynamic spatiotemporal expression patterns of transcription factors and miRNAs during development point to their stage- and tissue-specific functions. Recent studies have focused on miRNA functions during development; however, much remains to explore regarding how the expression of miRNAs is initiated and how dynamic miRNA expression patterns are achieved by transcriptional regulatory networks at different developmental stages. Here, we focused on the identification, regulation and function of miRNAs during the earliest stage of Drosophila development, when the maternal-to-zygotic transition (MZT) takes place. Eleven miRNA clusters comprise the first set of miRNAs activated in the blastoderm embryo. The transcriptional activator Zelda is required for their proper activation and regulation, and Zelda binding observed in genome-wide binding profiles is predictive of enhancer activity. In addition, other blastoderm transcription factors, comprising both activators and repressors, the activities of which are potentiated and coordinated by Zelda, contribute to the accurate temporal and spatial expression of these miRNAs, which are known to function in diverse developmental processes. Although previous genetic studies showed no early phenotypes upon loss of individual miRNAs, our analysis of the miR-1; miR-9a double mutant revealed defects in gastrulation, demonstrating the importance of co-activation of miRNAs by Zelda during the MZT.

In Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other arthropods, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. Recently, the cyclic nature of the pair-rule gene Tc-odd-skipped was demonstrated in the growth zone of Tribolium, indicating that a vertebrate-like segmentation clock is employed in the germband stage of its development. This suggests that two mechanisms might function in the same organism: a Drosophila-like mechanism in the blastoderm, and a vertebrate-like mechanism in the germband. Here, we show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, we show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clock-based segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Moreover, by tracking cells in live embryos and by analyzing mitotic profiles, we found that neither cell movement nor oriented cell division could explain the observed wave dynamics of Tc-eve. These results pose intriguing evolutionary questions, as Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms.

Summary The yolk syncytial layer (YSL) performs multiple critical roles during zebrafish development. However, little is known about the cellular and molecular mechanisms that underlie the formation of this important extraembryonic structure. Here, we demonstrate by timelapse confocal microscopy of a transgenic line expressing membrane-targeted GFP that the YSL forms as a result of the absence of cytokinesis between daughter nuclei at the tenth mitotic division and the regression of pre-existing marginal cell membranes, thus converting the former margin of the blastoderm into a syncytium. We show that disruption of components of the cytoskeleton induces the formation of an expanded YSL, and identify Rock1 as the regulator of cytoskeletal dynamics that lead to YSL formation. Our results suggest that the YSL forms as a result of controlled cytokinesis failure in the marginal blastomeres, and Rock1 function is necessary for this process to occur. Uncovering the cellular and molecular mechanisms underlying zebrafish YSL formation offers significant insight into syncytial development in other tissues as well as in pathological conditions.

Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin–syndecan–PIP2–Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development.

Integrin αV can form heterodimers with several β subunits to mediate cell-cell and cell-extracellular matrix interactions. During zebrafish gastrulation, αV is expressed maternally and zygotically. Here, we used a morpholino-mediated αV knockdown strategy to study αV function. Although αV morphants displayed vascular defects, they also exhibited left-right body asymmetry defects affecting multiple visceral organs. This was preceded by mislocalization of dorsal forerunner cells (DFCs) and malformation of the Kupffer's vesicle (KV) laterality organ. These defects were rescued with morpholino-resistant αV mRNA. Like αV, integrin β1b was expressed in DFCs, and β1b knockdown largely recapitulated the laterality phenotype of αV morphants. When tracked in real-time, individual DFCs of both morphants showed defects in DFC migration, preventing them from organizing into a KV of normal shape and size. Thus, we propose that αVβ1b mediates cellular interactions that are necessary for DFC clustering and movements necessary for Kupffer's vesicle formation, uncovering an early contribution of integrins to the regulation of vertebrate laterality.

In the short-germ beetle Tribolium castaneum, the head gap gene orthodenticle (Tc-otd) has been proposed to functionally substitute for bicoid, the anterior morphogen unique to higher dipterans. In this study we reanalyzed the function of Tc-otd. We obtained a similar range of cuticle phenotypes as in previously described RNAi experiments; however, we noticed unexpected effects on blastodermal cell fates. First, we found that Tc-otd is essential for dorsoventral patterning. RNAi depletion results in lateralized embryos, a fate map change that by itself can explain the observed loss of the anterior head, which is a ventral anlage in Tribolium. We find that this effect is due to diminished expression of short gastrulation (sog), a gene essential for establishment of the Decapentaplegic (Dpp) gradient in this species. Second, we found that gnathal segment primordia in Tc-otd RNAi embryos are shifted anteriorly but otherwise appear patterned normally. This anteroposterior (AP) fate map shift might largely be due to diminished zen-1 expression and is not responsible for the severe segmentation defects observed in some Tc-otd RNAi embryos. As neither Tc-sog nor Tc-zen-1 probably requires Otd gradient-mediated positional information, we posit that the blastoderm function of Tc-Otd depends on its initial homogeneous maternal expression and that this maternal factor does not provide significant positional information for Tribolium blastoderm embryos.

Drosophila undergoes a form of development termed long germ segmentation, where all segments are specified nearly simultaneously so that by the blastoderm stage, the entire body plan has been determined. This mode of segmentation is evolutionarily derived. Most insects undergo short or intermediate germ segmentation, where only anterior segments are specified early, and posterior segments are sequentially specified during germband elongation. These embryological differences imply that anterior and posterior segments might rely upon different molecular mechanisms. In Drosophila, embryos mutant for giant show a gap in the anterior as well fusions of several abdominal segments. In Tribolium, a short germ beetle, giant is required for segmental identity, but not formation, in gnathal segments and also for segmentation of the entire abdomen. This raises the possibility that giant might not act as a gap gene in short and intermediate germ insects. Oncopeltus fasciatus is an intermediate germ insect that is an outgroup to the clade containing Drosophila and Tribolium. We cloned the Oncopeltus homolog of giant and determined its expression and function during segmentation. We find that Oncopeltus giant is a canonical gap gene in the maxillary and labial segments and also plays a gap-like role in the first four abdominal segments. Our results suggest that giant was a bona fide gap gene in the ancestor of these insects with this role being lost in the lineage leading towards Tribolium. This highlights the conservation of anterior patterning and evolutionary plasticity of the genetic regulation controlling posterior segmentation, even in short and intermediate germ insects.

The formation of the mitotic spindle is controlled by the microtubule organizing activity of the centrosomes and by the effects of chromatin-associated Ran-GTP on the activities of spindle assembly factors. In this study we show that Mars, a Drosophila protein with sequence similarity to vertebrate hepatoma upregulated protein (HURP), is required for the attachment of the centrosome to the mitotic spindle. More than 80% of embryos derived from mars mutant females do not develop properly due to severe mitotic defects during the rapid nuclear divisions in early embryogenesis. Centrosomes frequently detach from spindles and from the nuclear envelope and nucleate astral microtubules in ectopic positions. Consistent with its function in spindle organization, Mars localizes to nuclei in interphase and associates with the mitotic spindle, in particular with the spindle poles, during mitosis. We propose that Mars is an important linker between the spindle and the centrosomes that is required for proper spindle organization during the rapid mitotic cycles in early embryogenesis.

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn(Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophiladespite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.

The Rel family transcription factor Dorsal patterns the dorsoventral axis of the Drosophila embryo by activating genes such as twistand snail and repressing genes such as decapentaplegic and zerknüllt. Dorsal represses transcription by recruiting the co-repressor Groucho. However, repression occurs only when Dorsal-binding sites are close to binding sites for other factors that also bind Groucho. The need for additional factors to assist Dorsal in repression may result from the intrinsically weak interaction between Dorsal and Groucho. To test this idea,we generated a Dorsal variant containing a high-affinity Groucho recruitment motif at its C terminus. As predicted, this variant functions as a dedicated repressor, silencing decapentaplegic and zerknülltwhile failing to activate twist and snail. We also converted Dorsal into a dedicated activator by replacing its weak Groucho-recruitment motif with heterologous activation domains. Although the dedicated activator alleles fail to repress decapentaplegic and zerknülltin the syncytial blastoderm embryo, they are able to pattern the dorsoventral axis. This indicates that dorsoventral patterning is not dependent upon Dorsal-mediated repression, reflecting the existence of redundant mechanisms to block Decapentaplegic signaling.

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here,we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. NasoniaHunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.

The pair-rule gene even-skipped is required for the initiation of metameric pattern in Drosophila. But Drosophila segmentation is evolutionarily derived and is not representative of most insects. Therefore, in order to shed light on the evolution of insect segmentation,homologs of the pair-rule gene even-skipped have been studied in several insect taxa. However, most of these studies have reported the expression eve but not its function. We report the isolation,expression and function of the homolog of Drosophila even-skippedfrom the intermediate germband insect Oncopeltus fasciatus. We find that in Oncopeltus, even-skipped striped expression initiates in a segmental and not pair-rule pattern. Weak RNAi suppression of Oncopeltus even-skipped shows no apparent pair-rule like phenotype, while stronger RNAi suppression shows deletion of nearly the entire body. These results suggest that in Oncopeltus, even-skipped is not acting as a pair-rule gene. In almost all insects, prior to its striped expression, even-skipped is expressed in a conserved broad gap-like domain but its function has been largely ignored. We find that this early broad domain is required for activation of the gap genes hunchback and Krüppel. Given the large RNAi deletion phenotype and its regulation of hunchback and Krüppel, even-skipped seems to act as an über-gap gene in Oncopeltus, indicating that it may have both upstream and downstream roles in segmentation.

Segmentation in long germband insects such as Drosophila occurs essentially simultaneously across the entire body. A cascade of segmentation genes patterns the embryo along its anterior-posterior axis via subdivision of the blastoderm. This is in contrast to short and intermediate germband modes of segmentation where the anterior segments are formed during the blastoderm stage and the remaining posterior segments arise at later stages from a posterior growth zone. The biphasic character of segment generation in short and intermediate germ insects implies that different formative mechanisms may be operating in blastoderm-derived and germband-derived segments. In Drosophila, the gap gene Krüppel is required for proper formation of the central portion of the embryo. This domain of Krüppel activity in Drosophila corresponds to a region that in short and intermediate germband insects spans both blastoderm and germband-derived segments. We have cloned the Krüppel homolog from the milkweed bug, Oncopeltus fasciatus (Hemiptera, Lygaeidae),an intermediate germband insect. We find that Oncopeltus Krüppelis expressed in a gap-like domain in the thorax during the blastoderm and germband stages of embryogenesis. In order to investigate the function of Krüppel in Oncopeltus segmentation, we generated knockdown phenotypes using RNAi. Loss of Krüppel activity in Oncopeltus results in a large gap phenotype, with loss of the mesothoracic through fourth abdominal segments. Additionally, we find that Krüppel is required to suppress both anterior and posterior Hox gene expression in the central portion of the germband. Our results show that Krüppel is required for both blastoderm-derived and germband-derived segments and indicate that Krüppel function is largely conserved in Oncopeltus and Drosophila despite their divergent embryogenesis.

A crucial step in generating the segmented body plan in Drosophilais establishing stripes of expression of several key segment-polarity genes,one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However,the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. We have used an ectopic expression system that allows for quantitative manipulation of expression levels to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1(slp1) as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz)transcription factor. By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. We furthermore find that Ftz modulates the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed(en). However, in the case of en the combination of Runt +Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation.

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos,the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.

The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented here, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, we show that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, we observe apoptosis in a third larval pharyngeal organ. Hence, our experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. Our conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis.

Transcriptional enhancers integrate positional and temporal information to regulate the complex expression of developmentally controlled genes. Current models suggest that enhancers act as computational devices, receiving multiple inputs from activators and repressors and resolving them into a single positive or a negative signal that is transmitted to the basal transcriptional machinery. We show that a simple, compact enhancer is capable of representing both repressed and activated states at the same time and in the same nucleus. This finding suggests that closely apposed factor binding sites, situated within compact cis-elements, can be independently interpreted by the transcriptional machinery, possibly through successive enhancer-promoter interactions. These results provide clear evidence that the computational functions usually ascribed to the enhancer itself are actually shared with the basal machinery. In contrast to the autonomous computer model of enhancer function, an information-display or `billboard' model of enhancer activity may better describe many developmentally regulated transcriptional enhancers.

Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Here we compare patterns of two related cellular responses, both signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Our data demonstrate that nuclear accumulation of the co-SMAD Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm,a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (SOG) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm.

Embryo segmentation has been studied extensively in the fruit fly,Drosophila. These studies have demonstrated that a mechanism acting with dual segment periodicity is required for correct patterning of the body plan in this insect, but the evolutionary origin of the mechanism, the pair-rule system, is unclear. We have examined the expression of the homologues of two Drosophila pair-rule genes, runt andpaired (Pax Group III), in segmenting embryos of the two-spotted spider mite (Tetranychus urticae Koch). Spider mites are chelicerates, a group of arthropods that diverged from the lineage leading toDrosophila at least 520 million years ago. In T. urticae,the Pax Group III gene Tu-pax3/7 was expressed during patterning of the prosoma, but not the opisthosoma, in a series of stripes which appear first in even numbered segments, and then in odd numbered segments. The miterunt homologue (Tu-run) in contrast was expressed early in a circular domains that resolved into a segmental pattern. The expression patterns of both of these genes also indicated they are regulated very differently from their Drosophila homologues. The expression pattern of Tu-pax3/7 lends support to the possibility that a pair-rule patterning mechanism is active in the segmentation pathways of chelicerates.

During segmentation of the Drosophila embryo, even skipped is required to activate engrailed stripes and to organize odd-numbered parasegments. A 16 kb transgene containing the even skipped coding region can rescue normal engrailed expression, as well as all other aspects of segmentation, in even skipped null mutants. To better understand its mechanism of action, we functionally dissected the Even-skipped protein in the context of this transgene. We found that Even-skipped utilizes two repressor domains to carry out its function. Each of these domains can function autonomously in embryos when fused with the Gal4 DNA-binding domain. A chimeric protein consisting only of the Engrailed repressor domain and the Even-skipped homeodomain, but not the homeodomain alone, was able to restore function, indicating that the repression of target genes is sufficient for even skipped function at the blastoderm stage, while the homeodomain is sufficient to recognize those target genes. When Drosophila Even skipped was replaced by its homologs from other species, including a mouse homolog, they could provide substantial function, indicating that these proteins can recognize similar target sites and also provide repressor activity. Using this rescue system, we show that broad, early even skipped stripes are sufficient for activation of both odd- and even-numbered engrailed stripes. Furthermore, these ‘unrefined’ stripes organize odd-numbered parasegments in a dose-dependent manner, while the refined, late stripes, which coincide cell-for-cell with parasegment boundaries, are required to ensure the stability of the boundaries.

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), we followed signaling directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp.

In the long-germ insect Drosophila melanogaster dorsoventral polarity is induced by localized Toll-receptor activation which leads to the formation of a nuclear gradient of the rel/ NF-κB protein Dorsal. Peak levels of nuclear Dorsal are found in a ventral stripe spanning the entire length of the blastoderm embryo allowing all segments and their dorsoventral subdivisions to be synchronously specified before gastrulation. We show that a nuclear Dorsal protein gradient of similar anteroposterior extension exists in the short-germ beetle, Tribolium castaneum, which forms most segments from a posterior growth zone after gastrulation. In contrast to Drosophila, (i) nuclear accumulation is first uniform and then becomes progressively restricted to a narrow ventral stripe, (ii) gradient refinement is accompanied by changes in the zygotic expression of the Tribolium Toll-receptor suggesting feedback regulation and, (iii) the gradient only transiently overlaps with the expression of a potential target, the Tribolium twist homolog, and does not repress Tribolium decapentaplegic. No nuclear Dorsal is seen in the cells of the growth zone of Tribolium embryos, indicating that here dorsoventral patterning occurs by a different mechanism. However, Dorsal is up-regulated and transiently forms a nuclear gradient in the serosa, a protective extraembryonic cell layer ultimately covering the whole embryo.

Formation of the three germ layers requires a series of inductive events during early embryogenesis. Studies in zebrafish indicate that the source of these inductive signals may be the extra-embryonic yolk syncytial layer (YSL). The characterization of genes encoding the nodal-related factor, Squint, and homeodomain protein, Bozozok, both of which are expressed in the YSL, suggested that the YSL has a role in mesendoderm induction. However, these genes, and a second nodal-related factor, cyclops, are also expressed in the overlying marginal blastomeres, raising the possibility that the marginal blastomeres can induce mesendodermal genes independently of the YSL. We have developed a novel technique to study signaling from the YSL in which we specifically eliminate RNAs in the YSL, thus addressing the in vivo requirement of RNA-derived signals from this region in mesendoderm induction. We show that injection of RNase into the yolk cell after the 1K cell stage (3 hours) effectively eliminates YSL transcripts without affecting ubiquitously expressed genes in the blastoderm. We also present data that indicate the stability of existing proteins in the YSL is unaffected by RNase injection. Using this technique, we show that RNA in the YSL is required for the formation of ventrolateral mesendoderm and induction of the nodal-related genes in the ventrolateral marginal blastomeres, revealing the presence of an unidentified inducing signal released from the YSL. We also demonstrate that the dorsal mesoderm can be induced independently of signals from the YSL and present evidence that this is due to the stabilization of β-catenin in the dorsal marginal blastomeres. Our results demonstrate that germ layer formation and patterning in zebrafish uses a combination of YSL-dependent and-independent inductive events.

Segmentation of a vertebrate embryo begins with the subdivision of the paraxial mesoderm into somites through a not-well-understood process. Recent studies provided evidence that the Notch-Delta and the FGFR (fibroblast growth factor receptor) signalling pathways are required for segmentation. In addition, the Mesp family of bHLH transcription factors have been implicated in establishing a segmental prepattern in the presomitic mesoderm. In this study, we have characterized zebrafish mesp-a and mesp-b genes that are closely related to Mesp family genes in other vertebrates. During gastrulation, only mesp-a is expressed in the paraxial mesoderm at the blastoderm margin. During the segmentation period, both genes are segmentally expressed in one to three stripes in the anterior parts of somite primordia. In fused somites (fss) embryos, in which all early somite boundary formation is blocked, initial mesp-a expression at the gastrula stage remains intact, but the expression of mesp-a and mesp-b is not detected during the segmentation period. This suggests that these genes are downstream targets of fss at the segmentation stage. Comparison with her1 expression (Müller, M., von Weizsäcker, E. and Campos-Ortega, J. A. (1996) Development 122, 2071-2078) suggests that, like her1, mesp genes are not expressed in primordia of the first several somites. Furthermore, we found that zebrafish her1 expression oscillates in the presomitic mesoderm. The her1 stripe, which first appears in the tailbud region, moves in a caudal to rostral direction, and it finally overlaps the most rostral mesp stripe. Thus, in the trunk region, both her1 and mesp transcripts are detected in every somite primordium posterior to the forming somites. Ectopic expression of Mesp-b in embryos causes a loss of the posterior identity within the somite primordium, leading to a segmentation defect. These embryos show a reduction in expression of the posterior genes, myoD and notch5, with uniform expression of the anterior genes, FGFR1, papc and notch6. These observations suggest that zebrafish mesp genes are involved in anteroposterior specification within the presumptive somites, by regulating the essential signalling pathways mediated by Notch-Delta and FGFR.

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. In this paper, we report the phenotypic analysis of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; it therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent. After gastrulation, positive autoregulation of col is limited to cells of anterior PS0. Conversely, heat-pulse induced ubiquitous expression of Col leads to disruption of the head skeleton. Together, these results indicate that col is required for establishment of the PS(-1)/PS0 parasegmental border and formation of the intercalary segment. Our data support neither a simple combinatorial model for segmental patterning of the head nor a direct activation of segment polarity gene expression by head-gap genes, but rather argue for the existence of parasegment-specific second order regulators acting in the head, at a level similar to that of pair-rule genes in the trunk.

Studies in Xenopus laevis and zebrafish suggest a key role for β-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear β-catenin demarcates the dorsalizing centers. We have asked whether β-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of β-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear β-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear β-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of β-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, β-catenin acts in the chick in a manner similar to that in fish and amphibia.

The selector homeoproteins are a highly conserved group of transcription factors found throughout the Eumetazoa. Previously, the Drosophila selector homeoproteins Eve and Ftz were shown to bind with similar specificities to all genes tested, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Here, we demonstrate that the expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve. Suspecting that the selector homeoproteins may affect many more genes than previously thought, we have characterized the expression of randomly selected genes at different stages of embryogenesis. At cellular blastoderm, 25-50% of genes whose transcription can be monitored are regulated by both Eve and Ftz. In late embryogenesis, 87% of genes are directly or indirectly controlled by most or all selector homeoproteins. We argue that this broad control of gene expression is essential to coordinate morphogenesis. Our results raise the possibility that each selector homeoprotein may directly regulate the expression of most genes.

During Drosophila gastrulation, mesodermal precursors are brought into the interior of the embryo by formation of the ventral furrow. The first steps of ventral furrow formation involve a flattening of the apical surface of the presumptive mesodermal cells and a constriction of their apical diameters. In embryos mutant for folded gastrulation (fog), these cell shape changes occur but the timing and synchrony of the constrictions are abnormal. A similar phenotype is seen in a maternal effect mutant, concertina (cta). fog encodes a putative secreted protein whereas cta encodes an α-subunit of a heterotrimeric G protein. We have proposed that localized expression of the fog signaling protein induces apical constriction by interacting with a receptor whose downstream cellular effects are mediated by the cta Gα protein. In order to test this model, we have ectopically expressed fog at the blastoderm stage using an inducible promoter. In addition, we have examined the constitutive activation of cta protein by blocking GTP hydrolysis using both in vitro synthesized mutant alleles and cholera toxin treatment. Activation of the fog/cta pathway by any of these procedures results in ectopic cell shape changes in the gastrula. Uniform fog expression rescues the gastrulation defects of fog null embryos but not cta mutant embryos, arguing that cta functions downstream of fog expression. The normal location of the ventral furrow in embryos with uniformly expressed fog suggests the existence of a fog-independent pathway determining mesoderm-specific cell behaviors and invagination. Epistasis experiments indicate that this pathway requires snail but not twist expression.

tailless encodes a transcription factor expressed in multiple domains in the developing embryo. Early and transient expression at the posterior pole is required to establish a domain from which the eighth abdominal segment, telson and posterior gut arise. Just a few nuclear cycles later, a brain-specific domain is initiated at the anterior; expression in this domain is maintained with complex modulations throughout embryogenesis. Expression of tailless in this domain is required to establish the most anterior region of the brain. To understand the function and regulation of these different domains of expression, we provide a detailed description of tailless expression in brain neuroblasts and show that this expression is not detectably regulated by the head gap genes buttonhead or orthodenticle, by the proneural gene lethal of scuteor by taillessitself. We show that approximately 6 kb of sequenced upstream regulatory DNA can drive lacZ expression in a pattern that mimics the full tailless embryonic expression pattern. Within this sequence we identify multiple modules responsible for different aspects of thetaillesspattern. In addition to identifying additional torso response elements that mediate early blastoderm polar expression, we show that the complex brain expression pattern is driven by a combination of modules; thus expression at a low level through-out the brain and at a high level in the dorsal medial portion of the brain and in the optic lobe, as well as neuro-blast-specific repression are mediated by different DNA regions.

14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as they are ubiquitously present in cells and found to associate with Raf-1 in vivo regardless of its activation state. We have analyzed the function of the Drosophila 14-3-3 gene leonardo (leo) in the Torso (Tor) receptor tyrosine kinase (RTK) pathway. In the syncytial blastoderm embryo, acti-vation of Tor triggers the Ras/Raf/MEK pathway that controls the transcription of tailless (tll). We find that, in the absence of Tor, overexpression of leo is sufficient to activate tll expression. The effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently identi-fied essential components of Drosophila RTK signaling pathways. Tor signaling is impaired in embryos derived from females lacking maternal expression of leo. We propose that binding to 14-3-3 by Raf is necessary but not sufficient for the activation of Raf and that overexpressed Drosophila 14-3-3 requires Ras1 to activate D-Raf.

In Drosophila intracellular gradients establish the pattern of segmentation by controlling gene expression during a critical syncytial stage, prior to cellularization. To investigate whether a similar mechanism may be exploited by other insects, we examined the timing of cellularization with respect to blastoderm formation in an insect with extreme short-germ development, the African desert locust, Schistocerca gregaria. Using light and electron microscopic techniques, we show that the islands of cytoplasm surrounding cleavage nuclei are largely isolated from their neighbours, allowing cleavage to proceed asynchronously. Within a short time of their arrival at the surface and prior to blastoderm formation, nuclei become surrounded by complete cell membranes that block the free uptake of dye (10,000 kDa) from the yolk. Our results imply that the formation of the blastoderm disc involves the aggregation of cells at the posterior pole of the egg and not the migration of nuclei within a syncytial cytoplasm. These findings suggest that the primary cleavage syncytium does not play the same role in patterning the locust embryo as it does in Drosophila. However, we do identify a syncytial nuclear layer that underlies the forming blastoderm and remains in continuity with the yolk.

The Drosophila gap-like segmentation genes orthodenticle, empty spiracles and buttonhead (btd) are expressed and required in overlapping domains in the head region of the blastoderm stage embryo. Their expression domains correspond to two or three segment anlagen that fail to develop in each mutant. It has been proposed that these overlapping expression domains mediate head metamerization and could generate a combinatorial code to specify segment identity. To test this model, we developed a system for targeted gene expression in the early embryo, based on region specific promoters and the flp-out system. Misex-pression of btd in the anterior half of the blastoderm embryo directed by the hunchback proximal promoter rescues the btd mutant head phenotype to wild-type. This indicates that, while btd activity is required for the formation of specific head segments, its ectopic expression does not disturb head development. We conclude that the spatial limits of btd expression are not instructive for metamerization of the head region and that btd activity does not contribute to a combinatorial code for specification of segment identity.

During Drosophila embryogenesis, each segment is subdivided into an anterior and a posterior compartment through the action of the engrailed gene. Compartmental boundaries bisect imaginal disc primordia which give rise to adult appendages. In early larval development, a short-range Hedgehog signal originating from the posterior compartment of the imaginal wing disc activates expression of genes including decapentaplegic (dpp) in a stripe running along the anterior-posterior compartment boundary. Secreted Dpp emanating from the A/P boundary of wing discs then acts as a secondary signal to organize the wing over large distances. The transcription factor encoded by spalt major (salm) gene, which is expressed in a broad wedge centered over the dpp stripe, is one target of Dpp signaling. In this manuscript, we show that the anterior edge of the salm expression domain abuts a narrow stripe of rhomboid (rho)-expressing cells corresponding to the L2 longitudinal vein primordium. hh mis-expression along the anterior wing margin induces a surrounding domain of salm expression, the anterior edge of which abuts a displaced rho L2 stripe. salm plays a key role in defining the position of the L2 vein since loss of salm function in mosaic patches induces the formation of ectopic L2 branches, which comprise salm− cells running along clone borders where salm− cells confront salm+ cells. These data suggest that salm determines the position of the L2 vein primordium by activating rho expression in neighboring cells through a locally non-autonomous mechanism. rho then functions to initiate and maintain vein differentiation. We discuss how these data provide the final link connecting the formation of a linear adult structure to the establishment of a boundary by the maternal Bicoid morphogen gradient in the blastoderm embryo.

The even-skipped (eve) pair-rule gene plays a key role in the establishment of the anterior-posterior segmental pattern of the Drosophila embryo. The continuously changing pattern of eve expression can be resolved into two phases. Early expression consists of seven broad stripes in the blastoderm embryo, while late expression, which occurs after cellularization, consists of narrow stripes with sharp anterior borders that coincide with the odd-numbered parasegment boundaries. Previous studies have shown that these two phases are controlled by separate classes of cis elements in the eve promoter. Early stripes are expressed by multiple stripe-specific elements under the control of maternal-effect genes and gap genes, while late stripes are expressed by a single regulatory element, the ‘late element’, under the control of pair-rule genes including eve itself. We report here that paired (prd), a pair-rule gene which had been considered to be below eve in the regulatory hierarchy of pair-rule genes, in fact plays a critical role in the regulation of late eve expression. Transgenic analysis shows that this regulation is largely mediated by an evolutionarily conserved sequence within the late element termed PTE (Paired Target Element). In vitro analysis shows that the Prd protein binds strongly to this sequence. Interestingly, PTE contains juxtaposed binding sites for the two DNAbinding domains of the Prd protein, the paired domain and the homeodomain. Mutagenesis of either binding site leads to significant reduction in the activity of the late element, indicating that both DNA-binding domains in the Paired protein are required for regulation.

In Drosophila equalization of the amounts of gene products produced by X-linked genes in the two sexes is achieved by hypertranscription of the single male X chromosome. This process, dosage compensation, is controlled by a set of male-specific lethal (msl) genes, that appear to act at the level of chromatin structure. The properties of the MSL proteins have been extensively studied in the polytene salivary gland chromosomes where they bind to the same set of sites along the male X chromosome in a co-dependent manner. Here we report experiments that show that the MSL proteins first associate with the male X chromosome as early as blastoderm stage, slightly earlier than the histone H4 isoform acetylated at lysine 16 is detected on the X chromosome. MSL binding to the male X chromosome is observed in all somatic tissues of embryos and larvae. Binding of the MSLs to the X chromosome is also interdependent in male embryos and prevented in female embryos by the expression of Sex-lethal (Sxl). A delayed onset of binding of the MSLs in male progeny of homozygous mutant msl-1 or mle mothers coupled with the previous finding that such males have an earlier lethal phase supports the idea that msl-mediated dosage compensation begins early in embryogenesis. Other results show that the maleless (MLE) protein on embryo and larval chromosomes differs in its reactivity with antibodies; the functional significance of this finding remains to be explored.

The identification and analysis of genes controlling segmentation in Drosophila melanogaster has opened the way for understanding similarities and differences in mechanisms of segmentation among the insects. Homologues of Drosophila segmentation genes have been cloned and their expression patterns have been analyzed in a variety of insects, revealing that the patterns of expression of many genes are conserved. Conserved expression patterns do not, however, necessarily reflect conserved gene function. To address gene function, we have conducted a screen for mutations that alter embryonic patterning of the beetle, Tribolium castaneum. One of the mutations isolated, godzilla, affects early steps in the segmentation process in the whole animal, like Drosophila pair-rule mutants. Another mutation, jaws, is novel: it caused both a dramatic homeotic transformation in the thorax and first abdominal segment as well as a deletion of most of the segments of the abdomen. In Tribolium and other intermediated germ band insects, the anterior segments of the embryo are determined in the syncytium of the blastoderm, whereas the abdominal segments proliferated in the cellular environment. Both the godzilla and jaws mutations affect segments that are formed in the syncytium differently from those that are formed after cellularization. These regionally specific phenotypes may reflect the different patterning mechanisms that must be employed by the anterior and posterior regions of an intermediated germ insect.

We have analysed the developmental defects in Drosophila embryos lacking a gelsolin-related protein encoded by the gene flightless I. Such embryos have previously been reported to gastrulate abnormally. We now show that the most dramatic defects are seen earlier, in actin-dependent events during cellularisation of the syncytial blastoderm, a process with similarities to cytokinesis. The blastoderm nuclei migrate to the periphery of the egg normally but lose their precise cortical positioning during cellularisation. Cleavage membranes are initially formed, but invaginate irregularly and often fail to close at the basal end of the newly formed cells. The association of actin with the cellularisation membranes is irregular, suggesting a role for flightless I in the delivery of actin to the actin network, or in its stabilisation.

We describe our analysis of primordial germ cell migration in Drosophila wild-type and mutant embryos using high resolution microscopy and primary culture in vitro. During migratory events the germ cells form transient interactions with each other and surrounding somatic cells. Both in vivo and in vitro they extend pseudopodia and the accompanying changes in the cytoskeleton suggest that actin polymerization drives these movements. These cellular events occur from the end of the blastoderm stage and are regulated by environmental cues. We show that the vital transepithelial migration allowing exit from the gut primordium and passage into the interior of the embryo is facilitated by changes in the structure of this epithelium. Migrating germ cells extend processes in different directions. This phenomenon also occurs in primary culture where the cells move in an unoriented fashion at substratum concentration-dependent rates. In vivo this migration is oriented leading germ cells to the gonadal mesoderm. We suggest that this guidance involves stabilization of states of an intrinsic cellular oscillator resulting in cell polarization and oriented movement.

The zebrafish is an excellent vertebrate model for the study of the cellular interactions underlying the patterning and the morphogenesis of the nervous system. Here, we report regional fate maps of the zebrafish anterior nervous system at two key stages of neural development: the beginning (6 hours) and the end (10 hours) of gastrulation. Early in gastrulation, we find that the presumptive neurectoderm displays a predictable organization that reflects the future anteroposterior and dorsoventral order of the central nervous system. The precursors of the major brain subdivisions (forebrain, midbrain, hindbrain, neural retina) occupy discernible, though overlapping, domains within the dorsal blastoderm at 6 hours. As gastrulation proceeds, these domains are rearranged such that the basic order of the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors is refined and becomes aligned with the primary axes of the embryo. Time-lapse video microscopy shows that the rearrangement of blastoderm cells during gastrulation is highly ordered. Cells near the dorsal midline at 6 hours, primarily forebrain progenitors, display anterior-directed migration. Cells more laterally positioned, corresponding to midbrain and hindbrain progenitors, converge at the midline prior to anteriorward migration. These results demonstrate a predictable order in the presumptive neurectoderm, suggesting that patterning interactions may be well underway by early gastrulation. The fate maps provide the basis for further analyses of the specification, induction and patterning of the anterior nervous system, as well as for the interpretation of mutant phenotypes and gene-expression patterns.