Pattern formation in the Drosophila embryo: allocation of cells to parasegments by even-skipped and fushi tarazu

The first sign of metamerization in the Drosophila embryo is the striped expression of pair-rule genes such as fushi tarazu (ftz) and even-skipped (eve). Here we describe, at cellular resolution, the development of ftz and eve protein stripes in staged Drosophila embryos. They appear gradually, during the syncytial blastoderm stage and soon become asymmetric, the anterior margins of the stripes being sharply demarcated while the posterior borders are undefined. By the beginning of germ band elongation, the eve and ftz stripes have narrowed and become very intense at their anterior margins. The development of these stripes in hairy-, runt-, eve-, ftz- and engrailed- embryos is illustrated. In eve- embryos, the ftz stripes remain symmetric and lack sharp borders. Our results support the hypothesis (Lawrence et al. Nature 328, 440-442, 1987) that individual cells are allocated to parasegments with respect to the anterior margins of the eve and ftz stripes.     

even-skipped determines the dorsal growth of motor axons in Drosophila

Axon pathfinding and target choice are governed by cell type-specific responses to external cues. Here, we show that in the Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and that this expression is necessary and sufficient to direct motor axons into the dorsal muscle field. Previously, it was shown that motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the ventral muscle field. Thus, even-skipped complements the function of islet, and together these two genes constitute a bimodal switch regulating axonal growth and directing motor axons to ventral or to dorsal regions of the muscle field.     

Regulation of cell adhesion in the Drosophila embryo by phosphorylation of the Cadherin-Catenin-Complex

Cell-culture studies indicate that tyrosine phosphorylation of the cadherin-catenin-complex (CCC) is one of the post-translational mechanism regulating E-cadherin-mediated cell adhesion. In this investigation, controlled application of a tyrosine phosphatase inhibitor (orthovanadate) and tyrosine kinase inhibitor (tyrphostin) to early Drosophila embryos, followed by biochemical assays and phenotypic analysis, has been utilized to address the mechanism by which tyrosine phosphorylation regulates E-cadherin-mediated cell adhesion in vivo. Our data suggest that, in the Drosophila embryo, β-catenin (Drosophila homolog Armadillo) is the primary tyrosine-phosphorylated protein in the CCC. The increase in tyrosine phosphorylation correlates with a loss of epithelial integrity and adherens junctions in the ectoderm of early embryos. Late application of the phosphatase inhibitor does not have this effect, presumably because of the formation of septate junctions in late embryos. Co-immunoprecipitation assays have demonstrated that tyrosine hyper-phosphorylation does not cause the dissociation of Drosophila (D)E-cadherin and α-catenin or Armadillo, suggesting that abrogation in adhesion is most likely attributable to the detachment of actin-associated proteins from the CCC. Finally, although the Drosophila epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is linked to the CCC and shows genetic interactions with DE-cadherin, we find that a constitutively active Drosophila EGFR construct does not cause any detectable changes in the level of tyrosine phosphorylation of Armadillo or destabilization of the CCC. This work was supported by UCLA USPHS National Research Service Award GM07185 to F.W., and NIH Grant NS 29367 to V.H.     

Axis specification in the Drosophila embryo.

Three genetic hierarchies control cell-fate specification in largely distinct regions of the antero-posterior axis of the Drosophila embryo, whereas a single hierarchy specifies dorso-ventral cell fates. Molecular genetic analysis of these hierarchies is leading to increased understanding of the nature of the regulatory circuitry that controls regional cell-fate specification.     

An essential role of even-skipped for homeotic gene expression in the Drosophila visceral mesoderm.

We have analysed homeotic gene expression in the embryonic visceral mesoderm of segmentation mutants by antibody staining against Ultrabithorax, Antennapedia and Sex combs reduced protein. We found that even-skipped (eve) function is crucially required for homeotic gene expression, whereas most other segmentation mutations have only minor effects on position and/or width of the homeotic expression domains in this germ layer. Analysis of pair-rule double mutants indicates that complete loss of homeotic gene activity in the visceral mesoderm, as observed in amorphic eve mutants, correlates with loss of engrailed (en) expression in the epidermis and loss of segmentation. We suggest that the establishment of parasegment borders, a consequence of eve expression and witnessed by subsequent en expression, is a necessary precondition for homeotic gene expression in the visceral mesoderm.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

Regulation of Easter activity is required for shaping the Dorsal gradient in the Drosophila embryo

Dorsoventral polarity of the Drosophila embryo requires maternal sp?tzle-Toll signaling to establish a nuclear gradient of Dorsal protein. The shape of this gradient is altered in embryos produced by females carrying dominant alleles of easter (ea(D)). The easter gene encodes a serine protease that generates processed Sp?tzle, which is proposed to act as the Toll ligand. By examining the expression domains of the zygotic genes zen, sog, rho and twist, which are targets of nuclear Dorsal, we show that the slope of the Dorsal gradient is progressively flattened in stronger ea(D) alleles. In the wild-type embryo, activated Easter is found in a high M(r) complex called Ea-X, which is hypothesized to contain a protease inhibitor. In ea(D) embryo extracts, we detect an Easter form corresponding to the free catalytic domain, which is never observed in wild type. These mutant ea(D) proteins retain protease activity, as determined by the production of processed Sp?tzle both in the embryo and in cultured Drosophila cells. These experiments suggest that the ea(D) mutations interfere with inactivation of catalytic Easter, and imply that this negative regulation is essential for generating the wild-type shape of the Dorsal gradient.     

hairy mediates dominant repression in the Drosophila embryo.

hairy encodes a bHLH repressor that regulates several developmental processes in Drosophila, including embryonic segmentation and neurogenesis. Segmentation repressors such as Krüppel and knirps have been shown to function over short distances, less than 50-100 bp, to inhibit or quench closely linked upstream activators. This mode of repression permits multiple enhancers to work independently of one another within a modular promoter. Here, we employ a transgenic embryo assay to present evidence that hairy acts as a dominant repressor, which can function over long distances to block multiple enhancers. hairy is shown to repress a heterologous enhancer, the rhomboid NEE, when bound 1 kb from the nearest upstream activator. Moreover, the binding of hairy to a modified NEE leads to the repression of both the NEE and a distantly linked mesoderm-specific enhancer within a synthetic modular promoter. Additional evidence that hairy is distinct from previously characterized embryonic repressors stems from the analysis of the gypsy insulator DNA. This insulator selectively blocks the hairy repressor, but not the linked activators, within a modified NEE. We compare hairy with previously characterized repressors and discuss the consequences of short-range and long-range repression in development.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interactions of Krüppel with other terminal genes. The gap genes tailless and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

Making stripes in the Drosophila embryo

The striped pattern of expression of the Drosophila primary pair rule genes is controlled by independent regulatory units that give rise to individual stripes. The different stripes seem to respond in a concentration-dependent manner to the different combinations of maternal and gap protein gradients found along the anterior-posterior axis of the early embryo. Thus, the initial periodicity appears to be generated by putting together a series of nonperiodic events.     

Pattern formation in the Drosophila embryo

Three plausible hypotheses about developmental commitments in the Drosophila embryo propose that: (1) a micromosaic of localized determinants in the egg trigger somatic commitments; (2) monotonic anterior-posterior and dorsal-ventral gradients in the egg specify positions by a series of threshold values; (3) sequential subdivision of the early embryo into 'anterior' or 'posterior' 'middle' or 'end', 'dorsal' or 'ventral', 'odd' or 'even' compartmental domains encodes the somatic commitment in each region in a combinatorial epigenetic code. Evidence in favour of such a combinatorial code includes its capacity to account for major features of transdetermination and for many single and coordinated homoeotic transformations. In particular, both these metaplasias often cause transformations between ectodermal tissues such as antenna and genitalia, whose anlagen lie far apart on the blastoderm fate map. This phenomenon is not naturally explained by monotonic gradient models. In contrast, not only transformation between distant regions of the fate map, but also the observed geometries of compartmental boundaries on the wing, and probable ones in the early embryo, are naturally explained by reaction-diffusion models. These systems form a discrete succession of differently shaped monotonic and nonmonotonic eigenfunction gradient patterns of the same morphogens, as the tissue containing the chemical system changes in size and shape, or in other parameters. The successive mirror symmetries in non-monotonic gradients predict that distant regions of the embryo make similar developmental commitments, and also predict specific classes of pattern mutants forming mirror symmetric structures along the embryo on a variety of length scales. Finally, reaction diffusion systems spontaneously generate transverse gradients of the underlying chemicals when more than one eigenfunction is amplified at once, and therefore specify two-dimensional positional information within domains. Although it is attractive, no feature of the combinatorial code hypothesis is verified. Current data relating to whether the sequential formation of compartmental boundaries actually reflects the commitment of the two isolated 'polyclones' to alternative fates, whether any genes act continuously to maintain disc commitments, and whether homoeotic mutants actually 'switch' disc determined states, are assessed.