Computational models for neurogenic gene expression in the Drosophila embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.     

Control and function of terminal gap gene activity in the posterior pole region of the Drosophila embryo.

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.     

Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg

 Genetically mosaic flies were constructed which lack a functional decapentaplegic (dpp) or wingless (wg) gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has previously been shown to be transcribed both ventrally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally – a result that was expected, given that wg is only expressed ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications confined to individual leg segments. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg-null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both dpp and wg are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene’s null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another. Received: 25 March 1996 / Accepted: 13 June 1996     

A regulatory code for neurogenic gene expression in the Drosophila embryo

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.     

Genetic analysis of the cellularization of the Drosophila embryo.

The synchronous cellularization of the Drosophila embryo at the blastoderm stage provides a unique system for studying the molecular mechanisms involved in cytokinesis, using genetical and biochemical approaches. The cellularization process requires the major components of the embryonic cytoskeleton that are deposited into the egg during oogenesis. Genetical analysis indicates that it requires also the products of additional maternally-acting genes, as well as that of a limited set of zygotically-acting genes. The cellularization defective phenotypes associated with small deficiencies uncovering these latter loci reveal specific steps within this complex process. The molecular analysis of these genes will ultimately provide meaningful insights into the normal process of cellularization. Among them, the serendipity alpha gene encodes a membrane-associated protein, which is exclusively accumulated during cellularization, and is required for the reorganization of the microfilaments as the onset of cellularization.     

Homeotic gene function in the muscles of Drosophila larvae

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 `abdominal' muscles and the gain of 11 `thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.     

The consequences of ubiquitous expression of the wingless gene in the Drosophila embryo.

The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.     

Quantitative genetic analysis of sleep in Drosophila melanogaster

Although intensively studied, the biological purpose of sleep is not known. To identify candidate genes affecting sleep, we assayed 136 isogenic P-element insertion lines of Drosophila melanogaster. Since sleep has been negatively correlated with energy reserves across taxa, we measured energy stores (whole-body protein, glycogen, and triglycerides) in these lines as well. Twenty-one insertions with known effects on physiology, development, and behavior affect 24-hr sleep time. Thirty-two candidate insertions significantly impact energy stores. Mutational genetic correlations among sleep parameters revealed that the genetic basis of the transition between sleep and waking states in males and females may be different. Furthermore, sleep bout number can be decoupled from waking activity in males, but not in females. Significant genetic correlations are present between sleep phenotypes and glycogen stores in males, while sleep phenotypes are correlated with triglycerides in females. Differences observed in male and female sleep behavior in flies may therefore be related to sex-specific differences in metabolic needs. Sleep thus emerges as a complex trait that exhibits extensive pleiotropy and sex specificity. The large mutational target that we observed implicates genes functioning in a variety of biological processes, suggesting that sleep may serve a number of different functions rather than a single purpose.     

The function of the neurogenic genes during epithelial development in the Drosophila embryo.

The complex embryonic phenotype of the six neurogenic mutations Notch, mastermind, big brain, Delta, Enhancer of split and neuralized was analyzed by using different antibodies and PlacZ markers, which allowed us to label most of the known embryonic tissues. Our results demonstrate that all of the neurogenic mutants show abnormalities in many different organs derived from all three germ layers. Defects caused by the neurogenic mutations in ectodermally derived tissues fell into two categories. First, all cell types that delaminate from the ectoderm (neuroblasts, sensory neurons, peripheral glia cells and oenocytes) are increased in number. Secondly, ectodermal tissues that in the wild type form epithelial structures lose their epithelial phenotype and dissociate (optic lobe, stomatogastric nervous system) or show significant differentiative abnormalities (trachea, Malpighian tubules and salivary gland). Abnormalities in tissues derived from the mesoderm were observed in all six neurogenic mutations. Most importantly, somatic myoblasts do not fuse and/or form an aberrant muscle pattern. Cardioblasts (which form the embryonic heart) are increased in number and show differentiative abnormalities; other mesodermal cell types (fat body, pericardial cells) are significantly decreased. The development of the endoderm (midgut rudiments) is disrupted in most of the neurogenic mutations (Notch, Delta, Enhancer of split and neuralized) during at least two stages. Defects occur as early as during gastrulation when the invaginating midgut rudiments prematurely lose their epithelial characteristics. Later, the transition of the midgut rudiments to form the midgut epithelium does not occur. In addition, the number of adult midgut precursor cells that segregate from the midgut rudiments is strongly increased. We propose that, at least in the ectodermally and endodermally derived tissues, neurogenic gene function is primarily involved in interactions among cells that need to acquire or to maintain an epithelial phenotype.     

A biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo

The article provides a biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo. Ventral furrow formation is the first large-scale morphogenetic movement in the fly embryo. It involves deformation of a uniform cellular monolayer formed following cellularisation, and has therefore long been used as a simple system in which to explore the role of mechanics in force generation. Here we use a quantitative framework to carry out a systematic perturbation analysis to determine the role of each of the active forces observed. The analysis confirms that ventral furrow invagination arises from a combination of apical constriction and apical-basal shortening forces in the mesoderm, together with a combination of ectodermal forces. We show that the mesodermal forces are crucial for invagination: the loss of apical constriction leads to a loss of the furrow, while the mesodermal radial shortening forces are the primary cause of the internalisation of the future mesoderm as the furrow rises. Ectodermal forces play a minor but significant role in furrow formation: without ectodermal forces the furrow is slower to form, does not close properly and has an aberrant morphology. Nevertheless, despite changes in the active mesodermal and ectodermal forces lead to changes in the timing and extent of furrow, invagination is eventually achieved in most cases, implying that the system is robust to perturbation and therefore over-determined.     

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.     

The Notch gene, adhesion, and developmental fate in the Drosophila embryo

The Notch locus in Drosophila encodes a transmembrane protein required for the determination of cell fate in ectodermal cells. When these cells are faced with a choice of two possible fates, Notch enables some of them to advance from the fate that represents a "default" state. The decision appears to involve both an interaction between cells and the presence of the Notch product on the surface of those advancing from the default state. The timing of gene action suggests that Notch plays a role in the stabilization of the choice of cell fate and that the Notch-mediated interaction occurs between presumptive epidermal cells. Several properties of the Notch product are characteristic of an adhesion molecule, and thus cell adhesion may play a role in the determination of cell fate.     

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.