Programming the Drosophila embryo

A critical step in understanding the mechanisms of development is in defining the steps at the molecular, cellular, and organismal levels in the developmental program for a given organism-so that given the egg one can predict not only how the embryo will develop but also how that embryo evolved from its ancestors. Using methods employed by chemists and engineers in modeling hierarchical systems, I have integrated current theory and experiment into a calculational method that can model early Drosophila embryogenesis on a personal computer. This quantitative calculation tool is simple enough to be useful for experimentalists in designing experiments yet detailed enough for theoreticians to derive new insights on the evolution of developmental genetic networks. By integrating the strengths of theoretical and experimental methods into a single engineering model that can compute the cascade of genetic networks in a real organism, I provide a new calculational tool that can apply current theory to current experimental data to study the evolution of developmental programs.     

Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin

A systematic approach to the clinical history, physical, and arthroscopic examination of patellofemoral disorders will lead to improved diagnostic accuracy and clinical treatment success. We review important aspects of physical and arthroscopic examination of patellofemoral disorders Basic and advanced physical examination techniques are presented, and their clinical significance is reviewed. Arthroscopic examination of the patellofemoral joint is used as an adjunct to physical examination to evaluate chondral lesions of the patella and femoral sulcus and to visualize patella tracking. Techniques to assess patellar tracking and the integrity of patellar restraints and to grade chondral lesions are outlined. Utilization of these techniques will improve clinical studies on the treatment of patellofemoral disorders.     

Ultrastructure and blood-nerve barrier of chordotonal organs in the Drosophila embryo

Chordotonal organs of Drosophila embryos have become models for studies of developmental biology and molecular genetics due to their consistent segmental placement and mutability. Our first goal was to find the origin and anatomical correlate of the blood-nerve barrier of this PNS proprioreceptor in wild type embryos. The concept of a blood-nerve barrier for the PNS of the Drosophila embryo is new, and the present data are the first in this regard. A second goal was to reveal the ultrastructure of these four-celled stretch receptors, focusing particularly on the 'core' of this organ: the bipolar neuron enclosed by a scolopale cell. These latter data have resulted in a graphic reconstruction of the chordotonal organ which reveals how the four consistent cells fit together. At Stage 13 we first observed a clearly recognizable scolopale cell with an enclosed neuron. Surprisingly, an operative blood-nerve barrier, comprised of occlusive pleated-sheet septate junctions, exists at this relatively early stage. A blood-brain barrier is not yet functioning in the CNS during this same stage, as the perineurium is not present until Stage 17. Cross-sectional views of a more mature chordotonal organ show that the neuron's inner segment has a 'tongue-in-groove' formation which fits the dendrite into the scolopale cell. Other newly discovered fine structural features are: hemidesmosomes linking individual scolopale rod bundles to the inner dendrite, and a cap cell matrix bonding with the tip of the ciliary dendrite. Functional aspects of these findings are discussed.     

The Drosophila EGF receptor controls the formation and specification of neuroblasts along the dorsal-ventral axis of the Drosophila embryo

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage it produces. Recent experiments indicate that the segmentation genes specify neuroblast identity along the AP axis. However, little is known as to the control of neuroblast identity along the DV axis. Here, I show that the Drosophila EGF receptor (encoded by the DER gene) promotes the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Specifically, I use molecular markers that identify particular neuroectodermal domains, all neuroblasts or individual neuroblasts, to show that in DER mutant embryos (1) intermediate column neuroblasts do not form, (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Furthermore, I show that active DER signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. In addition, I demonstrate that the concomitant loss of rhomboid and vein yield CNS phenotypes indistinguishable from DER mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that DER plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS.     

Regulation of a dpp target gene in the Drosophila embryo

EnvZ is an inner membrane protein present in Escherichia coli that is important for osmosensing and required for porin gene regulation. EnvZ is phosphorylated by intracellular ATP, and EnvZ-P phosphorylates OmpR, which then binds to the porin promoters to regulate their expression. An overexpressed, truncated form of the enzyme, EnvZ115, was used to characterize the kinase reaction in vitro. Using a filter binding assay, we report the first direct measurements of the kinase activity, including the apparent affinity for ATP of 200 microM. The phosphorylation reaction is dependent on MgCl2, and the phosphoenzyme has the expected stability of a phosphohistidine; i.e., it is stable in base and less stable in acid at room temperature. The addition of OmpR and ATP to solutions containing EnvZ resulted in an OmpR-stimulated, EnvZ-dependent ATPase activity that was not vanadate-sensitive. The in vivo kinase activity of EnvZ and two mutants that were deficient in porin expression were studied using an immune complex kinase reaction. Interestingly, a mutation located in the periplasmic domain of EnvZ exhibited kinase activity that was identical to that of the wild-type enzyme, while a mutation located close to the phosphorylation site showed a significant decrease in both kinase and phosphotransferase activities. These data provide support for models of EnvZ consisting of separate sensing and kinase domains.     

Ecdysone signaling cascade and regulation of Drosophila metamorphosis

Pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) regulate diverse biological responses during the life history of insects. Studies of the fruit fly, Drosophila melanogaster, have provided significant insights into the mechanisms underlying ecdysone mediated regulation of development. During the dramatic metamorphosis of Drosophila, ecdysone induces the histolysis of nearly all of the larval tissues and differentiation and morphogenesis of the structures composing the adult fly. These changes are mediated by a genetic signaling cascade that was first recognized as puffs in the giant polytene chromosomes of the salivary gland. This genetic regulatory cascade is composed of early and late genes that are intricately coordinated by changes in hormone titer. Early genes encode regulatory proteins that are involved in the proper regulation of late genes, which are thought to play a more direct role in development. The regulation and function of these genes is discussed in the context of the cell- and tissue-specific changes required for the reorganization of a larva to form an adult fly.     

Recruitment of new sites of synaptic transmission during the cAMP-dependent late phase of LTP at CA3-CA1 synapses in the hippocampus

Long-term potentiation at CA3-CA1 hippocampal synapses exhibits an early phase and a late phase, which can be distinguished by their underlying molecular mechanisms. Unlike the early phase, the late phase is dependent on both cAMP and protein synthesis. Quantal analysis of unitary synaptic transmission between a single presynaptic CA3 neuron and a single postsynaptic CA1 neuron suggests that, under certain conditions, the early phase of LTP involves an increase in the probability of release of a single quantum of transmitter from a single presynaptic release site, with no change in the number of quanta that are released or in postsynaptic sensitivity to transmitter. Here, we show that the cAMP-induced late phase of LTP involves an increase in the number of quanta released in response to a single presynaptic action potential, possibly due to an increase in the number of sites of synaptic transmission between a single CA3 and a single CA1 neuron.     

The cell cycle program in germ cells of the Drosophila embryo

Activation of metabotropic glutamate receptors (mGluRs) with 1-aminocyclopentane-1S,3R-dicarboxylic acid 20 min prior to tetanus facilitates, or "primes," subsequent induction of long-term potentiation (LTP; Cohen and Abraham, J Neurophysiol 1996;76:953-962). In the present study, we investigated the receptor specificity and associated second messenger pathways involved in the mGluR priming effect by using field potentials recorded from area CA1 of rat hippocampal slices. In controls, mild theta-burst or high-frequency (100 Hz) stimulation induced 16% and 21% LTP, respectively. A 10-min application of the group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) caused a transient depression of synaptic responses but a significant enhancement of subsequent LTP for both tetanus protocols (45% and 41% LTP, respectively). Maximal LTP, induced by stronger tetanization protocols, was not enhanced by DHPG, nor was mild LTP facilitated by post-tetanic application of DHPG. Priming with agonists selective for group II or III mGluRs had no effect on LTP. The mGluR antagonists L-2-amino-3-phosphonopropionic acid and 1-aminoindan-1,5-dicarboxylic acid inhibited the LTP facilitatory effect of DHPG but not the transient response depression, whereas alpha-methyl-4-carboxyphenylglycine produced the opposite effects. Priming with N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid did not facilitate LTP induction. Prior activation of muscarinic acetylcholine receptors produced at best a weak priming effect. Inhibition of phospholipase C by U-73122 completely abolished the priming of LTP by DHPG. We conclude that mGluR priming of LTP results from biochemical cascades triggered by activation of phospholipase C coupled to group I mGluRs.     

A photographic study of development in the living embryo of Drosophila melanogaster

The changes which can be seen occurring during the development of a living embryo of Drosophila melanogaster are described in detail, and represented photographically as a series of developmental stages. This provides an easy, but accurate technique for selecting eggs at precise developmental stages for experiments.     

Different core promoters possess distinct regulatory activities in the Drosophila embryo

There are numerous examples of shared enhancers interacting with just a subset of target promoters. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. Here, we employ a transgenic embryo assay to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer from the Bithorax complex (BX-C) preferentially activate TATA-containing promoters when challenged with linked TATA-less promoters. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between these two classes of promoters. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters. These results provide in vivo evidence that different core promoters possess distinct regulatory activities. We discuss the possibility that an invariant TFIID complex can adopt different conformations on the core promoter.     

Differential regulation of gastrulation and neuroectodermal gene expression by Snail in the Drosophila embryo

The initiation of mesoderm differentiation in the Drosophila embryo requires the gene products of twist and snail. In either mutant, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutant is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such possibility, we analysed a series of snail mutant alleles. We found that different neuroectodermal genes respond differently in various snail mutant background. Due to the differential response of target genes, one of the mutant alleles, V2, that has reduced Snail function showed an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. Together with the data showing that the expression of some ventral genes disappear in snail mutants, we propose that Snail may repress or activate another set of target genes that are required specifically for gastrulation.     

Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo

After gastrulation, progenitor cells of the cardiac, visceral and body wall musculature arise at defined positions within the mesodermal layer of the Drosophila embryo. The regulatory mechanisms underlying this process of pattern formation are largely unknown, although ablation experiments carried out in other insects indicate that inductive influences from ectodermal cells have major roles in embryonic mesoderm differentiation. An early and important event in the regional subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells. Genetic analysis has shown that this homeobox gene controls the formation of the visceral musculature and the heart from dorsal portions of the mesoderm. We now show that an inductive signal from dorsal ectodermal cells is required for activation of tinman in the underlying mesoderm and present evidence that Decapentaplegic (Dpp), a member of the transforming growth factor-beta superfamily, serves as a signalling molecule in this process. This demonstrates that the spatial expression of dpp in the ectoderm determines which cells of the mesoderm become competent to develop into visceral mesoderm and the heart.     

GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo

Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The eve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl-GAGA increases the stability of enhancer-promoter interactions by creating an open chromatin configuration at the core promoter.     

Localization of thoracic imaginal-disc precursor cells in the early embryo of Drosophila melanogaster

Our previous cell lineage analysis of the thoracic disc primordia of Drosophila showed that at the blastoderm and early gastrula stage, cells are not yet committed to form either larval or imaginal tissue (Meise and Janning, 1993). We have now refined our studies on the cell lineage and have mapped the imaginal primordia in the thoracic region. Homotopic transplantations of single cells within the thoracic region of blastoderm and early gastrula stages show that the precursor cells of thoracic imaginal discs are locally restricted to a small lateral area of the thoracic region. Clones labelling leg discs frequently included the Keilin's organs. Heterotopic transplantations along the dorsoventral axis indicate that cells within the thoracic region are not yet committed with respect to larval or imaginal tissue, their fate being dependent on the position where the transplanted cell had been deposited. On the other hand, cells taken from the abdominal anlagen and transplanted into the region of thoracic disc primordia could not participate in the formation of imaginal discs. This shows that, in contrast to the dorsoventral axis, determinative events had separated primordia along the anterior-posterior axis.     

Analysis of the genes involved in organizing the tail segments of the Drosophila melanogaster embryo

The metameric organization of the Drosophila melanogaster tail is obscured by developmental events that partially suppress or fuse some of its regions. To better define the developmental origins and segmental identities in the tail of the Drosophila embryo, we documented expression patterns and mutant phenotypes of several genes that play important roles in its morphogenesis. We documented the domains of engrailed (en), Abdominal-B (Abd-B) and caudal (cad) expression in the tail region. The staining pattern of cut (ct) was used to correlate the embryonic sense organs with their respective positions on the larval cuticle. The en patterns in different Bithorax-Complex (BX-C) Abd-B morphogenetic (m) and regulatory (r) mutants demonstrated that Abd-B functions to, among other things, suppress embryonic ventral epidermal structures on the posterior side of A8 to A9. Ventral epidermal structures were not added back into the en pattern in r- or BX-C- mutants, indicating that although the BX-C functions extend through A10, other non-BX-C genes must be required for development of this segment.