The Ultrabithorax gene of Drosophila and the specification of abdominal histoblasts.

Separation of the imaginal and larval developmental pathways in Drosophila occurs early in embryogenesis, resulting in the formation of imaginal discs and abdominal histoblast nests along the larval body wall. The dorsal and ventral histoblast nests within the first abdominal (A1) segment are shown not to be segmentally homologous with the metathoracic (T3) haltere and leg discs, respectively, since they occur at distinct dorso-ventral locations during normal development and can be found together within the same segment in mutants of the Bithorax complex (BX-C) where T3 is transformed towards A2-A4 or A1 towards T3. Several patterning abnormalities are also observed in BX-C mutants. A ventral shift in the A1 ventral nest occurs in partially transformed larvae harboring weak bithoraxoid (bxd) mutations; in more fully transformed larvae (Ubx1/Df) both the anterior dorsal and ventral nests are lost and instead a dorsal and ventral disc bud are formed. Dorso-ventral inversions in the pattern of the ventral nest occur in a random fashion throughout A1-A7 in response to an increase or decrease in the gene dosage of the BX-C. In gain-of-function mutants anterior dorsal histoblast cells form in the homologous anterior as well as the nonhomologous posterior portion of T3. Based on these and other findings it appears that the Ultrabithorax (Ubx) locus (and possibly abdominal-A and Abdominal-B) is required to steer ectodermal cells toward an imaginal histoblast rather than a larval cell fate at specific regions within the first abdominal segment.     

A position-specific cell surface antigen in the drosophila wing imaginal disc

The antibody produced by the hybrid cell line DK.1A4 recognizes an antigen present initially on all the epithelial cells of the D. melanogaster wing imaginal disc. This antigen becomes progessively restricted to cells in the dorsal region of the disc during the final larval instar. The presence of the antigen does not correlate with the specific adult structures to which the cells will eventually contribute, but rather with the position of the cells in the disc. In late discs, the line bounding the region in which the antigen persists corresponds to the boundary between the dorsal and ventral compartments as revealed by a clonal analysis of the undifferentiated disc. Together, these data suggest that the antigen's disappearance may be specific to the cells of the ventral compartment of the wing disc.     

The Iroquois homeobox genes function as dorsal selectors in the Drosophila head

The Iroquois complex (Iro-C) genes are expressed in the dorsal compartment of the Drosophila eye/antenna imaginal disc. Previous work has shown that the Iro-C homeoproteins are essential for establishing a dorsoventral pattern organizing center necessary for eye development. Here we show that, in addition, the Iro-C products are required for the specification of dorsal head structures. In mosaic animals, the removal of the Iro-C transforms the dorsal head capsule into ventral structures, namely, ptilinum, prefrons and suborbital bristles. Moreover, the Iro-C(-) cells can give rise to an ectopic antenna and maxillary palpus, the main derivatives of the antenna part of the imaginal disc. These transformations are cell-autonomous, which indicates that the descendants of a dorsal Iro-C(-) cell can give rise to essentially all the ventral derivatives of the eye/antenna disc. These results support a role of the Iro-C as a dorsal selector in the eye and head capsule. Moreover, they reinforce the idea that developmental cues inherited from the distinct embryonic segments from which the eye/antenna disc originates play a minimal role in the patterning of this disc.     

Fate map of the eye-antennal imaginal disc of the tumorous-head mutant of Drosophila melanogaster

In specific genetic backgrounds, a mutation in the tuh-3 gene results in the homeotic transformation of head structures to either leg disc derivatives or structures normally found in the extreme posterior end of wild-type animals. The origins of the homeotic structures were mapped to defined positions in the eye-antennal imaginal disc by transplanting abnormal regions of discs isolated from tuh-3 mutants into host mwh;e4 larvae. These metamorphosed implants were removed and differentiated structures were identified. Of 211 successfully recovered implants, 157 gave rise to homeotic tissue: abdominal tergite, male or female external genitalia and/or leg tissue. Transformations to abdominal tergite occurred primarily in cells taken from the eye region of the compound disc. Male and female genitalia arose most often in implants taken from the antennal portion of the disc, although some tissue taken from the lateral region of the eye disc also gave rise to external genitalia. Leg structures came exclusively from implants from the antennal region of the imaginal disc. These results suggest that cells from within specific regions of the eye-antennal compound disc are constrained in their developmental potential. An obvious constraint observed with this mutation is a dorsal/ventral one: Cells from the eye disc, a dorsal structure, primarily gave rise to other dorsal structures, abdominal tergite tissue. Cells from the antennal disc, a ventrally derived structure, primarily gave rise to other ventral structures including genital tissue and distal leg.     

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.     

Distinct Parasegmental and Imaginal Enhancers and the Establishment of the Expression Pattern of the Ubx Gene

The expression domain of the Ubx gene in Drosophila embryos is bounded by the product of the hb gene, acting as a repressor. We show that all Ubx fragments that bind Hb protein in vitro contain parasegmental enhancers active in the embryo in specific parasegmental patterns. We have found three new embryonic enhancer elements in the upstream region, in addition to the two previously identified. Each produces a pattern initially bounded at PS6 by Hb but sooner or later breaks down this boundary and begins to express in the anterior region. These enhancers do not respond to the long-term maintenance mediated by the Polycomb group of genes. They also cease functioning after germ band extension. Expression in imaginal tissues is due to a set of entirely separate and independent imaginal disc enhancers. These do not contain Hb binding sites and by themselves have no anterior/posterior positional information, although some distinguish between ventral and dorsal discs. A third kind of element, the Polycomb Response Element (PRE), has no enhancer activity but causes long-term maintenance of the expression domain of other enhancers present in the vicinity. The interaction of these elements results in the correct expression of Ubx in imaginal tissues.     

msh specifies dorsal cell fate in the Drosophila wing

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary (2) establishment of a lineage restriction boundary between compartments and (3) specification of dorsal cell fate. Here, we report that the homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development.     

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). Here, we uncover a new morphogenetic/cellular mechanism for disc eversion. We show that imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. We found that the JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells.     

Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes

The compound eye of D. melanogaster is a reiterative pattern of facets, each containing eight photoreceptor cells in a precise arrangement. This pattern is established in the eye imaginal disc during the third larval instar. A wave of morphogenesis sweeps from posterior to anterior across the disc, leaving in its wake organized clusters of photoreceptor cells. We have used monoclonal antibodies to highlight pattern elements that are not readily observable by other techniques. Monoclonal antibodies can be used to identify the molecules associated with particular patterns, providing links between observable structures and the genes. As an example, we present the purification and N-terminal sequence of a glycoprotein antigen specific to photoreceptor cells and their axons.     

Development of the Drosophila retina,a neurocrystalline lattice

Pattern formation in the Drosophila retina proceeds by the recruitment of cells, along a morphogenetic front, into a lattice. At the advancing front, marked by a dorso-ventral furrow in the eye imaginal disc, cells are organized into ommatidial precursors, each containing cells destined to become photoreceptors 2, 3, 4, 5, and 8. Behind the front, a mitotic wave produces photoreceptors 1, 6, and 7, plus the remaining cells needed to complete the ommatidia. During the third larval instar, the front sweeps anteriorly across the eye disc, leaving a highly ordered pattern in its wake. Preceding the dorso-ventral furrow is a groove that bisects the eye disc into dorsal and ventral halves and presumably plays a role in establishing the equatorial symmetry line. Cell lineage plays little role in pattern formation in the eye. Genetic mosaics show that the cells of each ommatidium are not derived from a single mother cell; the cells appear to be recruited at random at the morphogenetic front. Similarly, the mirror symmetry above and below the equator is not established by a clonal mechanism; a single clone can contribute cells to ommatidia on both sides of the equator.     

Plasma protein beta-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells

A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein beta-2-glycoprotein 1 (beta2GP1). beta2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of beta2GP1 to EC induced to expose PS. We also show that divalent 3G4-beta2GP1 complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric beta2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of beta2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of beta2GP1 for anionic phospholipids through formation of multivalent 3G4-beta2GP1 complexes.     

In-Vivo Imaging of the Drosophila Wing Imaginal Disc over Time: Novel Insights on Growth and Boundary Formation

In developmental biology, the sequence of gene induction and pattern formation is best studied over time as an organism develops. However, in the model system of Drosophila larvae this oftentimes proves difficult due to limitations in imaging capabilities. Using the larval wing imaginal disc, we show that both overall growth, as well as the creation of patterns such as the distinction between the anterior(A) and posterior(P) compartments and the dorsal(D) and ventral(V) compartments can be studied directly by imaging the wing disc as it develops inside a larva. Imaged larvae develop normally, as can be seen by the overall growth curve of the wing disc. Yet, the fact that we can follow the development of individual discs through time provides the opportunity to simultaneously assess individual variability. We for instance find that growth rates can vary greatly over time. In addition, we observe that mechanical forces act on the wing disc within the larva at times when there is an increase in growth rates. Moreover, we observe that A/P boundary formation follows the established sequence and a smooth boundary is present from the first larval instar on. The division of the wing disc into a dorsal and a ventral compartment, on the other hand, develops quite differently. Contrary to expectation, the specification of the dorsal compartment starts with only one or two cells in the second larval instar and a smooth boundary is not formed until the third larval instar.     

Syngeneic monoclonal anti-idiotype antibodies that bear the internal image of a human cytomegalovirus neutralization epitope

Two glycoproteins have been identified on human CMV that induce neutralizing antibody; an 86,000-Da glycoprotein and a 130,000-, 92,000-, and 50,000-Da glycoprotein coimmunoprecipitating complex that appears to be the gB homologue of HSV. We have produced syngeneic monoclonal anti-Id antibodies (mAb2) of the IgM isotype to a CMV-neutralizing monoclonal antibody (mAb1) that is known to bind to the 86,000-Da glycoprotein on the virion envelope. These mAb2 bear the internal image of the original viral antigen as shown by their ability to 1) recognize an interspecies idiotype in CMV-positive human antisera, 2) block mAb1 binding to CMV antigen, and 3) block CMV neutralization by mAb1 in vitro. Immunization of mice with both of these affinity chromatography-purified mAb2 stimulated the production of anti-anti-Id monoclonal antibodies (which we termed mAb3), which bound to the mAb2 by ELISA and neutralized CMV infectivity.     

Allocation and specification of the genital disc precursor cells in Drosophila

The adult structures of Drosophila melanogaster are derived from larval imaginal discs, which originate as clusters of cells within the embryonic ectoderm. The genital imaginal disc is composed of three primordia (female genital, male genital, and anal primordia) that originate from the embryonic tail segments A8, A9, and A10, respectively, and produce the sexually dimorphic genitalia and analia. We show that the genital disc precursor cells (GDPCs) are first detectable during mid-embryogenesis as a 22-cell cluster in the ventral epidermis. Analysis of mutant and double mutant phenotypes of embryonic patterning genes in the GDPCs, together with their expression patterns in these cells, revealed the following with respect to the origins and specification of the GDPCs. The allocation of the GDPCs from the ventral epidermis requires the function of ventral patterning genes, including the EGF receptor and the spitz group of genes. The ventral localization of the GDPCs is further restricted by the action of dorsal patterning genes. Along the anterior-posterior axis, several segment polarity genes (wingless, engrailed, hedgehog, and patched) are required for the proper allocation of the GDPCs. These segment polarity genes are expressed in some, but not all of the GDPCs, indicating that anterior and posterior compartments are not fully established in the GDPCs. In addition, we found that the three primordia of the larval genital disc have already been specified in the GDPCs by the coordinated actions of the homeotic (Hox) genes, abdominal-A, Abdominal-B, and caudal. By identifying how these different patterning networks regulate the allocation and primordial organization of the 22 embryonic precursors of the compound genital disc, we demonstrate that at least some of the organization of the larval disc originates as positional information in the embryo, thus providing a context for further studies on the development of the genital disc.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.