Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing

Appendage formation in insects and vertebrates depends upon signals from both the anterior-posterior and dorsal-ventral (DV) axes. In Drosophila, wing formation is organized symmetrically around the DV boundary of the growing wing imaginal disc and requires interactions between dorsal and ventral cells. Compartmentalization of the wing disc, dorsal cell behavior, and the expression of two dorsally expressed putative signaling molecules, fringe (fng) and Serrate (Ser), are regulated by the apterous selector gene. Here, we demonstrate that fng and Ser have distinct roles in a novel cell recognition and signal induction process. fng serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. Ser in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary.     

Pattern formation in epithelial development: the vertebrate limb and feather bud spacing

The ectoderm of the vertebrate limb and feather bud are epithelia that provide good models for epithelial patterning in vertebrate development. At the tip of chick and mouse limb buds is a thickening, the apical ectodermal ridge, which is essential for limb bud outgrowth. The signal from the ridge to the underlying mesoderm involves fibroblast growth factors. The non-ridge ectoderm specifies the dorsoventral pattern of the bud and Wnt7a is a dorsalizing signal. The development of the ridge involves an interaction between dorsal cells that express radical fringe and those that do not. There are striking similarities between the signals and genes involved in patterning the limb ectoderm and the epithelia of the Drosophila imaginal disc that gives rise to the wing. The spacing of feather buds involves signals from the epidermis to the underlying mesenchyme, which again include Wnt7a and fibroblast growth factors.     

Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb

Vertebrate limb outgrowth requires a structure called the apical ectodermal ridge, formation of which follows the previous establishment of the dorsoventral limb axis. Radical fringe is expressed in the dorsal ectoderm before the ridge appears, and is repressed by Engrailed-1, which is expressed in the ventral ectoderm. Misexpression of these genes indicates that a ridge is formed wherever there is a boundary between cells expressing and not expressing Radical fringe. Thus, as in Drosophila, Radical fringe positions the ridge at the dorsoventral limb boundary.     

Identification of a novel dual angiotensin II/vasopressin receptor

The development and patterning of the Drosophila wing relies on interactions between cell populations that have the anteroposterior (AP) axis and dorsoventral (DV) axis of the wing imaginal disc as frames of reference [1-3]. Each of these cell populations gives rise to a compartment - a group of cells that have their fates restricted by cell lineage - within which cells acquire specific identities through the expression of 'selector' genes [1,2,4]. The genes engrailed (en) and invected (inv), for example, label cells in the posterior compartment and mediate a set of cell interactions that direct the patterning and growth of the wing along the AP axis [1,2,4]. A similar situation has been proposed to exist across the DV axis, along with apterous (ap) as a dorsal selector gene [5], mediating cell interactions by regulating the expression of Serrate (Ser) [6] [7] and fringe (fng) [8]. In ap mutants, the wing is lost [5] [9], and here we report that this phenotype can be rescued by ectopic expression of either Ser or fng and that, surprisingly, the resulting wings have both dorsal and ventral cell fates.     

The expression and regulation of chick EphA7 suggests roles in limb patterning and innervation

Eph receptors and their ligands, the ephrins, have been implicated in early patterning and axon guidance in vertebrate embryos. Members of these families play pivotal roles in the formation of topographic maps in the central nervous system, the formation of brain commissures, and in the guidance of neural crest cells and motor axons through the anterior half of the somites. Here, we report a highly dynamic expression pattern of the chick EphA7 gene in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signalling in dorsal limb patterning and/or axon guidance. We carried out experimental manipulations in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene was downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalisation in response to the ectodermal signal. These results add a new gene to the dorsalisation signalling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery.     

The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing

Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein (Ap) in dorsal cells. Here we report that dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the DLDB/CHIP co-factor, and suggest that the Ap and DLDB/CHIP proteins form a multimeric functional complex. In support of this model, we show that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression.     

Dorsal-ventral signaling in limb development

In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal-ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal-ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.     

Hox9 genes and vertebrate limb specification

Development of paired appendages at appropriate levels along the primary body axis is a hallmark of the body plan of jawed vertebrates. Hox genes are good candidates for encoding position in lateral plate mesoderm along the body axis and thus for determining where limbs are formed. Local application of fibroblast growth factors (FGFs) to the anterior prospective flank of a chick embryo induces development of an ectopic wing, and FGF applied to posterior flank induces an ectopic leg. If particular combinations of Hox gene expression determine where wings and legs develop, then formation of additional limbs from flank should involve changes in Hox gene expression that reflect the type of limb induced. Here we show that the same population of flank cells can be induced to form either a wing or a leg, and that induction of these ectopic limbs is accompanied by specific changes in expression of three Hox genes in lateral plate mesoderm. This then reproduces, in the flank, expression patterns found at normal limb levels. Hox gene expression is reprogrammed in lateral plate mesoderm, but is unaffected in paraxial mesoderm. Independent regulation of Hox gene expression in lateral plate mesoderm may have been a key step in the evolution of paired appendages.     

Cutting edge: protective effects of notch-1 on TCR-induced apoptosis

The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions. Members of this family have been isolated from invertebrates as well as vertebrates. We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines. The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis. These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.     

Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo

Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates. In C. elegans, two DSL genes, lag-2 and apx-1, influence different cell fate decisions during development. Here we show that APX-1 can fully substitute for LAG-2 when expressed under the control of lag-2 regulatory sequences. In addition, we demonstrate that truncated forms lacking the transmembrane and intracellular domains of both LAG-2 and APX-1 can also substitute for endogenous lag-2 activity. Moreover, we provide evidence that these truncated forms are secreted and able to activate LIN-12 and GLP-1 ectopically. Finally, we show that expression of a secreted DSL domain alone may enhance endogenous LAG-2 signalling. Our data suggest ways that activated forms of DSL ligands in other systems may be created.     

Pattern formation in the imaginal wing disc of Drosophila melanogaster: fate map, regeneration and duplication

Fragments of the wing disc of Drosophila (fig. 2) were either injected into mature third instar larvae for immediate metamorphosis, or cultured in adult abdomens for seven days before being transferred to larvae for metamorphosis. The structures differentiated during metamorphosis were then analysed. The results of the first series of experiments were used to construct an accurate fate map of the disc, and those of the second series were used to determine the regenerative properties of the disc. The fate map (fig. 7) shows presumptive proximal parts (notum, pleura, and dorsal and ventral hinge) at the two ends of the disc, with presumptive distal wing parts in between. During metamorphosis the disc epithelium folds upon itself along the presumptive wing border, bringing dorsal and ventral wing and hinge surfaces into apposition. The wing surfaces occupy a much smaller relative area, and the hinge parts a much larger relative area, in the fate map than in the adult structure. The cultured fragments, in general, behaved in accordance with the rule that when two cut surfaces are created by cutting across the disc, regeneration occurs from one of the cut surfaces and duplication occurs from the other (fig. 14). It was possible to define a level in the longitudinal axis of the disc from which regeneration proceeds outwards. Cut surfaces facing away from this level show regeneration, while cut surfaces facing this level undergo duplication. Similar behavior was found for the transverse axis, and for two diagonal series of cuts. Some fragments with two cut edges could regenerate from one edge while duplicating from the other, whereas others could regenerate from two cut edges simultaneously. However, fragments with four cut edges showed incomplete regeneration, and a high tendency to duplicate even though regeneration in all directions might have been expected on the basis of the other experiments.     

Control of axis formation in Xenopus by the NF-kappa B-I kappa B system

We describe the isolation and analysis in Xenopus of Xrel2, a novel member of the NF-kappa B/Rel protein family that remains to be described in other vertebrates. We show that Xrel2 is expressed throughout development but with higher levels in pre-gastrula embryos. Like other NF-kappa B/Rel proteins, Xrel2 protein is able to bind DNA at a kappa B-Motif. Ectopic expression of Xrel2 disrupts normal morphogenesis at the early gastrula stages suggesting that the NF-kappa B/Rel family have developmental functions at stages earlier than previously thought. We also show that the Xrel2 over-expression phenotype can be rescued by co-expression of I kappa B-alpha and that ectopic expression of I kappa B-alpha or I kappa B-gamma alone has no effect on development. Finally, we show that Xrel2 does not divert animal caps from an ectodermal to a mesodermal cell fate. Overall, these results suggest that the NF-kappa B/Rel family does have key functions in early vertebrate development, however, there is not a simple conservation of the Drosophila dorsal pathway.     

Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain, but not limb development

The Engrailed-1 gene, En1, a murine homologue of the Drosophila homeobox gene engrailed (en), is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. An important question is whether, during evolution, the biochemical properties of En proteins have been conserved, revealing a common underlying molecular mechanism to their diverse developmental activities. To address this question, we have replaced the coding sequences of En1 with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither En2 nor en rescue the postnatal limb abnormalities that develop in rare En1 null mutants that survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally.     

Interactions among Delta, Serrate and Fringe modulate Notch activity during Drosophila wing development

The Notch signalling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signalling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signalling to the dorsoventral boundary of the developing wing blade. Here we report the results of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development. We find that Serrate is not required for the initiation of wing development but rather for the expansion and early patterning of the wing primordium. We provide evidence that, at the onset of wing development, Delta is under the control of apterous and might be the Notch ligand in this process. In addition, we find that Fringe function requires Su(H). Our results suggest that Notch signalling during wing development relies on careful balances between positive and dominant negative interactions between Notch ligands, some of which are mediated by Fringe.