Direct control of antennal identity by the spineless-aristapedia gene of Drosophila

Loss-of-function mutations in the spineless-aristapedia gene of Drosophila (ssa mutants) cause transformations of the distal antenna to distal second leg, deletions or fusions of the tarsi from all three legs, a general reduction in bristle size, and sterility. Because ssa mutants are pleiotropic, it has been suggested that ss+ has some rather general function and that the ssa antennal transformation is an indirect consequence of perturbations in the expression of other genes that more directly control antennal or second leg identity. Here we test whether the ssa transformation results from aberrant expression of Antennapedia (Antp), a homeotic gene thought to specify directly the identity of the second thoracic segment. We find that Antp-ssa mitotic recombination clones in the distal antenna behave identically to Antp+ ssa clones, and are transformed to second leg. This demonstrates that the ssa antennal transformation is independent of Antp+, and suggests that ss+ may itself directly define distal antennal identity. The results also reveal that Antp+ is not required for the development of distal second leg structures, as these develop apparently normally in Antp- ssa antennal clones. Because Antp- mutations cause deletions or transformations that are restricted to proximal structures, whereas ssa alleles cause similar defects that are distally restricted, we suggest that ss+ and Antp+ may play similar, but complementary, roles in the distal and proximal portions of appendages, respectively.     

The hernandez and fernandez genes of Drosophila specify eye and antenna

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of "eye-specifying" genes, among them eyeless, which can direct eye development ectopically. We report here the characterization of the hernandez and fernandez genes, expressed in the antennal and eye primordia of the eye-antenna imaginal disc. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: it can form distal antenna, activating genes like homothorax, spineless, and spalt, and it can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. Our results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically.     

Complex genetic interactions govern the temporal effects of <Emphasis Type="Italic">Antennapedia</Emphasis> on antenna-to-leg transformations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The putative regulatory relationships between Antennapedia (Antp), spalt major (salm) and homothorax (hth) are tested with regard to the sensitive period of antenna-to-leg transformations. Although Antp expression repressed hth as predicted, contrary to expectations, hth did not show increased repression at higher Antp doses, whereas salm, a gene downstream of hth, did show such a dose response. Loss of hth allowed antenna-to-leg transformations but the relative timing of proximal-distal transformations was reversed, relative to transformations induced by ectopic Antp. Finally, overexpression of Hth was only partially able to rescue transformations induced by ectopic Antp. These results indicate that there may be additional molecules involved in antenna/leg identity and that spatial, temporal and dosage relationships are more subtle than suspected and must be part of a robust understanding of molecular network behaviour involved in determining appendage identity in Drosophila melanogaster.     

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.     

Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Here, we analyze appendage genes in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic (dpp) expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax (hth) and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role of this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. We comment on the significance of this finding for attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, our results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies.     

Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.     

Regulation and function of Scr, exd, and hth in the Drosophila salivary gland

Salivary gland formation in the Drosophila embryo is dependent on the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when SCR is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, Scr mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that SCR also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Here, we show that two homeodomain-containing proteins encoded by the extradenticle (exd) and homothorax (hth) genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. We also show that Scr regulates the nuclear localization of EXD in the salivary gland primordia through repression of homothorax (hth) expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands.     

Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity

The Distal-less gene is known for its role in proximodistal patterning of Drosophila limbs. However, Distal-less has a second critical function during Drosophila limb development, that of distinguishing the antenna from the leg. The antenna-specifying activity of Distal-less is genetically separable from the proximodistal patterning function in that certain Distal-less allelic combinations exhibit antenna-to-leg transformations without proximodistal truncations. Here, we show that Distal-less acts in parallel with homothorax, a previously identified antennal selector gene, to induce antennal differentiation. While mutations in either Distal-less or homothorax cause antenna-to-leg transformations, neither gene is required for the others expression, and both genes are required for antennal expression of spalt. Coexpression of Distal-less and homothorax activates ectopic spalt expression and can induce the formation of ectopic antennae at novel locations in the body, including the head, the legs, the wings and the genital disc derivatives. Ectopic expression of homothorax alone is insufficient to induce antennal differentiation from most limb fields, including that of the wing. Distal-less therefore is required for more than induction of a proximodistal axis upon which homothorax superimposes antennal identity. Based on their genetic and biochemical properties, we propose that Homothorax and Extradenticle may serve as antenna-specific cofactors for Distal-less.     

The expression of the proximodistal axis patterning genes Distal-less and dachshund in the appendages of Glomeris marginata (Myriapoda: Diplopoda) suggests a special role of these genes in patterning the head appendages

The genes Distal-less, dachshund, extradenticle, and homothorax have been shown in Drosophila to be among the earliest genes that define positional values along the proximal-distal (PD) axis of the developing legs. In order to study PD axis formation in the appendages of the pill millipede Glomeris marginata, we have isolated homologues of these four genes and have studied their expression patterns. In the trunk legs, there are several differences to Drosophila, but the patterns are nevertheless compatible with a conserved role in defining positional values along the PD axis. However, their role in the head appendages is apparently more complex. Distal-less in the mandible and maxilla is expressed in the forming sensory organs and, thus, does not seem to be involved in PD axis patterning. We could not identify in the mouthparts components that are homologous to the distal parts of the trunk legs and antennnae. Interestingly, there is also a transient premorphogenetic expression of Distal-less in the second antennal and second maxillary segment, although no appendages are eventually formed in these segments. The dachshund gene is apparently involved both in PD patterning as well as in sensory organ development in the antenna, maxilla, and mandible. Strong dachshund expression is specifically correlated with the tooth-like part of the mandible, a feature that is shared with other mandibulate arthropods. homothorax is expressed in the proximal and medial parts of the legs, while extradenticle RNA is only seen in the proximal region. This overlap of expression corresponds to the functional overlap between extradenticle and homothorax in Drosophila.     

Spatial and temporal regulation of the homeotic selector gene Antennapedia is required for the establishment of leg identity in Drosophila

Antennapedia is one of the homeotic selector genes required for specification of segment identity in Drosophila. Dominant mutations that ectopically express Antennapedia cause transformation of antenna to leg. Loss-of-function mutations cause partial transformation of leg to antenna. Here we examine the role of Antennapedia in the establishment of leg identity in light of recent advances in our understanding of antennal development. In Antennapedia mutant clones in the leg disc, Homothorax and Distal-less are coexpressed and act via spineless to transform proximal femur to antenna. Antennapedia is negatively regulated during leg development by Distal-less, spineless, and dachshund and this reduced Antennapedia expression is needed for the proper development of distal leg elements. These findings suggest that the temporal and spatial regulation of the homeotic selector gene Antennapedia in the leg disc is necessary for normal leg development in Drosophila.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

Proximal fate marker homothorax marks the lateral extension of stalk‐eyed fly Cyrtodopsis whitei

The placement of eyes on insect head is an important evolutionary trait. The stalk‐eyed fly, Cyrtodopsis whitei, exhibits a hypercephaly phenotype where compound eyes are located on lateral extension from the head while the antennal segments are placed inwardly on this stalk. This stalk‐eyed phenotype is characteristic of the family Diopsidae in the Diptera order and dramatically deviates from other dipterans, such as Drosophila. Like other insects, the adult eye and antenna of stalk‐eyed fly develop from a complex eye‐antennal imaginal disc. We analyzed the markers involved in proximo‐distal (PD) axis of the developing eye imaginal disc of the stalk‐eyed flies. We used homothorax (hth) and distalless (dll), two highly conserved genes as the marker for proximal and distal fate, respectively. We found that lateral extensions between eye and antennal field of the stalk‐eyed fly's eye‐antennal imaginal disc exhibit robust Hth expression. Hth marks the head specific fate in the eye‐ and proximal fate in the antenna‐disc. Thus, the proximal fate marker Hth expression evolves in the stalk‐eyed flies to generate lateral extensions for the placement of the eye on the head. Moreover, during pupal eye metamorphosis, the lateral extension folds back on itself to place the antenna inside and the adult compound eye on the distal tip. Interestingly, the compound eye in other insects does not have a prominent PD axis as observed in the stalk‐eyed fly.     

Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb’hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb’wingless, Gb’dachshund, Gb’aristaless and Gb’Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb’hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb’hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb’hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.