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.     

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.     

Distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. A genetic hierarchy for specification of antennal identity has been partly elucidated. We report identification of a novel family of genes with roles in antennal development. The distal antenna (dan) and distal antenna-related (danr) genes encode novel nuclear proteins that are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore we suggest that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. We propose that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures.     

Functional analyses of tiptop and antennapedia in the embryonic development of Oncopeltus fasciatus suggests an evolutionary pathway from ground state to insect legs

In insects, selector genes are thought to modify the development of a default, or 'ground state', appendage into a tagma-specific appendage such as a mouthpart, antenna or leg. In the best described example, Drosophila melanogaster, the primary determination of leg identity is thought to result from regulatory interactions between the Hox genes and the antennal-specifying gene homothorax. Based on RNA-interference, a functional analysis of the selector gene tiptop and the Hox gene Antennapedia in Oncopeltus fasciatus embryogenesis is presented. It is shown that, in O. fasciatus, tiptop is required for the segmentation of distal leg segments and is required to specify the identity of the leg. The distal portions of legs with reduced tiptop develop like antennae. Thus, tiptop can act as a regulatory switch that chooses between antennal and leg identity. By contrast, Antennapedia does not act as a switch between leg and antennal identity. This observation suggests a significant difference in the mechanism of leg specification between O. fasciatus and D. melanogaster. These observations also suggest a significant plasticity in the mechanism of leg specification during insect evolution that is greater than would have been expected based on strictly morphological or molecular comparisons. Finally, it is proposed that a tiptop-like activity is a likely component of an ancestral leg specification mechanism. Incorporating a tiptop-like activity into a model of the leg-specification mechanism explains several mutant phenotypes, previously described in D. melanogaster, and suggests a mechanism for the evolution of legs from a ground state.     

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.     

Utilization ofDrosophila eye to probe the functions of two mammalian serine/threonine kinases, Snk and HsHPK

Here we report a quick functional analysis of two mammalian serine/threonine kinases, a serum inducible kinase (Snk) and Homo sapiens hepatoma protein kinase (HsHPK), using Drosophila eye as a model system. We generated transgenic fly lines carrying constructs of both kinases under control of the GAL upstream activating sequence (UAS). Each UAS line was then crossed to a line in which GAL4 expression was driven by one of the following promoters, eyeless (ey), glass or decapentaplegic. Thus, different kinase mutants can be ectopically expressed in a promoter-dependent manner. We observed that the ectopic expression of either the wild-type or active form of Snk driven by the glass promoter resulted in a rough-eye phenotype. Nevertheless, the ectopic expression of HsHPK under the control of the ey promoter resulted in a small-eye phenotype. The results of this study demonstrated that ectopic expression of these two mammalian genes could be achieved by the regulation of Drosophila promoters. In addition, the effects of these ectopically expressed genes on eye development could be an implication of their functions with respect to cell proliferation and differentiation. Thus, Drosophila eye, with the powerful genetic tools and vast information on eye development available, can be a useful system to probe the functions of mammalian genes in the postgenome era.     

The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation

Notch signaling controls formation of joints at leg segment borders and growth of the developing Drosophila leg. Here, we identify the odd-skipped gene family as a key group of genes that function downstream of the Notch receptor to promote morphological changes associated with joint formation during leg development. odd, sob, drm, and bowl are expressed in a segmental pattern in the developing leg, and their expression is regulated by Notch signaling. Ectopic expression of odd, sob, or drm can induce invaginations in the leg disc epithelium and morphological changes in the adult leg that are characteristic of endogenous invaginating joint cells. These effects are not due to an alteration in the expression of other genes of the developing joint. While odd or drm mutant clones do not affect leg segmentation, and thus appear to act redundantly, bowl mutant clones do perturb leg development. Specifically, bowl mutant clones result in a failure of joint formation from the distal tibia to tarsal segment 5, while more proximal clones cause melanotic protrusions from the leg cuticle. Together, these results indicate that the odd-skipped family of genes mediates Notch function during leg development by promoting a specific aspect of joint formation, an epithelial invagination. As the odd-skipped family genes are involved in regulating cellular morphogenesis during both embryonic segmentation and hindgut development, we suggest that they may be required in multiple developmental contexts to induce epithelial cellular changes.     

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene.     

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). Here, we investigate the function of these signaling pathways in this process. We find that organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Our analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. We propose that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

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.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

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.     

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.     

Limb type-specific regulation of bric a brac contributes to morphological diversity

The insect antenna and leg are considered homologous structures, likely to have arisen via duplication and divergence from an ancestral limb. Consistent with this, the antenna and leg are derived from primordia with similar developmental potentials. Nonetheless, the adult structures differ in both form and function. In Drosophila, one conspicuous morphological difference is that the antenna has fewer distal segments than the leg. We propose that this is due in part to the variations in the regulation of bric a brac. bric a brac is required for joint formation, and loss of bric a brac function leads to fusion of distal antennal and leg segments, resulting in fewer total segments. Here, we address how bric a brac is regulated to generate the mature expression patterns of two concentric rings in the antenna versus four concentric rings in the leg. We find that bric a brac expression is activated early throughout most of the Distal-less domain in both antenna and leg and subsequently is restricted to the distal portion and into rings. Although bric a brac expression in the antenna and in all four tarsal rings of the leg requires Distal-less, only the proximal three tarsal rings are Spineless-dependent. Thus bric a brac is regulated differentially even within a single appendage type. The restriction of bric a brac expression to the distal portion of the Distal-less domain is a consequence of negative regulation by distinct sets of genes in different limb types. In the leg, the proximal boundary of bric a brac is established by the medial-patterning gene dachshund, but dachshund alone is insufficient to repress bric a brac, and the expression of the two genes overlaps. In the antenna, the proximal boundary of bric a brac is established by an antenna-specifying gene, homothorax, in conjunction with dachshund and spalt, and there is much less overlap between the bric a brac and the dachshund domains. Thus tissue-specific expression of other patterning genes that differentially repress bric a brac accounts for antenna-leg differences in bric a brac pattern. We propose that the limb type-specific variations in expression of bric a brac repressors contribute to morphological variations by controlling distal limb segment number.