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.     

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.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

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.     

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.     

Comparative analysis of leg and antenna development in wild-type and homeotic<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The insect leg and antenna are thought to be homologous structures, evolved from a common ancestral appendage. The homeotic transformations of antenna to leg in Drosophila produced by mutation of the Hox gene Antennapedia are position-specific, such that every particular antenna structure is transformed into a specific leg counterpart. This has been taken to suggest that the developmental programmes of these two appendages are still similar. In particular, the mechanisms for the specification of a cell's position within the appendage would be identical, only their interpretation would be different and subject to homeotic gene control. Here we explore the degree of conservation between the developmental programmes of leg and antenna in Drosophila and other dipterans, in wild-type and homeotic conditions. Most of the appendage pattern-forming genes are active in both appendages, and their expression domains are partially conserved. However, the regulatory relationships and interactions between these genes are different, and in fact cells change their expression while undergoing homeotic transformation. Thus, the positional information, and the mechanisms which generate it, are not strictly conserved between leg and antenna; and homeotic genes alter the establishment of positional clues, not only their interpretation. The partial conservation of pattern-forming genes in both appendages ensures a predictable re-specification of positional clues, producing the observed positional specificity of homeotic transformations.     

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.     

Genetic interactions between the RhoA and Stubble-stubbloid loci suggest a role for a type II transmembrane serine protease in intracellular signaling during Drosophila imaginal disc morphogenesis

The Drosophila RhoA (Rho1) GTPase is essential for postembryonic morphogenesis of leg and wing imaginal discs. Mutations in RhoA enhance leg and wing defects associated with mutations in zipper, the gene encoding the heavy chain of nonmuscle myosin II. We demonstrate here that mutations affecting the RhoA signaling pathway also interact genetically with mutations in the Stubble-stubbloid (Sb-sbd) locus that encodes an unusual type II transmembrane serine protease required for normal leg and wing morphogenesis. In addition, a leg malformation phenotype associated with overexpression of Sb-sbd in prepupal leg discs is suppressed when RhoA gene dose is reduced, suggesting that RhoA and Sb-sbd act in a common pathway during leg morphogenesis. We also characterized six mutations identified as enhancers of zipper mutant leg defects. Three of these genes encode known members of the RhoA signaling pathway (RhoA, DRhoGEF2, and zipper). The remaining three enhancer of zipper mutations interact genetically with both RhoA and Sb-sbd mutations, suggesting that they encode additional components of the RhoA signaling pathway in imaginal discs. Our results provide evidence that the type II transmembrane serine proteases, a class of proteins linked to human developmental abnormalities and pathology, may be associated with intracellular signaling required for normal development.     

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.     

Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression

It has been shown in several organisms that multiple cis-regulatory modules (CRMs) of a gene locus can be active concurrently to support similar spatiotemporal expression. To understand the functional importance of such seemingly redundant CRMs, we examined two CRMs from the Drosophila snail gene locus, which are both active in the ventral region of pre-gastrulation embryos. By performing a deletion series in a ～25 kb DNA rescue construct using BAC recombineering and site-directed transgenesis, we demonstrate that the two CRMs are not redundant. The distal CRM is absolutely required for viability, whereas the proximal CRM is required only under extreme conditions such as high temperature. Consistent with their distinct requirements, the CRMs support distinct expression patterns: the proximal CRM exhibits an expanded expression domain relative to endogenous snail, whereas the distal CRM exhibits almost complete overlap with snail except at the anterior-most pole. We further show that the distal CRM normally limits the increased expression domain of the proximal CRM and that the proximal CRM serves as a `damper' for the expression levels driven by the distal CRM. Thus, the two CRMs interact in cis in a non-additive fashion and these interactions may be important for fine-tuning the domains and levels of gene expression.     

The Polar-Lethal Ovum Mutant Gene Maps to the Distal Portion of Mouse Chromosome 11

Genome imprinting is the process by which identical alleles at a particular locus may be rendered functionally different depending on the sex of the parent contributing the allele. While several mutations in imprinted genes have been defined, no variants in the regulatory system that gives rise to imprinting have been described. Here we report our genetic analysis of the behavior of the interstrain, polar, embryonic-lethal phenotype known as the "DDK syndrome." We have mapped the interstrain, polar-lethal region of the genome to the distal portion of mouse chromosome 11, near the Xmv-42 locus. We propose that the lethal phenotype is not caused by a standard mutation, but by aberrant imprinting of a gene within this region.     

Genetic modifier screens in Drosophila demonstrate a role for Rho1 signaling in ecdysone-triggered imaginal disc morphogenesis

Drosophila adult leg development provides an ideal model system for characterizing the molecular mechanisms of hormone-triggered morphogenesis. A pulse of the steroid hormone ecdysone at the onset of metamorphosis triggers the rapid transformation of a flat leg imaginal disc into an immature adult leg, largely through coordinated changes in cell shape. In an effort to identify links between the ecdysone signal and the cytoskeletal changes required for leg morphogenesis, we performed two large-scale genetic screens for dominant enhancers of the malformed leg phenotype associated with a mutation in the ecdysone-inducible broad early gene (br1). From a screen of >750 independent deficiency and candidate mutation stocks, we identified 17 loci on the autosomes that interact strongly with br1. In a complementary screen of approximately 112,000 F1 progeny of EMS-treated br1 animals, we recovered 26 mutations that enhance the br1 leg phenotype [E(br) mutations]. Rho1, stubbloid, blistered (DSRF), and cytoplasmic Tropomyosin were identified from these screens as br1-interacting genes. Our findings suggest that ecdysone exerts its effects on leg morphogenesis through a Rho1 signaling cascade, a proposal that is supported by genetic interaction studies between the E(br) mutations and mutations in the Rho1 signaling pathway. In addition, several E(br) mutations produce unexpected defects in midembryonic morphogenetic movements. Coupled with recent evidence implicating ecdysone signaling in these embryonic morphogenetic events, our results suggest that a common ecdysone-dependent, Rho1-mediated regulatory pathway controls morphogenesis during the two major transitions in the life cycle, embryogenesis and metamorphosis.     

Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome

Summary Hunter syndrome is a human X-linked disorder caused by deficiency of the lysosomal exohydrolase iduronate-2-sulphatase (IDS). The consequent accumulation of the mucopolysaccharides dermatan sulphate and heparan sulphate, in the brain and other tissues, often results in death before adulthood. There is, however, a broad spectrum of severity that has been attributed to different mutations of the Hunter syndrome gene. We have used an IDS cDNA clone to localise the IDS gene to Xq28, distal to the fragile X mutation (FRAXA). One-third of Hunter syndrome patients had various deletions or rearrangements of their IDS gene, proving that different mutations are common in this condition. Deletions of the IDS gene can include a conserved locus that is tightly linked to FRAXA, suggesting that deletion of nearby genes may contribute to the variable clinical severity noted in Hunter syndrome. The cDNA clone was also shown to span the X chromosome breakpoint in a female Hunter syndrome patient with an X;autosome translocation.     

Zinc deficiency and its inherited disorders -a review

Zinc is an essential trace element required by all living organisms because of its critical roles both as a structural component of proteins and as a cofactor in enzyme catalysis. The importance of zinc in human metabolism is illustrated by the effects of zinc deficiency, which include a diminished immune response, reduced healing and neurological disorders. Furthermore, nutritional zinc deficiency can be fatal in newborn or growing animals. While zinc deficiency is commonly caused by dietary factors, several inherited defects of zinc deficiency have been identified. Acrodermatitis enteropathica is the most commonly described inherited condition found in humans. In several of the few cases that have been reported, this disorder is associated with mutations in the hZIP4 gene, a member of the SLC39 family, whose members encode membranebound putative zinc transporters. Mutations in other members of this family or in different genes may account for other cases of acrodermatitis in which defects in hZIP4 have not been detected. Another inherited form of zinc deficiency occurs in the lethal milk mouse, where a mutation in ZnT4 gene, a member of the SLC30 family of transmembrane proteins results in impaired secretion of zinc into milk from the mammary gland. A similar disorder to the lethal milk mouse occurs in humans. In the few cases studied, no changes in ZnT4 orthologue, hZnT4, were detected. This, and the presence of several minor phenotypic differences between the zinc deficiency in humans and mice, suggests that the human condition is caused by defects in genes that are yet to be identified. Taking into account the fact that there are no definitive tests for zinc deficiency and that this disorder can go undiagnosed, plus the recent identification of multiple members of the SCL30 and SLC39, it is likely that mutations in other genes may underlie additional inherited disorders of zinc deficiency.     

Cytogenetic analysis of two ecdysone-regulated puffs 62C and 62E in Drosophila melanogaster

Genetic analysis has been performed to reveal vital genes around two puffs, a late 62C puff and an early-late 62E puff. Their roles in hormonal regulatory mechanisms have been estimated. A locus represented by four lethal mutations has been found in the vicinity of the 62E puff. The mutants display disturbed puffing, which suggests the involvement of this locus in hormonal regulatory mechanisms. In the 62C puff region. 26 mutations have been found that proved to be allelic to mutations in the D-Titin gene. The giant D-Titin gene is essential for the sarcomeric organization of striated muscles. According to the results of in situ hybridization with polytene chromosomes, the D-Titin gene occupies the entire 62C puff. The phenotypic characteristics of the novel mutants suggest that this protein is polyfunctional, and its role is not restricted to processes in the muscular tissue. It may also be involved in the morphogenesis of leg imaginal disks, and it is necessary for condensation and separation of sister chromatids during mitosis. Mutations in the ecdysone-induced BR-C and E74 genes cause disturbances similar to those found in this study. In addition, mutations of these genes can affect the D-Titin gene activity, which suggests that the three genes are involved in similar morphogenetic and myogenetic processes.     

Characterization of mutations that feminize gametophytes of the fern Ceratopteris.

Gametophytes of the fern Ceratopteris are either male or hermaphroditic. Their sex is epigenetically determined by the pheromone antheridiogen, which is secreted by the hermaphrodite and induces male and represses female development in other young, sexually undetermined gametophytes. To understand how antheridiogen represses the development of female traits at the genetic level, 16 new mutations that feminize the gametophyte in the presence of antheridiogen were identified and characterized. Seven are very tightly linked to the FEM1 locus previously described. Nine others define another locus named NOTCHLESS1 (NOT1), as several of the not1 mutants lack a meristem notch. Some not1 mutations also affect sporophyte development only when homozygous, indicating that the not1 mutations are recessive and that NOT1 is also required for normal sporophyte development. The epistatic interactions among FEM1, NOT1, and other sex-determining genes are described. This information was used to expand the genetic model of the sex-determining pathway in Ceratopteris. On the basis of this model, we can say that the presence of antheridiogen leads to the activation of the FEM1 gene, which not only promotes the differentiation of male traits, but also represses female development by activating the NOT1 gene. NOT1 represses the TRA genes necessary for the development of female traits in the gametophyte.     

Functional stability of the aristaless gene in appendage tip formation during evolution

The appendages of an insect are subdivided into distinct segments or podomeres. Many genes responsible for the regionalization of the growing limb into subdomains have been isolated from Drosophila. So far, only one gene is known in the leg that is solely required for specifying the distal-most pattern element—the pretarsal claw. In Drosophila, the gene aristaless is expressed in the centre of the antennal and leg imaginal disc that represents the most distal position of appendages, and in a proximal region. When Drosophila aristaless function is impaired, antennae and legs develop without their distal-most structures—the arista and the claw. We describe here the analysis of aristaless in the beetle Tribolium—an insect that shows a different, more ancestral mode of appendage formation than Drosophila. In Tribolium, appendages grow out continuously during embryogenesis, and no imaginal discs are formed. Tribolium aristaless (Tc-al) expression starts midway during appendage elongation, and is seen in a distal and a proximal position of head and trunk appendages. At the end of embryogenesis, Tc-al is seen in four expression domains in the leg, in the dorsal epidermis, and ventrally in every segment in lateral groups of cells, presumably the histoblasts. Like in the Drosophila adult, Tc-al is required in the larva for the formation of the most distal structures of the leg and the antenna as revealed by RNAi experiments. We conclude that aristaless is evolutionarily robust, meaning that it has retained its expressional and functional characteristics, although a heterochronic change of the process of appendage elongation took place towards the evolution of the highly derived diptera.Edited by D. Tautz