Aldehyde oxidase activity in the tumorous-head strain of Drosophila melanogaster

Gross aldehyde oxidase activity from the egg-stage through 10-day-old adults and distribution of the enzyme in eye-antennal imaginal discs in third instar larvae were determined for the tumorous-head strain of Drosophila melanogaster. Aldehyde oxidase activity of several laboratory strains was measured for comparative purposes. Aldehyde oxidase activity was 100% higher during embryogenesis in tuh(ASU) eggs than in Oregon-R-C eggs. A second period of elevated aldehyde oxidase activity was observed during metamorphosis where tuh(ASU) pupae averaged 65% more enzyme activity than Oregon-R-C. Therefore, during determination and differentiation of the eye-antennal imaginal disc, the tuh(ASU) strain possesses a high aldehyde oxidase activity. Wild-type Drosophila melanogaster antennal imaginal discs are aldehyde oxidase positive, whereas attached eye imaginal discs are apparently aldehyde oxidase negative. A sample of eye-antennal imaginal discs from tuh(ASU) third instar larvae revealed that either one or both eye discs of 64% of the larvae were aldehyde oxidase positive. Aldehyde oxidase activity may be correlated with the homoeotic transformation in parts of the eye disc.     

The role of the peripodial membrane in the morphogenesis of the eye-antennal disc ofDrosophila melanogaster

Summary The early morphogenesis of the eye-antennal disc ofDrosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial membrane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.     

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.     

Fate map of the eye-antennal imaginal disc in the stalk-eyed fly <Emphasis Type="Italic">Cyrtodiopsis dalmanni</Emphasis>

Hypercephaly, in the form of lateral extensions of the head capsule, is observed in several families of Diptera. A particularly extreme form is found in diopsid stalk-eyed flies, in which both eyes and antennae are laterally displaced at the end of eyestalks. We have studied the developmental basis of this exaggerated morphology in Cyrtodiopsis dalmanni. Diopsid eye-antennal imaginal discs are divided into anterior and posterior portions, which are joined by a narrow "disc-stalk" of intervening tissue. We established a fate map for this disc by cutting it into fragments and culturing them in vivo by injecting them into host larvae. The adult eye and dorsal head capsule structures, including the eyestalk and the ocelli, are derived from the posterior portion of the disc, while ventral adult structures such as the antenna and the palpus are derived from the anterior portion of the disc. Thus both posterior and anterior disc portions give rise to structures that are widely separated in the adult head. Moreover, structures that are adjacent in the adult are derived from different regions of the disc. These results confirm and extend previous conclusions about regional identity in diopsid eye-antennal discs that were based on the analysis of molecular markers.     

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.     

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.     

Developmental changes of the antenna and its neurons in the silkworm,Bombyx mori,with special regard to larval-pupal transformation

The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.     

Timing of Wingless signalling distinguishes maxillary and antennal identities in Drosophila melanogaster

The Drosophila adult head mostly derives from the composite eye-antenna imaginal disc. The antennal disc gives rise to two adult olfactory organs: the antennae and maxillary palps. Here, we have analysed the regional specification of the maxillary palp within the antennal disc. We found that a maxillary field, defined by expression of the Hox gene Deformed, is established at about the same time as the eye and antennal fields during the L2 larval stage. The genetic program leading to maxillary regionalisation and identity is very similar to the antennal one, but is distinguished primarily by delayed prepupal expression of the ventral morphogen Wingless (Wg). We find that precociously expressing Wg in the larval maxillary field suffices to transform it towards antennal identity, whereas overexpressing Wg later in prepupae does not. These results thus indicate that temporal regulation of Wg is decisive to distinguishing maxillary and antennal organs. Wg normally acts upstream of the antennal selector spineless (ss) in maxillary development. However, mis-expression of Ss can prematurely activate wg via a positive-feedback loop leading to a maxillary-to-antenna transformation. We characterised: (1) the action of Wg through ss selector function in distinguishing maxillary from antenna; and (2) its direct contribution to identity choice.     

The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis.

Ultraspiracle (usp) encodes the Drosophila cognate of RXR, the human retinoid X receptor. To examine how RXR subfamily members function in development, we have undertaken a phenotypic analysis of usp mutants. usp is required at multiple stages of development for functions that occur in a wide variety of tissues. usp is required in the eye-antennal imaginal disc for normal eye morphogenesis and in the somatic and germline tissues of adult females for fertilization, eggshell morphogenesis and embryonic development. An unusual sunken eye phenotype with marked ventral-dorsal polarity appears to be caused by a lack of usp function in the imaginal disc cells that reside between the eye and antennal anlage. The usp functions include cell autonomous and non-cell autonomous components, suggesting that usp controls the production of factors important for both cell-cell communication and cellular differentiation. These usp signalling pathways have mechanistic parallels to steroid and retinoid action in developing vertebrate tissues.     

Fusion ofDrosophila eye-antennal imaginal discs during differentiation in vitro

Summary Pairs of eye-antennal discs, attached to the cephalic ganglia, were cultured in vitro with a concentration of -ecdysone optimal for imaginal differentiation. The eye-antennal discs fused to form a vesicle inside which the antennae were partially everted, and on the inner surface of which imaginal structures differentiated. The epithelium of the discs was continuous, and an integrated pattern of bristles and hairs differentiated in vitro. In particular, the median ocellus, a unified structure derived partially from each disc, differentiated normally.     

Decapentaplegic head capsule mutations disrupt novel peripodial expression controlling the morphogenesis of the Drosophila ventral head

Drosophila adult structures derive from imaginal discs, which are sacs with apposed epithelial sheets, the disc proper (DP) and the peripodial epithelium (PE). The Drosophila TGF-beta family member decapentaplegic (dpp) contributes to the development of adult structures through expression in all imaginal discs, driven by enhancers from the 3' cis-regulatory region of the gene. In the eye/antennal disc, there is 3' directed dpp expression in both the DP and PE associated with cell proliferation and eye formation. Here, we analyze a new class of dpp cis-regulatory mutations, which specifically disrupt a previously unknown region of dpp expression, controlled by enhancers in the 5' regulatory region of the gene and limited to the PE of eye/antennal discs. These are the first described Drosophila mutations that act by solely disrupting PE gene expression. The mutants display defects in the ventral adult head and alter peripodial but not DP expression of known dpp targets. However, apoptosis is observed in the underlying DP, suggesting that this peripodial dpp signaling source supports cell survival in the DP.     

rap gene encodes Fizzy-related protein (Fzr) and regulates cell proliferation and pattern formation in the developing Drosophila eye-antennal disc

The rap (retina aberrant in pattern) gene encodes the Fizzy-related protein (Fzr), which as an activator of the ubiquitin ligase complex; APC/C (anaphase promoting complex/cyclosome) facilitates the cell cycle stage-specific degradation of cyclins. Loss-of-function mutations in rap cause unscheduled accumulation of cyclin B in the developing eye imaginal disc, resulting in additional mitotic cycles and defective patterning of the developing Drosophila eye. Targeted mis-expression of rap/fzr in the eye primordial cells causes precocious cell cycle exit, and smaller primordial eye fields, which either eliminate or drastically reduce the size of the adult eye. Although mitosis is inhibited in the mis-expression animals, cells with abnormally large nuclei form tumor-like structures from continued endoreplication, cell growth and retinal differentiation. Interestingly, overexpression of Rap/Fzr in the eye primordia also increases the size of the antennal primordium resulting in the induction of ectopic antennae. These results suggest that Rap/Fzr plays an essential role in the timely exit of precursor cells from mitotic cycles and indicate that mechanisms that regulate cell cycle exit are critical during pattern formation and morphogenesis.     

Expression of <Emphasis Type="Italic">defective proventriculus</Emphasis> during head capsule development is conserved in <Emphasis Type="Italic">Drosophila</Emphasis> and stalk-eyed flies (<Emphasis Type="Italic">Diopsidae</Emphasis>)

Hypercephaly, in the form of lateral extensions of the head capsule, is observed in several families of Diptera. A particularly exaggerated form is found in Diopsid stalk-eyed flies, in which both eyes and antennae are laterally displaced at the end of stalks. The processes of early development and specification of the head capsule in stalk-eyed flies are similar to those in Drosophila melanogaster. In Drosophila the homeobox gene ocelliless (oc) shows a mediolateral gradient of expression across the region of the eye-antennal imaginal disc that gives rise to the head capsule and specifies the development of different head structures. The genes and developmental mechanisms that subsequently define head shape in Drosophila and produce hypercephaly in stalk-eyed flies remain unclear. To address this, we performed an enhancer trap screen for Drosophila genes expressed in the same region as oc and identified the homeobox gene defective proventriculus (dve). In the eye-antennal imaginal disc, dve is coexpressed with oc in the region that gives rise to the head capsule and is active along the medial edge of the antennal disc and in the first antennal segment. Analyses of dve expression in mutant eye-antennal discs are consistent with it acting downstream of oc in the development of the head capsule. We confirm that orthologues of dve are present in a diverse panel of five stalk-eyed fly species and analyse patterns of dve sequence variation within the clade. Our results indicate that dve expression and sequence are both highly conserved in stalk-eyed flies.M. Carr and I. Hurley contributed equally to this work.     

Dramatic changes in patterning gene expression during metamorphosis are associated with the formation of a feather-like antenna by the silk moth, Bombyx mori

Many moths use sex pheromones to find their mates in the dark. Their antennae are well developed with lateral branches to receive the pheromone efficiently. However, how these structures have evolved remains elusive, because the mechanism of development of these antennae has not been studied at a molecular level. To elucidate the developmental mechanism of this type of antenna, we observed morphogenesis, cell proliferation, cell death and antennal patterning gene expression in the branched antenna of the silk moth, Bombyx mori. Region-specific cell proliferation and almost ubiquitous apoptosis occur during early pupal stages and appear to shape the lateral branch cooperatively. Antennal patterning genes are expressed in a pattern largely conserved among insects with branchless antennae until the late 5th larval instar but most of them change their expression dramatically to a pattern prefiguring the lateral branch during metamorphosis. These findings imply that although antennal primordium is patterned by conserved mechanisms before metamorphosis, most of the antennal patterning genes are reused to form the lateral branch during metamorphosis. We propose that the acquisition of a new regulatory circuit of antennal patterning genes may have been an important event during evolution of the sensory antenna with lateral branches in the Lepidoptera.     

Segregation of lens and olfactory precursors from a common territory: cell sorting and reciprocity of Dlx5 and Pax6 expression

Cranial placodes are focal regions of columnar epithelium next to the neural tube that contribute to sensory ganglia and organs in the vertebrate head, including the olfactory epithelium and the crystalline lens of the eye. Using focal dye labelling within the presumptive placode domain, we show that lens and nasal precursors arise from a common territory surrounding the anterior neural plate. They then segregate over time and converge to their final positions in discrete placodes by apparently directed movements. Since these events closely parallel the separation of eye and antennal primordia (containing olfactory sensory cells) from a common imaginal disc in Drosophila, we investigated whether the vertebrate homologues of Distalless (Dll) and Eyeless (Ey), which determine antennal and eye identity in the fly, play a role in segregation of lens and nasal precursors in the chick. Dlx5 and Pax6 are initially co-expressed by future lens and olfactory cells. As soon as presumptive lens cells acquire columnar morphology all Dlx family members are down-regulated in the placode, while Pax6 is lost in the olfactory region. Lens precursor cells that express ectopic Dlx5 never acquire lens-specific gene expression and are excluded from the lens placode to cluster in the head ectoderm. These results suggest that the loss of Dlx5 is required for cells to adopt a lens fate and that the balance of Pax6 and Dlx expression regulates cell sorting into appropriate placodal domains.     

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.     

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.