Pattern regulation during the development of the dorsal abdomen in the flesh fly,Sarcophaga agryostoma

Summary In dipteran flies the adult abdominal epidermis is formed from small nests of diploid histoblast cells which spread out and replace the larval epidermis during metamorphosis. The pattern of nest outgrowth and fusion in Sarcophaga shows that the large dorsal hemitergite is normally formed by the two dorsal nests, the spiracle nest and part of the ventral nest (which also forms the hemisternite). By rotating the dorsal histoblast nests, we demonstrate that the adult segment border lies between the flexible intersegmental membrane (ISM) and the naked anterior strip of tergite, the acrotergite. Deletion of histoblast nests often results in a corresponding deletion of adult structures, accompanied by enlargement of adjacent structures within the segment and in neighbouring segments. Pattern formation is not strictly coupled to cell division (as in imaginal discs), since the nests remaining after an ablation, in spreading to fill vacant areas, generate more cells and larger structures than normal. Nest deletions can also result in regeneration, with remaining nests forming additional structures in the dorsal-ventral or anterior-posterior axis of the segment. The deletion of strips of anterior and intersegmental larval epidermis without histoblasts results in the formation of double-posterior duplications of the adult hemitergite. Although these operations damage adjacent histoblast nests, several features of the results suggest that the duplications arise from the interaction (after healing) of histoblasts with larval cells which they would not normally encounter, leading to the intercalation of histoblast cells bearing intervening anterior-posterior positional values. A similar process of intercalation may occur in normal development, as the histoblasts spread from their local origins across the larval epidermal sheet, replacing the larval cells to form the entire epidermis of the adult segment. Offprint requests to: V. French     

DECIPHERING THE EVOLUTIONARY HISTORY AND DEVELOPMENTAL MECHANISMS OF A COMPLEX SEXUAL ORNAMENT: THE ABDOMINAL APPENDAGES OF SEPSIDAE (DIPTERA)

Male abdomen appendages are a novel trait found within Sepsidae (Diptera). Here we demonstrate that they are likely to have evolved once, were lost three times, and then secondarily gained in one lineage. The developmental basis of these appendages was investigated by counting the number of histoblast cells in each abdominal segment in four species: two that represented the initial instance of appendage evolution, one that has secondarily gained appendages, and one species that did not have appendages. Males of all species with appendages have elevated cell counts for the fourth segment, which gives rise to the appendages. In Perochaeta dikowi, which reacquired the trait, the females also have elevated cell count on the fourth segment despite the fact that females do not develop appendages. The species without appendages has similar cell counts in all segments regardless of sex. These results suggest that the basis for appendage development is shared in males across all species, but the sexual dimorphism is regulated differently in P. dikowi.     

Pattern regulation in the ventral histoblasts of the housefly: induction of sternal pattern abnormalities by mechanical wounding of larval epidermal cells

In higher Diptera, two nests of diploid cells called the ventral histoblasts, located one on either side of each abdominal segment among the polytene larval epidermal cells, give rise to the sternite and its surrounding pleura. During metamorphosis of the insect, these two groups of cells migrate and meet with each other in the midventral region of the developing adult. The cuticular pattern elements and pigmentation in the fifth sternite of the male housefly, when compared to those of other segments as well as the tergites of both sexes, are quite distinct. The above-mentioned features, coupled with the smaller number and predictable occurrence of one of the pattern elements in this sternite, viz, the primary forceps, help one to determine the developmental potential of the histoblast nest and the regulation of its potential which occur at the time of fusion of the two contralateral nests of this segment. A simple operation of slitting the larval epidermal cells (LEC) in a hemisegment in the vicinity of the histoblast nest or extirpation or rotation of a small rectangular piece of LEC between the ventral nest and the midventral line produced pattern abnormalities including mirror image duplication in the hemisternite. An analysis of these pattern abnormalities in the different segments and, in particular, in the fifth segment provides a dynamic picture of the formation of the median sternite. Further, these abnormalities indicate the significance of the presence of the intervening pleural cells between the confronting hemisternites under experimental conditions. Thus, each of the fifth ventral nests has the developmental potential to form more than half of the final sternite pattern. Possible mechanisms for the formation of the normal median sternite during metamorphosis and for the formation of duplicated hemisternites and their fusion products under experimental conditions are discussed in light of current models of pattern regulation.     

Regulation and metamorphosis of the abdominal histoblasts ofDrosophila melanogaster

Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.     

The Ultrabithorax gene of Drosophila and the specification of abdominal histoblasts.

Separation of the imaginal and larval developmental pathways in Drosophila occurs early in embryogenesis, resulting in the formation of imaginal discs and abdominal histoblast nests along the larval body wall. The dorsal and ventral histoblast nests within the first abdominal (A1) segment are shown not to be segmentally homologous with the metathoracic (T3) haltere and leg discs, respectively, since they occur at distinct dorso-ventral locations during normal development and can be found together within the same segment in mutants of the Bithorax complex (BX-C) where T3 is transformed towards A2-A4 or A1 towards T3. Several patterning abnormalities are also observed in BX-C mutants. A ventral shift in the A1 ventral nest occurs in partially transformed larvae harboring weak bithoraxoid (bxd) mutations; in more fully transformed larvae (Ubx1/Df) both the anterior dorsal and ventral nests are lost and instead a dorsal and ventral disc bud are formed. Dorso-ventral inversions in the pattern of the ventral nest occur in a random fashion throughout A1-A7 in response to an increase or decrease in the gene dosage of the BX-C. In gain-of-function mutants anterior dorsal histoblast cells form in the homologous anterior as well as the nonhomologous posterior portion of T3. Based on these and other findings it appears that the Ultrabithorax (Ubx) locus (and possibly abdominal-A and Abdominal-B) is required to steer ectodermal cells toward an imaginal histoblast rather than a larval cell fate at specific regions within the first abdominal segment.     

Negative growth control of mitotically active imaginal cells (histoblasts) of the abdominal epidermis during metamorphosis of the housefly

On the ventral side of each pupal abdominal segment of the housefly, there is a pair of histoblast nests, each containing about 600 diploid cells. These cells, during adult development, divide, replace intervening polytene larval epidermal cells (LEC), and form both the median sternite and the surrounding pleura of the adult segment. Since the histoblast nests and the LEC form a contiguous layer, we examined the role of these two types of cells in regulating the mitotic potential of the histoblasts during development of the median sternite. Two experimental approaches were used: deletion of one of the nests by thermocautery; and by disturbance of the continuity of the monolayered epidermis by thermocautery of, or topical application of heptanol on, the midventral LEC. Ablation of one of the contralateral nests resulted in a mirror image duplication of the hemisternite and pleura by the surviving nest. Disturbance of the continuity of the LEC produced mirror image duplication of the hemisternal pattern by each of the contralateral nests. From these results, we propose that the contralateral ventral nests mutually downregulate their mitotic potential by secreting regulatory factor(s) to produce the normal median sternite pattern and surrounding pleura. We also suggest that these chemicals act in a paracrine fashion, possibly through gap junctions in the LEC. © 1994 Wiley-Liss, Inc.     

Segment- and nest-specific determination in the histoblasts of the housefly

Summary Using a novel grafting procedure for histoblasts, we have transplanted the fifth dorsal or ventral histoblast nests to heterotopic positions in the abdomen of the prepupa of the housefly to find out how rigid are these imaginai cells in their commitment to form their respective segmental pattern. Our results clearly show that these histoblasts survive in their new positions and form patterns according to their original determined state.     

Embryogenesis of a springtail,Tomocerus ishibashii (Collembola,tomoceridae): External morphology

The external features of the developing embryos of the springtail, Tomocerus ishibashii, are described. The clypeolabral anlage arises as a single, unpaired swelling. The entognathy is completed by the ventral growth of the tergal anlagen of mandibular, maxillary, and labial segments. These anlagen also form the posterior part of the cranium. The palpi of maxilla and labium are homologous with the telopodites, and proximal parts of these head appendages are homologous with the coxopodites. The sternal element of the labial segment does not participate in the postmentum formation. The anlagen of abdominal appendages appear in the first to the fourth abdominal segments. The first, third, and fourth appendage anlagen form the ventral tube, tenaculum, and furcula, respectively. The fused proximal parts of the first, third, and fourth appendage anlagen are homologous with the coxopodites, and the distal parts which do not fuse are homologous with the telopodites. The anlagen of the second abdominal appendages become flattened and spread over the ventral side of this segment. The ventral structures of the first four abdominal segments are appendicular in origin.     

Nonautonomous apoptosis is triggered by local cell cycle progression during epithelial replacement in Drosophila

Tissue remodeling involves collective cell movement, and cell proliferation and apoptosis are observed in both development and disease. Apoptosis and proliferation are considered to be closely correlated, but little is known about their coordinated regulation in physiological tissue remodeling in vivo. The replacement of larval abdominal epidermis with adult epithelium in Drosophila pupae is a simple model of tissue remodeling. During this process, larval epidermal cells (LECs) undergo apoptosis and are replaced by histoblasts, which are adult precursor cells. By analyzing caspase activation at the single-cell level in living pupae, we found that caspase activation in LECs is induced at the LEC/histoblast boundary, which expands as the LECs die. Manipulating histoblast proliferation at the LEC/histoblast boundary, either genetically or by UV illumination, indicated that local interactions with proliferating histoblasts triggered caspase activation in the boundary LECs. Finally, by monitoring the spatiotemporal dynamics of the S/G₂/M phase in histoblasts in vivo, we found that the transition from S/G₂ phases is necessary to induce nonautonomous LEC apoptosis at the LEC/histoblast boundary. The replacement boundary, formed as caspase activation is regulated locally by cell-cell communication, may drive the dynamic orchestration of cell replacement during tissue remodeling.     

The structure of the normal puparium and in segmentation disorders in Drosophila

In Drosophila puparium is a cuticular mould of external structures formed by the larval cells and can, therefore, preserve traces of developmental defects which appeared in embryogenesis. In this way ontogenetic relationship between embryonic defects and developmental defects in adult individuals can be established. The normal pattern of segmentation in puparia of Drosophila melanogaster was established by comparing segmentation defects in adult flies and corresponding puparia. Boundaries of segment and compartments have been determined, imaginal rudiments of abdominal segments (nests of histoblasts) and rudiments of trachea have been localized on puparia.     

Characterization of abdominal appendages in the sawfly, Athalia rosae (Hymenoptera), by morphological and gene expression analyses

Larvae of the sawfly, Athalia rosae, have remarkable abdominal prolegs. We analyzed the morphogenesis of appendages and the expression of decapentaplegic and Distal-less genes during embryonic development to characterize the origin of prolegs. Proleg primordia in abdominal segments A1–A9 appeared shortly after the inner lobes (endites) of gnathal appendages were formed. These were located on the ventral plates, medioventral to the appendages of the other segments in light of serial homology. Nothing was seen where the main axis of the appendage should develop in abdominal segments. The primordia in A1 and A9 disappeared before larval hatching. Anal prolegs appeared separate from cerci, the main axes of appendages, which were formed temporarily in A11. The expression of decapentaplegic, which reflects the primary determination of appendages, was detected in the lateral juxtaposition with the prolegs. Distal-less was expressed in the main axes of appendages, protruding endites and the cerci, but not in prolegs and anal prolegs or the gnathal endites which do not protrude. These findings suggest a possibility that the abdominal and anal prolegs of A. rosae are outgrowths of ventral plates which derived from coxopodal elements, but not main axes of appendages.     

The result of evolutionary limb loss on the complement of motor neurons in a crayfish limb nerve revealed by serial homology

Many macruran decapod crustaceans show sexual dimorphism of abdominal appendages adapted for use as secondary reproductive organs. Not only does the Australian crayfish, Cherax destructor, show no external, abdominal dimorphism, but both males and females have lost the pleopods of the first abdominal segment entirely. The first nerves of the abdominal ganglia of crayfish and lobsters carry the axons of the pleopod motor neurons. We used intracellular cobalt infusion into the first nerves of the first and second abdominal ganglia to reveal the motor neuron complement of these ganglia in males and females. The first nerves of the second abdominal ganglia of both males and females have approximately 37 motor neurons associated with them. The homologous nerves in the first abdominal segment, where there are no pleopods, have only 8 or 9 motor neurons associated with them. The evolutionary implications of this difference are discussed.     

AN ANATOMICAL STUDY OF THE ABDOMINAL NERVOUS AND MUSCULAR SYSTEMS OF DRAGONFLY (AESHNIDAE) NYMPHS

The musculature of the fourth to eighth abdominal segments is typically composed of twenty pairs of segmental muscles associated with the body wall. In the first to third and ninth and tenth segments certain modifications to the basic plan occur in association with the abdominal-thoracic junction, the respiratory apparatus and the anal appendages. In some segments there are also paired muscles associated with the alimentary canal. Two large transverse muscles are present in the abdomen. There are eight abdominal ganglia, the first seven of which each give rise to three pairs of lateral nerves, the eighth to five pairs. In addition there are ten median abdominal nerves. The innervation fields of the various nerves are described. The first three pairs of lateral nerves of the last ganglion are homologous with the lateral nerves of the other abdominal ganglia; the fourth pair innervates most of segment nine; and the fifth pair innervates the remainder of segment nine, segment ten and the anal appendages. Certain of the abdominal muscles are innervated by branches from two different nerve roots. In segments six and seven the anterior point of attachment of the longitudinal stretch receptors is normally different from that in the other abdominal segments. This is discussed in the light of the types of movement which involve the abdomen and it seems apparent that these receptors are affected not only by swimming and abdominal flexion, as are the other longitudinal stretch receptors, but also by respiratory movements. Two distinct types of epidermal sensilla are present on the abdomen, spines and hairs. The former are the more numerous on the body, the latter on the anal appendages.     

Role of abd-A and Abd-B in Development of Abdominal Epithelia Breaks Posterior Prevalence Rule

Hox genes that determine anteroposterior body axis formation in all bilaterians are often found to have partially overlapping expression pattern. Since posterior genes dominate over anterior Hox genes in the region of co-expression, the anterior Hox genes are thought to have no function in such regions. In this study we show that two Hox genes have distinct and essential functions in the same cell. In Drosophila, the three Hox genes of the bithorax complex, Ubx, abd-A and Abd-B, show coexpression during embryonic development. Here, we show that in early pupal abdominal epithelia, Ubx does not coexpress with abd-A and Abd-B, while abd-A and Abd-B continue to coexpress in the same nuclei. The abd-A and Abd-B are expressed in both histoblast nest cells and larval epithelial cells of early pupal abdominal epithelia. Further functional studies demonstrate that abd-A is required in histoblast nest cells for their proliferation and suppression of Ubx to prevent first abdominal segment like features in posterior segments while in larval epithelial cells it is required for their elimination. We also observed that these functions of abd-A are required in its exclusive as well as the coexpression domain with that of Abd-B. The expression of Abd-B is required in histoblast nest cells for their identity while it is dispensable in the larval epithelial cells. The higher level of Abd-B in the seventh abdominal segment, that down-regulates abd-A expression, leads this segment to be absent in males or of smaller size in females. We also show that abd-A in histoblast nest cells positively regulates expression of wingless for the formation of the abdominal epithelia. Our study reveals an exception to the rule of posterior prevalence and shows that two different Hox genes have distinct functions in the same cell, which is essential for the development of abdominal epithelia.     

Selection on bristle length has the ability to drive the evolution of male abdominal appendages in the sepsid fly Themira biloba

Many exaggerated and novel traits are strongly influenced by sexual selection. Although sexual selection is a powerful evolutionary force, underlying genetic interactions can constrain evolutionary outcomes. The relative strength of selection vs. constraint has been a matter of debate for the evolution of male abdominal appendages in sepsid flies. These abdominal appendages are involved in courtship and mating, but their function has not been directly tested. We performed mate choice experiments to determine whether sexual selection acts on abdominal appendages in the sepsid Themira biloba. We tested whether appendage bristle length influenced successful insemination by surgically trimming the bristles. Females paired with males that had shortened bristles laid only unfertilized eggs, indicating that long bristles are necessary for successful insemination. We also tested whether the evolution of bristle length was constrained by phenotypic correlations with other traits. Analyses of phenotypic covariation indicated that bristle length was highly correlated with other abdominal appendage traits, but was not correlated with abdominal sternite size. Thus, abdominal appendages are not exaggerated traits like many sexual ornaments, but vary independently from body size. At the same time, strong correlations between bristle length and appendage length suggest that selection on bristle length is likely to result in a correlated increase in appendage length. Bristle length is under sexual selection in T. biloba and has the potential to evolve independently from abdomen size.     

A clonal analysis of tergite development in Drosophila of ultraabdominal and paradoxical genotypes.

The developmental parameters of homeotic second abdominal anlage cells in flies with Ultraabdominal and paradoxical genotypes are compared with those of normal second abdominal anlage cells through the use of induced mitotic recombination to mark the clonal descendants of single anlage cells. Homeotic and normal second abdominal anlage cells show the same pattern of mitotic activity during development. The homeotic second abdominal anlage cells with Ultraabdominal genotype proliferate to the same extent as normal anlage cells during hemitergite formation. However, the proliferation of homeotic second abdominal anlage cells with paradoxical genotype is decreased due to the failure of some daughter cells either to divide or to differentiate normally. The number of anlage cells in a homeotic second abdominal histoblast with Ultraabdominal genotype is slightly smaller and more variable than that in a normal second abdominal histoblast. The number of anlage cells in a homeotic second abdominal histoblast with paradoxical genotype is much smaller and much more variable than that in a normal second abdominal histoblast. These results are discussed in relation to mechanisms governing cell determination. In addition, some aspects of pattern formation in incomplete homeotic second abdominal hemitergites are presented and discussed.     

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis

The fusion of epithelial sheets is an essential morphogenetic event. Here, we study the development of the abdomen of Drosophila as a model of bounded epithelia expansion and uncover a complex multistep process for the generation of the adult epidermis from histoblasts, founder cells that replace the larval cells during metamorphosis. We find that histoblasts experience a biphasic cell cycle and emit apical projections that direct their invasive planar intercalation in between larval cells. Coordinately, the larval cells extrude from the epithelia by apical constriction of an actomyosin ring and as a consequence die by apoptosis and are removed by circulating haemocytes. We demonstrate that the proliferation of histoblasts and the death of larval cells are triggered by two independent extrinsic Ecdysone hormonal pulses. Finally, we show that histoblast spreading and the death of larval cells depend on a mutual exchange of signals and are non-autonomous processes.     

Influence of RNAi knockdown for E-complex genes on the silkworm proleg development

Larvae of many holometabolous insects possess abdominal appendages called prolegs. Lepidoptera larvae have prolegs in the segments A3-A6. Functions of Lepidoptera hox genes on these abdominal appendages development is still a controversial issue. In this article, we report the use of double strand RNA (dsRNA)-mediated interference (RNAi) to dissect the function of some hox genes, specifically E-complex genes Ubx, abd-A, and Abd-B, in the ventral appendage development of the Lepidoptera silkworm, Bombyx mori. We found that Ubx RNAi caused leg identity in A1 segment, abd-A RNAi caused severe defect of abdominal prolegs and Abd-B RNAi allowed proleg identity in more posterior abdominal segments. These results confirm that Lepidoptera hox genes Ubx and Abd-B have evolved the repressing function to ventral appendage development, which is similar to those of Drosophila. However, Lepidoptera abd-A might have been modified distinctively during evolution, and has important roles in directing the development of prolegs.     

Changing gears in the Cell Cycle: Histoblasts and beyond

Although the molecular elements controlling cell cycle progression are well established, the mechanisms regulating how cell proliferation is triggered in response to extrinsic stimuli and how cell divisions change speed, particularly in stem or tumor cells or regenerative tissues, are poorly understood. One exceptional model system in which these events are precisely defined is Drosophila abdominal morphogenesis, in which stem-like histoblasts build the adult epidermis at metamorphosis by undergoing a series of sequential transitions from a non-proliferative to a growing, and finally to an invasive epithelium. We have recently uncovered in histoblasts an internal logic modulating cell cycle transitions that should constitute a reference paradigm for the study of other equivalent processes in stem cell, cancer or developmental biology.