Identification, characterization and cDNA cloning of two novel proteins secreted into the external space of the regenerating leg of Periplaneta americana

We have isolated two proteins (Rap 60 and Rap 40) that were expressed specifically in regenerating legs of the American cockroach (Periplaneta americana). These proteins appear to be derived from a precursor protein with 443 amino acid residues termed regeneration-associated protein (RAP). Rap 60 and Rap 40 corresponded to Glu 223 to Ser 443 and Glu 223 to Glu 366 of RAP, respectively. Hence, Rap 40 is the N-terminal part of Rap 60. These proteins contained 13 repeats of a novel motif, [Glu/Asp-Glu/Asp-Val/Ala-Lys]. The gene encoding RAP was shown to be expressed during embryogenesis and by newly-formed epidermal cells of regenerating legs. Rap 60 and Rap 40 were secreted into the external space of regenerating leg saccules, where they accumulated, suggesting that they are components of extracellular matrix of regenerating leg saccules.     

Development of the adult leg epidermis in <Emphasis Type="Italic">Manduca sexta</Emphasis>: contribution of different larval cell populations

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.Edited by D. Tautz     

Purification and localization of p10, a novel protein that increases in nymphal regenerating legs of Periplaneta americana (American cockroach).

Content of a protein with a molecular mass of 10 kDa (p10) was found to increase significantly in regenerating legs of the American cockroach (Periplaneta americana). This protein was purified to homogeneity from a homogenate of regenerating legs. Partial amino acid sequencing indicated that p10 is a novel protein. Immunoblotting showed that its content in regenerating legs was 30 times that in normal legs, and decreased significantly when leg regeneration was complete. An immunofluorescence study revealed that p10 localizes exclusively on the external side of newly formed epidermis of regenerating legs.     

Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster

Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins {FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)]} are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates.

Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLO^Dm). FLO^Dm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLO^Dm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLO^Dm message in D. melanogaster embryos reveals that expression of FLO^Dm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLO^Dm gene is localized to chromosomal region 52 B1–B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.     

Involvement of hedgehog,wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs,a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.     

Insertional mutagenesis screening identifies the <Emphasis Type="Italic">zinc finger homeodomain 2</Emphasis> (<Emphasis Type="Italic">zfh2</Emphasis>) gene as a novel factor required for embryonic leg development in <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

The genetic control of leg development is well characterized in the fly Drosophila melanogaster. These control mechanisms, however, must differ to some degree between different insect species to account for the morphological diversity of thoracic legs in the insects. The legs of the flour beetle Tribolium castaneum differ from the Drosophila legs in their developmental mode as well as in their specific morphology especially at the larval stage. In order to identify genes involved in the morphogenesis of the Tribolium larval legs, we have analyzed EGFP enhancer trap lines of Tribolium. We have identified the zfh2 gene as a novel factor required for normal leg development in Tribolium. RNA interference with zfh2 function leads to two alternative classes of leg phenotype. The loss of a leg segment boundary and the generation of ectopic outgrowths in one class of phenotype suggest a role in leg segmentation and segment growth. The malformation of the pretarsal claw in the second class of phenotype suggests a role in distal development and the morphogenesis of the claw-shaped morphology of the pretarsus. This suggests that zfh2 is involved in the regulation of an unidentified target gene in a concentration-dependent manner. Our results demonstrate that enhancer trap screens in T. castaneum have the potential to identify novel gene functions regulating specific developmental processes.     

The effect of streptococcus on the persistence of Brugia malayi and on the production of elephantiasis in cats

Cats were exposed to 200 Brugia malayi larvae on one hind foot over a 3 week period. Six weeks after the initial exposure to B. malayi, 10 of the cats were challenged on both hind legs with a Group G streptococcus. The remaining 10 cats were not exposed to the streptococcus. Following bacterial challenge, the B. malayi-infected leg of 9 of 10 cats displayed sequelae including erysipelas and abscesses. In addition, 5 of the affected legs had an elephantoid appearance, both by gross observation and as seen at necropsy 10 weeks after the initial B. malayi infection. The contralateral, uninfected leg of each cat remained normal in appearance. Histologic processing and examination of the elephantoid tissue showed it to be collagen; eosinophils and mast cells were plentiful in the collagen matrix. In the controls, only 1 animal displayed erysipelas and no abscesses were seen. Lymphedema seen in the B. malayi-infected leg of 5 control cats was less extensive than in uninfected cats challenged with streptococci and at necropsy no significant collagen matrix was evident. The location and number of worms in the lymphatics were noted. This study demonstrated that secondary microbial infections can contribute to the causation of elephantiasis under certain circumstances and that developing B. malayi were in some way adversely affected by the streptococcal involvement of the filaria-infected lymphatics.     

Comparative Density of Hair Sensilla on the Legs of Cavernicolous and Epigean Harvestmen (Arachnida: Opiliones)

To allow an animal to behave appropriately, the location of sensorial structures is expected to be related to their function. As the different leg pairs of arachnids may have different functions (probing x supporting the body), one could expect them to have a different density of sensilla. Moreover, different regions of the same leg (dorsal, lateral, and ventral) would also be expected to have different densities of sensilla, according to the use of each region (e.g., the ventral part is often in contact with the substrate while the dorsal part is not). As cavernicolous animals are expected to be more sensitive than their epigean relatives, one could also expect a different density of sensilla when comparing cavernicolous and epigean animals. Using three epigean and three cavernicolous species of harvestmen (Arachnida, Opiliones), this study aimed at describing the morphology of hair sensilla on the legs and answering three questions: (1) Are there differences in the density of hair sensilla between the dorsal, lateral and ventral regions of each leg pair of the same individual? (2) Are there differences in the density of hair sensilla between the leg pairs of the same individual? (3) Are there differences in the density of hair sensilla when comparing the leg pairs of individuals of cavernicolous and non-cavernicolous species? The tarsi and metatarsi of all right legs of the six studied species were analyzed under a scanning electron microscope. The results (P < 0.05) showed that, in general: the ventral region of the tarsus was denser in sensilla trichodea than the lateral and dorsal regions, particularly on legs I and II; the density of sensilla chaetica did not differ on legs III and IV, but was greater on the dorsal region of legs I and II; the ventral part of legs I had the higher density of sensilla trichodea of the four pairs, whereas the second pair had the lower density; Holcobunus citrinus (Eupnoi) was the species with higher density of sensilla trichodea, on all legs; the cavernicolous species had a lower density of sensilla than the epigean species. The results are tentatively related to harvestmen behavior.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

Spitz/EGFr signalling via the Ras/MAPK pathway mediates the induction of bract cells in Drosophila legs

In the development of Drosophila, the activation of the EGFr pathway elicits different cellular responses at different times and in different tissues. A variety of approaches have been used to identify the mechanisms that confer this response specificity. We have analysed the specification of bract cells in Drosophila legs. We observed that mechanosensory bristles induced bract fate in neighbouring epidermal cells, and that the RAS/MAPK pathway mediated this induction. We have identified Spitz and EGFr as the ligand and the receptor of this signalling, and by ubiquitous expression of constitutively activated forms of components of the pathway we have found that the acquisition of bract fate is temporally and spatially restricted. We have also studied the role of the poxn gene in the inhibition of bract induction in chemosensory bristles.     

Expression of the A12 form of acetylcholinesterase by developing avian leg muscle cells in vivo and during differentiation in primary cell cultures

The accumulation of the molecular forms of acetylcholinesterase (AChE) has been studied in leg muscles during embryonic chick development and in cell cultures initiated with myoblasts obtained from embryos at different stages of development. The collagen-tailed, A₁₂ form appears in leg muscles as soon as day 5 in ovo. An early excision of the lumbar zone of the neural tube at day 2 1/2 in ovo severely delayed the morphological development. In leg muscles dissected at day 12 in ovo from operated embryos, we found that the total amount of AChE activity and particularly the proportion of A₁₂ form were dramatically reduced.

Muscle cells were grown in vitro in a medium supplemented with fetal calf serum. In these conditions, chick muscle cells unequivocally synthesize the A₁₂ form when they originated from muscles which accumulated this form in vivo. In contrast, myoblasts obtained from 5-day old embryo leg muscles did not produce the A₁₂ form either in aneural cultures or in the presence of nerve cells. In relation with previous observations concerning chick myogenesis, we discuss the possibility that this difference reflects the existence of two types of myoblasts. This hypothesis would also explain the results of cocultures performed with nerve cells and normal or demedullated leg muscle myoblasts.     

Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Zebrafish keratin 8 is expressed at high levels in the epidermis of regenerating caudal fin

Cytokeratins are structural proteins of the intermediate filament family and are mainly expressed in epithelial cells. In several vertebrates it has been shown that keratin 8 is expressed in simple epithelial tissues, some non-epithelial tissue and in hyper-proliferative tissues during development and tumor transformation. We previously cloned and characterised the zebrafish (Danio rerio) homologous cytokeratin 8 cDNA (zfk8) which was described as an epidermal marker during zebrafish development. It has been found that the zfk8 gene is normally expressed in simple epithelia in embryonic and mature zebrafish. Using whole-mount in situ hybridisation, we show in this report that expression of zfk8 is tightly linked to the regeneration of caudal fin and exclusively observed in epidermal cells. It is strongly expressed in the epidermis overlaying the inter-rays zone of regenerating caudal fin. Our results indicate that in zebrafish, cytokeratin 8 is a suitable epidermal marker during regeneration.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.     

Temperature modulates epidermal cell size in Drosophila melanogaster

Most ectotherms show increased body size at maturity when reared under colder temperatures. In principle, temperature could produce this outcome by influencing growth, proliferation and/or death of epidermal cells. Here we investigated the effects of rearing temperature on the cell size and cell number in the wing blade, the basitarsus of the leg and the cornea of the eye of Drosophila melanogaster from two populations at opposite ends of a South American latitudinal cline. We found that, in both strains of D. melanogaster and in both sexes, a decrease in rearing temperature increases the size of the wings, legs and eyes through an effect on epidermal cell size, with no significant change in cell number. Our results indicate that temperature has a consistent effect on cell size in the Drosophila epidermis and this may also apply to other cell types. In contrast, the evolutionary effects of temperature on the different organs are not consistent. We discuss our findings in the context of growth control in Drosophila.