Expression of engrailed in embryos of a beetle and five dipteran species with special reference to the terminal regions

The engrailed expression in embryos of a beetle, four midges and a fly has been analysed with special reference to the terminal regions. In all six species the segmental expression pattern is very similar but variability occurs in the clypeolabrum, foregut and hindgut. In some cases, segmental engrailed expression seems to be extended into the hind- and/or foregut. The engrailed expression of these species is compared with published data from other insects. Correspondence to: U. Schmidt-Ott     

<Emphasis Type="Italic">Engrailed</Emphasis> is expressed in larval development and in the radial nervous system of <Emphasis Type="Italic">Patiriella</Emphasis> sea stars

We documented expression of the pan-metazoan neurogenic gene engrailed in larval and juvenile Patiriella sea stars to determine if this gene patterns bilateral and radial echinoderm nervous systems. Engrailed homologues, containing conserved En protein domains, were cloned from the radial nerve cord. During development, engrailed was expressed in ectodermal (nervous system) and mesodermal (coeloms) derivatives. In larvae, engrailed was expressed in cells lining the larval and future adult coeloms. Engrailed was not expressed in the larval nervous system. As adult-specific developmental programs were switched on during metamorphosis, engrailed was expressed in the central nervous system and peripheral nervous system (PNS), paralleling the pattern of neuropeptide immunolocalisation. Engrailed was first seen in the developing nerve ring and appeared to be up-regulated as the nervous system developed. Expression of engrailed in the nerve plexus of the tube feet, the lobes of the hydrocoel along the adult arm axis, is similar to the reiterated pattern of expression seen in other animals. Engrailed expression in developing nervous tissue reflects its conserved role in neurogenesis, but its broad expression in the adult nervous system of Patiriella differs from the localised expression seen in other bilaterians. The role of engrailed in patterning repeated PNS structures indicates that it may be important in patterning the fivefold organisation of the ambulacrae, a defining feature of the Echinodermata.     

Segmentation in the crustacean Artemia: engrailed staining studied with an antibody raised against the Artemia protein

We have studied the process of post-embryonic segmentation in the anostracan crustacean Artemia franciscana using a specific antibody raised against the engrailed protein of this organism. Three cephalic segments are specified during embryonic development, before larval hatching, whilst trunk (thoracic) segmentation begins after the first stage free-swimming nauplius larva has emerged from the dormant cyst. Thus, cephalic and trunk segmentation seem to be at least in part independent and superimposed processes. Trunk stripes of engrailed expression are added one at a time as segments are generated from the posterior growth zone. The first detectable decision in engrailed expression is the establishment of a line of engrailed-expressing cells, interpretable as delineating the parasegmental boundary. The subsequent widening of engrailed stripes is not correlated with cell lineage events but is probably mediated by the combination of inheritance of the active state and recruitment of new cells into the engrailed-expressing stripe.     

Development of the Deformed protein pattern in the embryo of the honeybee Apis mellifera L. (Hymenoptera)

Summary We have raised antiserum against part of the Deformed (Dfd) protein of the honeybee and describe here the expression pattern of the Dfd protein during honeybee embryogenesis. Dfd protein is first stained in the prospective gnathal region of the cellular blastoderm. This circumferential band corresponds to the distribution of Dfd mRNA described earlier, and to the blastodermal Dfd expression pattern in Drosophila. Using an antibody against the engrailed (en) protein of Drosophila, we found that at the beginning of gastrulation Dfd expression in the honeybee, as in Drosophila, is restricted to the future intercalary, mandibular and maxillary segments. During gastrulation, the mesodermal nuclei loose the Dfd label gradually from anterior to posterior, and in the ectoderm the most posterior ventral cells loose Dfd while retaining en staining; thus, in contrast to what has been described for Drosophila, the posterior Dfd expression border seems to move forward ventrally to the parasegmental boundary within the maxillary segment. In the late germ band, the lateral tips of the Dfd-expressing band are connected across the dorsal side by a row of amnion cells with strongly staining large nuclei. After dorsal closure, a narrow stripe of Dfd-staining dorsal cells behind the neck region may indicate that the maxillary segment contributes to the dorsal body wall posterior to the head capsule. Thus, apart from some minor deviations, the Dfd expression pattern in the honeybee strongly resembles that in Drosophila prior to head involution. This is compatible with the assumption that head involution (which is a special adaption in higher dipterans) ensues after a rather conserved course of early head development in which Dfd appears to play a basic role. Offprint requests to: R. Fleig     

<Emphasis Type="Italic">hedgehog</Emphasis> is a segment polarity gene in a crustacean and a chelicerate

The evolution of arthropod segmentation has been studied by comparing expression patterns of pair-rule and segment polarity genes in various species. In Drosophila, the formation and maintenance of the parasegmental boundaries depend on the interactions between the wingless (wg), engrailed (en) and hedgehog (hh) genes. Until now, the expression pattern of hh has not been analysed to such a great extent as en or wg. We report the cloning and expression analysis of hh genes from Euscorpius flavicaudis, a chelicerate, and Artemia franciscana, a branchiopod crustacean. Our data provide evidence that hh, being expressed in the posterior part of every segment, is a segment polarity gene in both organisms. Additional hh expression sites were observed in the rostrum and appendages of Euscorpius and in the gut of Artemia. From the available data on hh expression in various bilaterians, we review the various hypotheses on the evolution of hh function and we suggest an ancestral role of hh in proctodeum specification and gut formation.Edited by D. Tautz     

Light-dependent PER-like proteins in the cephalic ganglia of an apterygote and a pterygote insect species

Antibodies targeted to a highly conserved tetradecapeptide region of the pivotal biological clock protein PER detect in the firebrat Thermobia domestica a 115-kDa protein and in the cockroach Periplaneta americana a 110-kDa protein that are present in the cytoplasm of a small set of brain cells. A similar cytoplasmic reaction occurs with antisera to the whole PER protein of Drosophila melanogaster, but these antisera also react with numerous cell nuclei. On western blots, they detect an 80-kDa antigen in T. domestica and 70- and 80-kDa antigens in P. americana. No indication of antigen translocation between cell nuclei and cytoplasm was found. Nuclear staining is maintained at a high constant level in T. domestica held at a 12:12 h light:dark photoperiod (LD) or in continuous light, but disappears rapidly in response to extended darkness. In P. americana under LD conditions, the number of immunoreactive nuclei and their staining intensity fluctuate in parallel, with maximal staining late in the day. The circadian changes are maintained in continuous light but all staining vanishes in continuous darkness. A 6-h light pulse in early night of an LD cycle induces maximal staining after about 10 h, suggesting that the effect of light on nuclear PER-like expression is indirect. The behaviour of nuclear antigens is opposite to that of the cytoplasmic PER-like proteins that persist in constant darkness and disappear in constant light. Under LD conditions, the cytoplasmic PER-like antigen cycles in T. domestica but remains at a steady level in P. americana. The sensitivity to photoregime suggests that both the nuclear and the cytoplasmic PER-like antigens are components of the biological clock.R. Závodská and H. Sehadová contributed equally to this work     

Engrailed expression and body segmentation in the honeybee Apis mellifera

Summary Honeybee embryos were stained with a monoclonal antibody raised against the Drosophila engrailed protein. The antibody was found to label rows of nuclei in the transverse grooves that form the earliest external sign of metameric germ band organization. These grooves demarcate metameric units about seven cell rows wide, of which about three rows with reduced apical cell surfaces account for the grooves. The en stripes appear in the grooves as soon as these form and grow from one to about four cells in width and thus completely overlap the groove. During the rudimentary germ band retraction, the grooves shift slightly backwards relative to both the en stripes and the trachdeal pits. The spatio-temporal pattern by which the series of grooves and stripes arises is quite striking. Both become visible first in the gnathal and thoracic regions, then in the pregnathal parts of the head and in the abdomen. The stripes arise essentially in an antero-posterior sequence. In addition, the earliest stripes to form display a pattern of alternating intensities whereas the later stripes, those in the abdomen, arise with approximately equal strength. The latter trait was earlier observed in the grasshopper, while the former is known from Drosophila where, however, the strong stripes correspond to the weak stripes in the honeybee.     

Analysis of neural elements in head-mutant Drosophila embryos suggests segmental origin of the optic lobes

We describe the development of 20 sensory organs in the embryonic Drosophila head, which give rise to 7 sensory nerves of the peripheral nervous system (PNS), and 4 ganglia of the stomatogastric nervous system (SNS). Using these neural elements and the optic lobes as well as expression domains of the segment polarity gene engrailed in the wild-type head of Drosophila embryos as markers we examined the phenotype of different mutants which lack various and distinct portions of the embryonic head. In the mutants, distinct neural elements and engrailed expression domains, serving as segmental markers, are deleted. These mutants also affect the optic lobes to various degrees. Our results suggest that the optic lobes are of segmental origin and that they derive from the ocular segment anteriorly adjacent to the antennal segment of the developing head.     

The involvement of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">wingless</Emphasis> during segmentation in the onychophoran <Emphasis Type="Italic">Euperipatoides kanangrensis</Emphasis> (Peripatopsidae: Onychophora) (Reid 1996)

As the putative sister group to the arthropods, onychophorans can provide insight into ancestral developmental mechanisms in the panarthropod clade. Here, we examine the expression during segmentation of orthologues of wingless (Wnt1) and engrailed, two genes that play a key role in defining segment boundaries in Drosophila and that appear to play a role in segmentation in many other arthropods. Both are expressed in segmentally reiterated stripes in all forming segments except the first (brain) segment, which only shows an engrailed stripe. Engrailed is expressed before segments are morphologically visible and is expressed in both mesoderm and ectoderm. Segmental wingless expression is not detectable until after mesodermal somites are clearly distinct. Early engrailed expression lies in and extends to both sides of the furrow that first demarcates segments in the ectoderm, but is largely restricted to the posterior part of somites. Wingless expression lies immediately anterior to engrailed expression, as it does in many arthropods, but there is no precise cellular boundary between the two expression domains analogous to the overt parasegment boundary seen in Drosophila. Engrailed stripes extend along the posterior part of each limb bud, including the antenna, while wingless is restricted to the distal tip of the limbs and the neurectoderm basal to the limbs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Head segmentation in the embryo of the Colorado beetleLeptinotarsa decemlineata as seen with anti-en immunostaining

Segmentation in the head of the embryo of the Colorado beetleLeptinotarsa decemlineata is described on the basis of anti-engrailed (en) immunostaining of germ band stages. Six segmental units can be identified with this technique. Three segmentalen stripes can be distinguished in the gnathal region, a weak stripe interrupted medially shows the intercalary segment rudiment, a pair of oblique stripes indicate the antennal segment, and one pair of preantennalen spots are taken to indicate a sixth segment. In the broad head lobes of the beetle the spacing of the six segmental units as demarcated byen regions is similar to that in other parts of the germ band. The results are discussed with respect to old and new data concerning the number of head segments and origin of the compound eye in insects.     

The product of the Drosophila melanogaster segment polarity gene armadillo is highly conserved in sequence and expression in the housefly Musca domestica

Summary Segmental pattern in Drosophila melanogaster is set up via a set of cell-cell interactions mediated by the products of the segment polarity genes. Among these is the armadillo gene, whose product seems to be required for the reception of an intercellular signal encoded by the wingless gene. As part of our effort to relate the structure of the armadillo protein to its function within the cell, we have examined the evolutionary conservation of the armadillo gene during insect evolution. We have cloned the armadillo gene from the housefly, Musca domestica, which diverged from Drosophila 100 million years ago. The Musca protein is 97.5% identical to that in Drosophila, while the noncoding sequences have diverged extensively. This remarkable degree of conservation at the protein level is mirrored in the expression pattern of the armadillo protein. Antibodies against the Drosophila protein cross-react with a Musca protein of the appropriate size. We have also used these antibodies to show that the Musca armadillo protein has a pattern of expression in larval and adult tissues similar to that of Drosophila armadillo. We discuss the implications of conservation of structure and expression for the cellular role of the armadillo protein and its mammalian homologs.Offprint requests to: M. Peifer     

Requirement ofwingless signaling andengrailed action in the development and differentiation of reproductive system inDrosophila

The segment polarity geneswingless (wg) andengrailed (en) have been shown to play important roles in pattern formation at different stages ofDrosophila development in the thoracic imaginai discs. We have studied the patterns of expression of these genes in genital discs from wild type larvae, pupae and pharate adults and also from hetero-allelic mutant combinations of these genes. Our results suggest that these genes play vital roles in the normal development and differentiation of genital discs and gonads. In the absence of normalwg oren functions, the flies showed a complete lack of internal accessory reproductive organs and specific defects in the external genitalia. In addition, the testes in such males were small, rounded and with an abnormal cellular organization, although the ovaries in females appeared normal. Temperature shift experiments using the conditional mutant allele ofwg, (wg ^IL-114 ) indicated a requirement ofwg signaling from second instar onwards for normal development and differentiation of the accessory reproductive organs. Using a heat-shock allele (Hs-wg) we also show that the spatially regulated expression ofwg as a pre-requisite for normal development and differentiation. Based on the expression patterns ofen andhedgehog (hh) we suggest that even in the genital disc development and differentiation the action ofen is mediated throughhh.     

The salivary secretion of spinning larvae ofRhynchosciara americana

Summary The secretion present at the lumen of the salivary glands of spinning larvae ofRhynchosciara americana was studied cytochemically and with microspectrophotometry and fluorescence and quantitative polarization microscopy. It was found that structural proteins, including glycoproteins and lipoproteins, occur in this secretion. Findings involving spectral absorption profiles after xylidine ponceau staining, patterns of birefringence and dispersion of birefringence, and lack of dichroism after xylidine ponceau staining and of blue fluorescence after ANS staining are highly suggestive that the secretion ofR. americana differs from classical silks not only in terms of composition but also of macromolecular array. The silk secretion ofR. americana also appears to differ from that of another sciarid,Bradysia spatitergum. Part of the glycoproteins present at the glandular lumen is assumed to be extruded from cells of the posterior zone of the glands, whereas other glycoproteins (or their glycidic radicals) are probably removed from fat body cells via cells of the anterior zone of the glands. The salivary secretion of the spinning larvae ofR. americana contains calcium and is devoid of acid glycosaminoglycans.     

The expression of two engrailed-related genes in an apterygote insect and a phylogenetic analysis of insect engrailed-related genes

 Homologues of the Drosophila segment polarity gene engrailed have been cloned from many insect species, as well as other arthropods and non-arthropods. We have cloned partial cDNAs of two engrailed homologues, which we call engrailed-related genes, from the phylogenetically basal insect, Thermobia domestica (Order Thysanura) and possibly as many as four engrailed-related genes from the phylogenetically intermediate insect, Oncopeltus fasciatus (Order Hemiptera). Previous to our findings, only single engrailed-related homologues had been found in phylogenetically intermediate insect species (Tribolium and Schistocerca) and in the crustacean Artemia, while two engrailed-related homologues have been found in more derived orders (Hymenoptera and the engrailed and invected genes of lepidopterans and dipterans). Consequently, we performed a phylogenetic analysis of insect engrailed-related genes to determine whether insects ancestrally had one or two engrailed-related genes. We have found evidence of concerted evolution among engrailed-related paralogues, however, that masks the true phylogenetic history of these genes; the phylogeny may only be decipherable, therefore, by examining the presence or absence of engrailed-specific and invected-specific motifs, which will require cloning the full length cDNAs from more species. In addition, we examined the embryonic expression pattern of the two Thermobia engrailed-related genes; like Drosophila engrailed and invected, they are expressed in very similar patterns, but show one temporal difference in pregnathal segments that correlates with the tentative phylogenetic placement of the genes. Thermobia engrailed-related expression also confirms that the dorsal ridge is an ancient structure in insects. Received: 4 May 1998 / Accepted: 2 August 1998     

Embryonic expression of the single Tribolium engrailed homolog

We have cloned and sequenced the single Tribolium homolog of the Drosophila engrailed gene. The predicted protein contains a homeobox and several domains conserved among all engrailed genes identified to date. In addition it contains several features specific to the invected homologs of Bombyx and Drosophila, indicating that these features most likely were present in the ancestral gene in the common ancestor of holometabolous insects. We used the cross-reacting monoclonal antibody, 4D9, to follow the expression of the Engrailed protein during segmentation in Tribolium embryos. As in other insects, Engrailed accumulates in the nuclei of cells along the posterior margin of each segment. The first Engrailed stripe appears as the embryonic rudiment condenses. Then as the rudiment elongates into a germ band, Engrailed stripes appear in an anterior to posterior progression, just prior to morphological evidence of the formation of each segment. As in Drosophila (a long germ insect), expression of engrailed in Tribolium (classified as a short germ insect) is preceeded by the expression of several homologous segmentation genes, suggesting that similar genetic regulatory mechanisms are shared by diverse developmental types. © 1994 Wiley-Liss, Inc.     

Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs ofApterous genotypes inDrosophila melanogaster

Summary The aldehyde oxidase staining pattern in wing discs ofDrosophila melanogaster bearing the genotypesap ^blt /ap ^blt andap ^blt andap ^blt /ap ⁷³ⁿ showns changes from the wild-type pattern. Extensive areas of the presumptive dorsal posterior wing blade, which are normally unstained, have enzyme activity in these mutants. In wings of these genotypes, dorsal posterior structures are replaced by dorsal anterior wing structures. A strong correlation has been found between the frequencies of various staining patterns in the discs and the extent of transformation in the cuticular structures of the wing, which is consistent with the idea that aldehyde oxidase activity can be used as an indicator in the wing disc of this transformation. Unlike the homoeotic mutationengrailed, apterous has not been interpreted as a selector gene yet the work reported here shows thatapterous alleles can cause changes resembling those of theengrailed phenotype both in aldehyde oxidase staining behaviour and in the cuticular transformation.     

Identification of two Drosophila TGF-β family members in the grasshopper Schistocerca americana

Intercellular signaling molecules of the transforming growth factor- (TGF-) superfamily are required for pattern formation in many multicellular organisms. The decapentaplegic (dpp) gene of Drosophila melanogaster has several developmental roles. To improve our understanding of the evolutionary diversification of this large family we identified dpp in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. Our analyses indicate a single dpp locus in D. melanogaster and S. americana, suggesting that dpp copy number does not correlate with increasing genome size. Another TGF- superfamily member, the D. melanogaster gene 60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and 60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. Our results for the TGF- superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members.     

Ascobotryozyma americana gen. nov. et sp. nov. and its anamorph Botryozyma americana, an unusual yeast from the surface of nematodes

A new teleomorphic genus Ascobotryozyma, with a single species, A. americana, is proposed. Its anamorph is a Botryozyma that differs from the type species, B. nematodophila, on distributional, physiological, and molecular criteria; it is described as Botryozyma americana, anam. sp. nov. Ascobotryozyma is characterized by globose asci bearing four lunate ascospores. Fusion of thallus cells precedes ascus formation. Ascobotryozyma americana was isolated from the surface of nematodes (Panagrellus dubius) associated with galleries of the poplar borer (Saperda calcarata) in trembling aspen (Populus tremuloides) in eastern Washington, USA. The teleomorph has not been produced in pure culture.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

Evolution of <Emphasis Type="Italic">Hox3</Emphasis> and <Emphasis Type="Italic">ftz</Emphasis> in arthropods: insights from the crustacean <Emphasis Type="Italic">Daphnia pulex</Emphasis>

The Drosophila melanogaster genes zerknüllt (zen) and fushi tarazu (ftz) are members of the Hox gene family whose roles have changed significantly in the insect lineage and thus provide an opportunity to study the mechanisms underlying the functional evolution of Hox proteins. We have studied the expression of orthologs of zen (DpuHox3) and ftz (Dpuftz) in the crustacean Daphnia pulex (Branchiopoda), both of which show a dynamic expression pattern. DpuHox3 is expressed in a complex pattern in early embryogenesis, with the most anterior boundary of expression lying at the anterior limit of the second antennal segment as well as a ring of expression around the embryo. In later embryos, DpuHox3 expression is restricted to the mesoderm of mandibular limb buds. Dpuftz is first expressed in a ring around the embryo following the posterior limit of the mandibular segment. Later, Dpuftz is restricted to the posterior part of the mandibular segment. This is the first report of expression of a Hox3 ortholog in a crustacean, and together with Dpuftz data, the results presented here show that Hox3 and ftz have retained a Hox-like expression pattern in crustaceans. This is in accordance with the proposed model of Hox3 and ftz evolution in arthropods and allows a more precise pinpointing of the loss of ftz “Hox-like behaviour”: in the lineage between the Branchiopoda and the basal insect Thysanura.