Embryonic expression of the engrailed homologue of Rhynchosciara americana

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.     

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.     

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.     

Formation of the arthropod labrum by fusion of paired and rotated limb-bud-like primordia

The nature and origin of the arthropod labrum is a matter much under dispute. We show here that in Tribolium castaneum (Herbst, 1797) the labrum develops from two individual primordia, termed labral buds. Expression of the genes decapentaplegic (dpp) and wingless (wg) in these buds is identical to the buds of the metameric appendages (e.g. thoracic legs), except that the patterns are reversed. We propose that this reversal is the result of the rotation of the labral buds through an angle of approximately 180°. We also for the first time study dpp and wg expression in the fully developed labrum of older embryonic stages. Here, gene expression patterns show that the labrum proper is formed by fusion of the labral buds along their dorsal sides, while their ventral sides are facing outward forming the lateral sides of the fused labrum. Furthermore, we show that there are very similar patterns in another arthropod species, the spider Cupiennius salei (Keyserling, 1877), although in this species the labrum develops as a single structure and not from two separate primordia. However, in C. salei the expression of engrailed is also reversed in addition to the reversal of dpp and wg expression: engrailed is expressed in the anterior half of the labrum, and not in the posterior half like in the remaining appendages. Our results suggest that the arthropod labrum is derived evolutionarily from paired limb-bud-like primordia by rotation and fusion, and that this process is recapitulated ontogenetically to a different extent in different arthropod species.     

<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.     

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.     

<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     

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.     

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.     

Primordium specific requirement of the homeotic gene fork head in the developing gut of the Drosophila embryo

Summary The homeotic gene fork head (fkh) of Drosophila melanogaster promotes terminal as opposed to segmental development in the ectodermal parts of the gut. Molecular analysis revealed that fkh expression is not restricted to the ectodermal parts of the gut, but is detectable in a variety of other tissues. Therefore, the phenotype of fkh mutant embryos was re-examined using molecular probes as tissue specific markers. With the exception of the nervous system, which was not studied, phenotypic effects were found in all tissues expressing fkh protein in the wild-type. Particularly, these tissues include all components of the gut in the Drosophila embryo: the foregut and hindgut, the midgut and the yolk nuclei. The defects observed in the gut of fkh mutant embryos are primordium specific.     

The pleiohomeotic functions as a negative regulator of <Emphasis Type="Italic">Drosophila even-skipped</Emphasis> gene during embryogenesis

Polycomb group (PcG) proteins maintain the spatial expression patterns of genes that are involved in cell-fate specification along the anterior-posterior (A/P) axis. This repression requires cis-acting silencers, which are called PcG response elements (PREs). One of the PcG proteins, Pleiohomeotic (Pho), which has a zinc finger DNA binding protein, plays a critical role in recruiting other PcG proteins to bind to PREs. In this study, we characterized the effects of a pho mutation on embryonic segmentation. pho maternal mutant embryos showed various segmental defects including pair-rule gene mutant patterns. Our results indicated that engrailed and even-skipped genes were misexpressed in pho mutant embryos, which caused embryonic segment defects.     

Terminal versus segmental development in the Drosophila embryo: the role of the homeotic gene fork head

Summary Mutations of the homeotic gene fork head (fkh) of Drosophila transform the non-segmented terminal regions of the embryonic ectoderm into segmental derivatives: Pre-oral head structures and the foregut are replaced by post-oral head structures which are occasionally associated with thoracic structures. Posterior tail structures including the hindgut and the Malpighian tubules are replaced by post-oral head structures associated with anterior tail structures. The fkh gene shows no maternal effect and is required only during embryogenesis. The phenotypes of double mutants indicate that fkh acts independently of other homeotic genes (ANT-C, BX-C, spalt) and caudal. In addition, the fkh domains are not expanded in Polycomb (Pc) group mutant embryos. Ectopic expression of the homeotic selector genes of the ANT-C and BX-C in Pc group mutant embryos causes segmental transformations in terminal regions of the embryo only in the absence of fkh gene activity. Thus, fkh is a region-specific homeotic rather than a selector gene, which promotes terminal as opposed to segmental development. Offprint requests to: Institut für Biologie II (Genetik), Universität Tübingen, Auf der Morgenstelle 28, D-7400 Tübingen, Federal Republic of Germany     

Hedgehog signaling pathway function conserved in Tribolium segmentation

In Drosophila, maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity genes. Wingless and Engrailed appear to have similar roles in both short and long germ segmentation, but relatively little is known about the extent to which Hedgehog signaling is conserved. In a companion study to the Tribolium genome project, we analyzed the expression and function of hedgehog, smoothened, patched, and cubitus interruptus orthologs during segmentation in Tribolium. Their expression was largely conserved between Drosophila and Tribolium. Parental RNAi analysis of positive regulators of the pathway (Tc-hh, Tc-smo, or Tc-ci) resulted in small spherical cuticles with little or no evidence of segmental grooves. Segmental Engrailed expression in these embryos was initiated but not maintained. Wingless-independent Engrailed expression in the CNS was maintained and became highly compacted during germ band retraction, providing evidence that derivatives from every segment were present in these small spherical embryos. On the other hand, RNAi analysis of a negative regulator (Tc-ptc) resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar larval cuticles. These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos. These results suggest that the en–wg–hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect.     

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.     

Two engrailed-related genes in the cockroach: cloning, phylogenetic analysis, expression and isolation of splice variants

engrailed-related genes have been isolated in numerous taxa. Within the insects, some species have a single engrailed-related gene whilst others have two copies, raising the question of when and how often gene duplications have occurred. Here we report the cloning, in the cockroach Periplaneta americana, of two engrailed-related genes Pa-en1 and Pa-en2. By comparing conserved domains and by carrying out a phylogenetic analysis, we conclude that these two genes are likely to be the product of a recent duplication in the cockroach lineage. Pa-en1 and Pa-en2 are co-expressed during early embryogenesis and their segmental pattern of expression appears in an anterior-posterior progression. We have also isolated potential splice variants of Pa-en2 which lack some regulatory domains. The roles these splice variants may play in regulating developmental processes are discussed. Received: 6 August 1999 / Accepted: 4 April 2000     

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     

Histological changes in intestine of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) following in vitro exposure to pathogenic and probiotic bacterial strains

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 × 10⁴ or 6 × 10⁶ viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer’s solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut. I. Salinas thanks Fundacion Seneca for a PhD studentship.     

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

On the anatomy and taxonomy of Cerebratulus hepaticus Hubrecht, 1879 (Nemertini) from the Mediterranean (Banyuls-sur-Mer)

The anatomy of an insufficiently described species of heteronemertine, Cerebratulus hepaticus, is redescribed. The species is characterized by, inter alia, a cephalic lacuna with strands of longitudinal muscle fibres, a proboscis with three muscle layers, neurochord cells in the brain, a dermis in the foregut region with no or few longitudinal muscle fibres which is separated from the outer longitudinal muscle layer by connective tissue. The taxon is compared with two other Cerebratulus species, C. fuscus and C. marginatus. The material was collected at Banyuls-sur-Mer (France) in the Mediterranean.