Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.     

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.     

Cloning and expression of the engrailed.a gene of the barnacle Sacculina carcini

 Cirripedia (barnacles) constitute a crustacean monophyletic taxon which is very well defined by several synapomorphies. In particular, all cirripedes are composed of six thoracic segments, but are devoid of any complete abdominal segment. This body plan is preserved in the adult in non-parasitic groups, while the parasitic rhizocephalan cirripedes completely lose arthropodian segmentation at the adult stage. These traits make them a particularly favourable model for studying the formation and maintenance of segmental identity. For the above reasons, it seemed worthwhile to look at the segmentation gene engrailed in a cirripede. A complete engrailed.a cDNA was isolated from larvae of the rhizocephalan cirripede Sacculina carcini. Its expression was monitored during larval development by use of the monoclonal antibody MAb4D9 directed against the Drosophila homologous proteins. The Sacculina engrailed.a gene is expressed during the second and third larval stages in stripes within a posterior area corresponding to the presumptive trunk segments. Surprisingly, these stripes appear in a posterior to anterior sequence. Six engrailed.a stripes characterize the thoracic segments of the cirripedean ground plan. Received: 18 June 1998 / Accepted: 24 October 1998     

The segmentation cascade in the centipede Strigamia maritima: involvement of the Notch pathway and pair-rule gene homologues

The centipede Strigamia maritima forms all of its segments during embryogenesis. Trunk segments form sequentially from an apparently undifferentiated disk of cells at the posterior of the germ band. We have previously described periodic patterns of gene expression in this posterior disc that precede overt differentiation of segments, and suggested that a segmentation oscillator may be operating in the posterior disc. We now show that genes of the Notch signalling pathway, including the ligand Delta, and homologues of the Drosophila pair-rule genes even-skipped and hairy, show periodic expression in the posterior disc, consistent with their involvement in, or regulation by, such an oscillator. These genes are expressed in a pattern of apparently expanding concentric rings around the proctodeum, which become stripes at the base of the germ band where segments are emerging. In this transition zone, these primary stripes define a double segment periodicity: segmental stripes of engrailed expression, which mark the posterior of each segment, arise at two different phases of the primary pattern. Delta and even-skipped are also activated in secondary stripes that intercalate between primary stripes in this region, further defining the single segment repeat. These data, together with observations that Notch mediated signalling is required for segment pattern formation in other arthropods, suggest that the ancestral arthropod segmentation cascade may have involved a segmentation oscillator that utilised Notch signalling.     

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.     

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.     

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.     

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system

 Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity. Received: 10 October 1997 / Accepted: 12 December 1997     

Expression pattern of annelid <Emphasis Type="Italic">Zic</Emphasis> in embryonic development of the oligochaete <Emphasis Type="Italic">Tubifex tubifex</Emphasis>

Embryonic expression of a Zic homologue (Ttu-Zic) was examined in the oligochaete annelid Tubifex tubifex. The body plan of T. tubifex is characterized by obvious segmentation in the ectoderm and mesoderm. Ttu-Zic expression is detected in the mesodermal germ band and a subset of micromere descendants. Ttu-Zic is transiently expressed in primary m-blast cells (i.e., founder cells of mesodermal segments) as early as the time of their birth from M teloblasts. During its development, each mesodermal segment experiences two additional phases of Ttu-Zic expression. Ttu-Zic expression in micromere descendants is seen on the anterior surfaces of embryos undergoing teloblastogenesis; subsequently, these cells proliferate to form bilateral clusters, which then become internalized. Finally, clusters of Ttu-Zic-expressing cells are found in the center of the prostomium, corresponding to the cerebral ganglion. The Ttu-Zic expression profile in the early embryogenesis of T. tubifex may be homologous to those of evolutionarily distant animals.     

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.     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

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     

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.     

<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.     

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     

Gene expression during postembryonic segmentation in the centipede <Emphasis Type="Italic">Lithobius peregrinus</Emphasis> (Chilopoda,Lithobiomorpha)

Postembryonic segmentation (anamorphosis) is widespread among arthropods, but only partially known as for its developmental mechanics and control. Studies on developmental genetics of segmentation in anamorphic arthropods are mostly limited to the germ band stage, during early phases of embryonic development. This work presents the first data on the postembryonic expression of a segmentation gene in a myriapod. Using real-time PCR, we analyzed engrailed expression patterns during the anamorphic stages of the centipede Lithobius peregrinus. A variation pattern in en RNA level during anamorphosis suggests that gene expression is precisely modulated during this period of development and that engrailed is mainly expressed in the posterior part of the body, in the newly differentiating segments of each stage. As anamorphosis is possibly the primitive segmentation mode in arthropods, the postembryonic en expression pattern documented here provides evidence for a conservation of en role in ontogeny, across the embryonic/postembryonic boundary, as well as in phylogeny, across the same boundary, but in the opposite direction, from primitive postembryonic expression to the more derived expression in clades with exclusively embryonic segmentation.     

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.     

Ancestral Patterning of Tergite Formation in a Centipede Suggests Derived Mode of Trunk Segmentation in Trilobites

Trilobites have a rich and abundant fossil record, but little is known about the intrinsic mechanisms that orchestrate their body organization. To date, there is disagreement regarding the correspondence, or lack thereof, of the segmental units that constitute the trilobite trunk and their associated exoskeletal elements. The phylogenetic position of trilobites within total-group Euarthropoda, however, allows inferences about the underlying organization in these extinct taxa to be made, as some of the fundamental genetic processes for constructing the trunk segments are remarkably conserved among living arthropods. One example is the expression of the segment polarity gene engrailed, which at embryonic and early postembryonic stages is expressed in extant panarthropods (i.e. tardigrades, onychophorans, euarthropods) as transverse stripes that define the posteriormost region of each trunk segment. Due to its conservative morphology and allegedly primitive trunk tagmosis, we have utilized the centipede Strigamia maritima to study the correspondence between the expression of engrailed during late embryonic to postembryonic stages, and the development of the dorsal exoskeletal plates (i.e. tergites). The results corroborate the close correlation between the formation of the tergite borders and the dorsal expression of engrailed, and suggest that this association represents a symplesiomorphy within Euarthropoda. This correspondence between the genetic and phenetic levels enables making accurate inferences about the dorsoventral expression domains of engrailed in the trunk of exceptionally preserved trilobites and their close relatives, and is suggestive of the widespread occurrence of a distinct type of genetic segmental mismatch in these extinct arthropods. The metameric organization of the digestive tract in trilobites provides further support to this new interpretation. The wider evolutionary implications of these findings suggest the presence of a derived morphogenetic patterning mechanism responsible for the reiterated occurrence of different types of trunk dorsoventral segmental mismatch in several phylogenetically distant, extinct and extant, arthropod groups.     

<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 zebrafish Hairy/Enhancer-of-split-related gene her6 is segmentally expressed during the early development of hindbrain and somites

Several vertebrate genes of the Hairy/Enhancer-of-split (HES) family are involved in paraxial mesoderm segmentation and intersomitic boundary establishment/maintenance. Here, we show that the zebrafish hairy-related gene, her6, highly homologous to the mammalian and chicken HES-1 genes, is expressed in the posterior part of each segmented somite and in stripes in the anterior presomitic mesoderm (PSM), and also in a dynamic, segmentally restricted pattern during hindbrain segmentation, with all rhombomeres expressing her6 at different time points and at different levels.