Comparative gene expression in the heads of Drosophila melanogaster and Tribolium castaneum and the segmental affinity of the Drosophila hypopharyngeal lobes

SUMMARY Drosophila melanogaster has long played an important role in debates surrounding insect and arthropod head segmentation. It is surprising, therefore, that one important feature of Drosophila head segmentation has remained controversial: namely the position of the boundary between the intercalary and mandibular segments. The Drosophila embryonic head has a pair of structures lying behind the stomodeum known as the hypopharyngeal lobes. Traditionally they have been seen as part of the intercalary segment. More recent work looking at the position of the lobes relative to various marker genes has been somewhat equivocal: segment polarity gene expression has been used to argue for a mandibular affinity of these lobes, while the expression of the anterior-most hox gene labial ( lab ) has supported an intercalary affinity. We have addressed the question of the segmental affinity of the hypopharyngeal lobes by conducting a detailed comparison of gene expression patterns between Drosophila and the red flour beetle Tribolium castaneum , in which the intercalary segment is unambiguously marked out by lab . We demonstrate that there is a large degree of conservation in gene expression patterns between Drosophila and Tribolium , and this argues against an intercalary segment affinity for the hypopharyngeal lobes. The lobes appear to be largely mandibular in origin, although some gene expression attributed to them appears to be associated with the stomodeum. We propose that the difficulties in interpreting the Drosophila head result from a topological shift in the Drosophila embryonic head, associated with the derived process of head involution.     

Evolution of the pair rule gene network: Insights from a centipede

Comparative studies have examined the expression and function of homologues of the Drosophila melanogaster pair rule and segment polarity genes in a range of arthropods. The segment polarity gene homologues have a conserved role in the specification of the parasegment boundary, but the degree of conservation of the upstream patterning genes has proved more variable. Using genomic resources we identify a complete set of pair rule gene homologues from the centipede Strigamia maritima, and document a detailed time series of expression during trunk segmentation. We find supportive evidence for a conserved hierarchical organisation of the pair rule genes, with a division into early- and late-activated genes which parallels the functional division into primary and secondary pair rule genes described in insects. We confirm that the relative expression of sloppy-paired and paired with respect to wingless and engrailed at the parasegment boundary is conserved between myriapods and insects; suggesting that functional interactions between these genes might be an ancient feature of arthropod segment patterning. However, we find that the relative expression of a number of the primary pair rule genes is divergent between myriapods and insects. This corroborates suggestions that the evolution of upper tiers in the segmentation gene network is more flexible. Finally, we find that the expression of the Strigamia pair rule genes in periodic patterns is restricted to the ectoderm. This suggests that any direct role of these genes in segmentation is restricted to this germ layer, and that mesoderm segmentation is either dependent on the ectoderm, or occurs through an independent mechanism.     

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.     

Evolutionary plasticity of collier function in head development of diverse arthropods

The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as “tritocerebral segment.” In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment.     

Spatial regulation of segment polarity gene expression in the anterior terminal region of the Drosophila blastoderm embryo

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene.     

The myth of intercalary segment in insect head

Embryonic development of the head of Oxyrhachis tarandus (Membracidae) has been investigated in detail to settle the controversy of head segmentation and to refute the occurrence of an intercalary segment. The head is formed from six distinct elements: the prostominal lobe, the paired cephalic lobes, the antennal segment and the three noncontroversial gnathal segments. The prostomial lobe, which possesses a neuromere and a pair of coelomic cavities, represents the first body segment, called the prostomial segment. The tritocerebral lobes of the brain and the stomatogastric nervous system, consisting of a frontal ganglion, clypeolabral nerves, and the recurrent nerve etc., develop from the neuromere of the prostomial lobe. The tritocerebrum thus belongs to the prostomial segment rather than to an imaginary intercalary segment and mainly represents the ganglionic center of the stomatogastric nervous system in the brain. Frons, clypeus, and labrum develop from the outer wall of the prostomial lobulate plate, whereas the epipharyngeal wall, including the cibarial pump, develops from its inner wall. The presence of three coelomic cavities and of three distinct neural masses in the cephalic lobes during the initial stages of development shows that they have developed by the fusion of three distinct segments during the long phylogenetic history of insects. The portion of the germ band presently considered as the intercalary segment is actually the sternal part of the antennal segment. The neural cells located in this region give rise to the deutocerebrum by shifting forward, around the stomodaeum, and always leaving a commissure behind. The intercalary segment is thus a complete illusion. The antennal segment is postoral in the beginning and bears a pair of coelomic cavities, but later on it shifts forward and its sternal part invaginates into the stomodaeum.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.     

Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus

Arthropods and vertebrates display a segmental body organisation along all or part of the anterior-posterior axis. Whether this reflects a shared, ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates, segments are formed sequentially by a segmentation 'clock' of oscillating gene expression involving Notch pathway components. Recent studies in spiders and basal insects have suggested that segmentation in these arthropods also involves Notch-based signalling. These observations have been interpreted as evidence for a shared, ancestral gene network for insect, arthropod and bilaterian segmentation. However, because this pathway can play multiple roles in development, elucidating the specific requirements for Notch signalling is important for understanding the ancestry of segmentation. Here we show that Delta, a ligand of the Notch pathway, is not required for segment formation in the cricket Gryllus bimaculatus, which retains ancestral characteristics of arthropod embryogenesis. Segment patterning genes are expressed before Delta in abdominal segments, and Delta expression does not oscillate in the pre-segmental region or in formed segments. Instead, Delta is required for neuroectoderm and mesectoderm formation; embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus, what initially appear to be 'segmentation phenotypes' can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods, and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.     

Expression of engrailed-family genes in the jumping bristletail and discussion on the primitive pattern of insect segmentation

It has been shown that segmentation in the short-germ insects proceeds by a two-step mechanism. The anterior region is simultaneously segmented in a manner similar to that in Drosophila, which is apparently unique to insects, and the rest of the posterior region is segmented sequentially by a mechanism involving a segmentation clock, which is derived from the common ancestor of arthropods. In order to propose the evolutionary scenario of insect segmentation, we examined segmentation in the jumping bristletail, the basalmost extant insect. Using probes for engrailed-family genes for in situ hybridization, we found no sign of simultaneous segmentation in the anterior region of the jumping bristletail embryos. All segments except the anteriormost segment are formed sequentially. This condition shown in the jumping bristletail embryos may represent the primitive pattern of insect segmentation. The intercalating formation of the intercalary segment is assumed to be a synapomorphic trait shared among all insects after the branching of the jumping bristletail.     

Embryonic development of the scorpionfly Panorpa emarginata Cheng with special reference to external morphology (Mecoptera: Panorpidae)

The Mecoptera are thought to be one of the most primitive groups in the Holometabola, but their embryology is rarely studied. By means of scanning electron microscopy, we studied the external features of the embryo of the scorpionfly Panorpa emarginata in middle and late development. The embryo remains in the superficial position until hatching. Embryonic development can be divided into 10 stages along with the first‐instar larva. The external features are described from the germ band to the first‐instar larva, with special reference to the components and segmentation of the head, the segmentation of abdomen and the formation of abdominal prolegs. Our results confirm that the head consists of an anterior‐most acron and six trunk segments: the labral, antennal, intercalary, mandibular, maxillary, and labial segments. The labrum is confirmed to derive from the paired appendages. Our observations also provide additional direct evidence that the abdominal prolegs are not serially homologous with the thoracic legs. The presence of the eleventh abdominal segment is clarified. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.     

Pair-rule genes cooperate to activate en stripe 15 and refine its margins during germ band elongation in the D. melanogaster embryo

Patterns of gene expression have been well documented during embryogenesis for the Drosophila melanogaster trunk segments. The same is not the case for the terminal segments. Here, gene expression patterns are followed during embryogenesis in the caudal segments (A8-A10 and the anal plate), with special attention paid to the novel regulation of engrailed (en). Chosen for this study are the pair-rule genes even-skipped (eve), fushi tarazu (ftz), runt (run), hairy (h), paired (prd) and odd-skipped (odd), and the segment polarity gene (en). The results demonstrate a progressive and coupled translocation of gene expression distally for all genes studied, suggesting that the most posterior segments are determined later than trunk segments.     

Expression of en and wg in the embryonic head and brain of Drosophila indicates a refolded band of seven segment remnants.

Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, we found evidence that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) all of which contribute cells to neuromeres in the central nervous system. Until completion of germ band retraction, the four pregnathal segment remnants and their corresponding neuromeres become arranged in an S-shape. We discuss published evidence for seven head segments and morphogenetic movements during head formation in various insects (and crustaceans).     

Fate-mapping in the procephalic region of the embryonic Drosopbila head

Using intracellular horseradish peroxidase injection we traced the developmental fate of early gastrula cells of the procephalic region in the stage 16/17 embryo. Morphogenetic movements in the developing brain are described in three dimensions. The results are related to head segmentation, and an early gastrula fate map of pregnathal head segments is proposed.