Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.     

Isolation and embryonic expression of an abdominal-A-like gene from the lepidopteran, Manduca sexta.

Using sequence homology to the Drosophila Antennapedia gene, we isolated a homeobox-containing gene from the lepidopteran, Manduca sexta. Sequence analysis and in situ hybridizations to tissue sections suggest that the Manduca gene encodes a lepidopteran homologue of the Drosophila Bithorax complex gene abdominal-A. The predicted amino acid sequence of a 76 amino acid region that includes the homeobox and the regions immediately flanking it are identical between the Manduca and Drosophila genes. Northern blots reveal that the manduca abd-A gene is expressed first in the early embryo and continues to be expressed throughout later embryonic and larval stages. In situ hybridizations show that the posterior half of the first abdominal segment marks the anterior border of the Manduca abd-A expression. This expression pattern demonstrates the conservation of parasegments as domains of gene activity in the lepidopteran embryo. The Manduca abd-A expression extends from the posterior half of the first abdominal segment through the tenth abdominal segment, a domain that is greater than that of the Drosophila abd-A expression, and reflects the difference in visible segment number between the two insects.     

Functional similarity in appendage specification by the Ultrabithorax and abdominal-A Drosophila HOX genes.

In Drosophila, the Ultrabithorax, abdominal-A and Abdominal-B HOX genes of the bithorax complex determine the identity of part of the thorax and the whole abdomen. Either the absence of these genes or their ectopic expression transform segments into the identity of different ones along the antero-posterior axis. Here we show that misexpression of Ultrabithorax, abdominal-A and, to some extent, Abdominal-B genes cause similar transformations in some of the fruitfly appendages: antennal tissue into leg tissue and wing tissue into haltere tissue. abdominal-A can fully, and Abdominal-B partially, substitute for Ultrabithorax in haltere development. By contrast, when ectopically expressed, the three genes specify different segments in regions of the main body axis like notum or abdomen. Insects may have originally used the HOX genes primarily to specify this main body axis. By contrast, the homeotic requirement to form appendages is, in some cases, non-specific.     

Homeotic gene expression in the locust Schistocerca: An antibody that detects conserved epitopes in ultrabithorax and abdominal-A proteins

To investigate what role homeotic genes may play in morphological evolution, we are comparing homeotic gene expression in two very different insects, Drosophila (Diptera) and Schistocerca (Orthoptera). In this paper we describe a monoclonal antibody, FP6.87, that recognizes the products of both the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes in Drosophila, via an epitope common to the carboxy terminal region of these two proteins. This antibody recognizes nuclear antigens present in the posterior thorax and abdomen of Schistocerca. We infer that it recognizes the Schistocerca homolog of UBX protein, and probably also of ABD-A. As the distribution of Schistocerca ABD-A protein is already known, we can use this reagent to map the expression of Schistocerca UBX in the thorax and anterior abdomen, where ABD-A is not expressed. Both the general domain, and many of the details, of UBX exp ression are remarkably conserved compared with Drosophila. Thus UBX expression extends back from T2 in the ectoderm (including the CNS), but only from A1 in the mesoderm. As noted for other bithorax complex genes in Schistocerca, expression begins in the abdomen, at or shortly before the time of segmentation. It only later spreads anteriorly to the thorax. For much of embryogene-sis, the expression of UBX in the thoracic epidermis is largely restricted to the T3 limb. Inthis limb, UBX is strikingly regulated, in a complex pattern that reflects limb segmentation. Reviewing these and earlier observations, we conclude that evolutionary changes affect both the precise regulation of homeotic genes within segments, and probably also the spectrum of downstream genes that respond to homeotic gene expression in a given tissue. Overall domains of homeotic gene expression appear to be well conserved between different insect groups, though a change in the extent and timing of homeotic gene expression may underlie the modification of the posterior abdomen in different insect groups. © 1994 Wiley-Liss, Inc.     

Conservation of a functional hierarchy between mammalian and insect Hox/HOM genes.

We have generated several transgenic Drosophila strains containing different mouse Hox genes under heat shock control and studied how their generalized expression affects Drosophila larval patterns. We find that they have spatially restricted effects which correlate with their genetic order and expression pattern in the mouse; as they are expressed more posteriorly in the mouse, they have more extensive effects in Drosophila. The generalized expressions of Hoxd-8 and d-9 modify Drosophila anterior head segment(s), but have no effect in the rest of the body. Hoxd-10 expression affects head and thorax, but not the abdomen. Finally, Hoxd-11 alters head, thorax not the abdomen. Finally, Hoxd-11 alters head, thorax and abdomen. The developmental effect of the Hox genes consists of a homeotic transformation of the affected segment(s), which exhibit a 'ground' pattern similar to that obtained in the absence of homeotic information, suggesting that Hox genes are able to inactivate Drosophila homeotic genes, but do not specify a pattern of their own. A partial exception is Hoxd-11 which, even though it has a general suppressing effect, can also activate the resident Abdominal-B and empty spiracles genes in ectopic positions. Our results strongly suggest a general conservation of the functional hierarchy of homeotic genes that correlates with genetic order and expression patterns.     

Expression patterns of the homeotic genes Scr, Antp, Ubx, and abd-A during embryogenesis of the cricket Gryllus bimaculatus

We have studied embryogenesis of the two-spotted cricket Gryllus bimaculatus as an example of a hemimetabolous, intermediate germ insect, which is a phylogenetically basal insect and may retain primitive features. We observed expression patterns of the orthologs of the Drosophila homeotic genes, Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A) during embryogenesis and compared the expression patterns of these genes with the more basal thysanuran insect, Thermobia domestica (the firebrat), and the derived higher dipteran insect, Drosophila melanogaster. Although Scr is expressed commonly in the presumptive posterior maxillary and labial segment in all three insects, the thoracic expression domains vary. Antp is expressed similarly in the three thoracic segments, the limbs, and the anterior abdominal region among these three insects. The early Antp expression in the firebrat and cricket obeys a segmental register in all three thoracic segments, while in Drosophila its initial expression appears in parasegments 4 and 6. Ubx is expressed in the metathoracic (T3) and abdominal segments similarly in the three insects, whereas the expression pattern in the T3 leg differs among them. abd-A is expressed in the posterior compartment of the first abdominal segment and the remaining abdominal segments in all three insects, although its posterior border varies among them.     

Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation

While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.     

The role of homeotic genes in determining the segmental pattern of chordotonal organs in Drosophila

The homeotic genes are instrumental in establishing segment-specific characteristics. In Drosophila embryos there is ample evidence that the homeotic genes are involved in establishing the differences in the pattern of sense organs between segments. The chordotonal organs are compound sense organs made up of several stretch receptive sensilla. A set of serially homologous chordotonal organs, lch3 in the 1st thoracic segment, dch3 in the 2nd and 3rd thoracic segments and lch5 in abdominal segments 1 to 7, is composed of different numbers of sensilla with different positions and orientations. Here we examine this set of sense organs and a companion set, vchA/B and veh1, in the wild type and mutants for Sexcombs reduced, Antennapedia, Ultrabithorax, and abdominal-A, using immunostaining. Mutant phenotypes indicate that Ultrabithorax and abdominal-A in particular influence the formation of these sense organs. Differential expression of abdominal-A and Ultrabithorax within compartments of individual parasegments can precisely modulate the types of sense organs that will arise from a segment.     

Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system.

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677-2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.     

Compartmental modulation of abdominal Hox expression by engrailed and sloppy-paired patterns the fly ectoderm

In Drosophila, segmentation genes partition the early embryo into reiterative segments along the anterior-posterior axis, while Hox genes assign segments their identities. Each segment is also subdivided into distinct anterior (A) and posterior (P) compartments based on the expression of the engrailed (en) segmentation gene. Differences in Hox expression often correlate with compartmental boundaries, but the genetic basis for these differences is not well understood. In this study, we extend previous results to describe a genetic circuit that controls the differential expression of two Hox genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), within the A and P compartments of the abdominal ectoderm. Consistent with earlier findings, we show that en is essential for high Abd-A levels and low Ubx levels in the P compartment, whereas sloppy-paired (slp) is required for high Ubx levels in the A compartment. Overall, these results demonstrate that the compartmental expression of Ubx and abd-A is established through a repressive regulatory network between en, slp, Ubx and abd-A. We also show that abd-A expression in the P compartment is important for the formation of abdominal-specific cell types, suggesting that en and slp modulation of Hox expression within the A and P compartments is essential for embryonic patterning.     

Pax group III genes and the evolution of insect pair-rule patterning

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. We have investigated the expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, we show that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. We also show that two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect.     

Genetic analysis of the homeotic gene complex (HOM-C) in the beetle Tribolium castaneum

Our laboratories have undertaken both genetic and molecular studies of the homeotic gene complex (HOM-C) of the beetle Tribolium castaneum, and this paper discusses results from our genetic analyses. We describe here the adult phenotypes and complementation behavior of over 50 new mutations. Many of these homeotic phenotypes resemble those of Drosophila melanogaster, but few precisely parallel the segmental transformations seen in this fly. Analysis of putative loss-of-function mutations affecting the head and thorax suggests that the maxillopedia and Cephalothorax genes most closely resemble proboscipedia and Sex combs reduced of Drosophila. In the abdomen, putative loss-of-function alleles of Abdominal affect a domain corresponding to those of the combined abdominal-A and Abdominal-B genes of Drosophila. In contrast to the situation in flies, Abdominal loss-of-function variants in Tribolium cause anteriorward transformations in A3-A5a, but posteriorward transformations in A5p-A7. The implications of the differences in developmental strategies evolved in Tribolium vs Drosophila are discussed.