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.     

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.     

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.     

Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis

The morphological patterns in the adult cuticle of Drosophila are determined principally by the homeotic genes of the bithorax and Antennapedia complexes. We find that many of these genes become indiscriminately active in the adult epidermis when the Pc gene is eliminated. By using the Pc3 mutation and various BX-C mutant combinations, we have generated clones of imaginal cells possessing different combinations of active homeotic genes. We find that, in the absence of BX-C genes, Pc- clones develop prothoracic patterns; this is probably due to the activity of Sex combs reduced which overrules Antennapedia. Adding contributions of Ultrabithorax, abdominal-A and Abdominal-B results in thoracic or abdominal patterns. We have established a hierarchical order among these genes: Antp less than Scr less than Ubx less than abd-A less than Abd-B. In addition, we show that the engrailed gene is ectopically active in Pc- imaginal cells.     

The developmental effect of overexpressing a Ubx product in Drosophila embryos is dependent on its interactions with other homeotic products

We report the developmental effects of expressing an Ultrabithorax (Ubx) product under the hsp70 promoter. Heat induction gives rise to a high, ubiquitous expression of Ubx product that lasts for several hours. We find that whether or not the overexpression of Ubx has a developmental effect on a particular body region of the larva depends on the interactions with the resident homeotic genes. In head and thorax the Ubx product overrides Sex combs reduced, Antennapedia, and probably other homeotic genes and dictates its own developmental program. In abdominal segments A1-A8 the overexpressed Ubx product establishes a normal pattern, alone (A1) or in combination with abdominal-A (A2-A4) and Abdominal-B (A5-A8), indicating that the excess of product is irrelevant. In segment A9 the highly expressed Ubx product is phenotypically suppressed by the r product of Abdominal-B. The presence of high levels of Ubx protein is also irrelevant in the telson.     

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.     

Homeotic gene expression in the visceral mesoderm of Drosophila embryos.

The visceral mesoderm adhering to the midgut constitutes an internal germ layer of the Drosophila embryo that stretches along most of the anteroposterior axis (parasegment 2-13). Most cells of the midgut visceral mesoderm express exclusively one of five homeotic genes. Three of these genes, Antennapedia, Ultrabithorax and abdominal-A are active in parasegmental domains characteristic for this germ layer as they are nonoverlapping and adjacent. The common boundaries between these domains depend on mutual regulatory interactions between the three genes. The same genes function to control gut morphogenesis. Two further homeotic genes Sex combs reduced and Abdominal-B are expressed at both ends of the midgut visceral mesoderm, although absence of their expression does not appear to affect gut morphogenesis. There are no regulatory interactions between these two and the other homeotic genes. As a rule, the anterior limit of each homeotic gene domain in the visceral mesoderm is shifted posteriorly by one parasegment compared to the ectoderm. The domains result from a set of regulatory processes that are distinct from the ones ruling in other germ layers.     

The Anatomy of the Tarsi of Schistocerca gregaria Forskål

Summary The tarsus of S. gregaria is divided into three units (here called segments) and an arolium set between a pair of claws. The first segment bears three pairs of pulvilli in the fore and middle legs, and one pair and two single pulvilli in the hind legs. Segment two bears a pair of pulvilli, segment three one long pulvillus and the arolium a similar pad on the undersurface. The outer layers of the arolium pad differ from those of the pulvilli in possibly lacking an epicuticle and in having a layer of cuticle which, unlike the corresponding layer in the pulvilli, does not stain with protein stains. The claws and dorsal surfaces bear trichoid sensilla, basiconic sensilla and campaniform sensilla. Smaller basiconic sensilla and canal sensilla occur on the proximal part of the pulvilli, and basiconic sensilla on the arolium undersurface. Internally the cuticle is modified in the arolium and pulvilli so that rods of probably chitin and resilin are formed. This would impart flexibility to the undersurfaces whilst retaining some degree of rigidity which might prevent damage to the small and delicate sense organs on the pulvilli. The tip of the arolium is specialised for adhesion, and there are two large neurones internally which could conceivably monitor attachment or detachment of the tip. There are chordotonal organs in segment three, and several other large neurones throughout the tarsus, some of which are associated with the slings of tissue holding the apodeme in a ventral position. Gland cells occurring in the dorsal epidermis of the adult mature male are also briefly described.     

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.     

The design and analysis of a homeotic response element

We have shown that the 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides us with an assay to analyze the sequence requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent EXD 5'-TGAT core site are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A(-)mut, that on the consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. We observed, however, that the bx1:A(-)mut site was regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions.     

Sensory organs of the thoracic legs of the moth Manduca sexta

Summary The thoracic legs of the moth Manduca sexta acquire a new form and develop a new complement of sensory organs and muscles during metamorphosis from larva to adult. Because of our interest in the reorganization of neural circuitry and the acquisition of new behaviors during metamorphosis, we are characterizing sensory elements of larval and adult legs so that we may determine the contribution of new sensory inputs to the changes in behaviors. Here we describe the sensory structures of adult legs using scanning electron microscopy to view the external sensilla and cobalt staining to examine innervation by underlying sensory neurons. We find that, in contrast to larval legs, the adult legs are covered with a diverse array of sensilla. All three pairs of thoracic legs contain scattered, singly innervated scalelike sensilla. Campaniform sensilla occur singly or in clusters near joints. Hair plates, consisting of numerous singly innervated hairs, are also present near joints. Other more specialized sensilla occur on distal leg segments. These include singly innervated spines, two additional classes of singly innervated hairs, and three classes of multiply innervated sensilla. Internal sensory organs include chordotonal organs, subgenual organs, and multipolar joint receptors.     

Molecular analysis of the deletion mutants in the E homeotic complex of the silkworm Bombyx mori.

The E loci in Bombyx mori are expected to contain a homeotic gene complex specifying the identities of the larval abdominal segments. However, the molecular structure of this complex remains to be determined. We have started to analyze the structural changes in the E complex mutations. We used three newly isolated Bombyx homeobox genes as probes. These genes are probably homologues of the Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) in the Drosophila bithorax complex, because the amino-acid sequences of the homeobox regions in these Bombyx genes are almost identical to those of Drosophila genes. We found that the Bombyx Ubx and abd-A genes are deleted in the EN chromosome, and the Bombyx abd-A gene is deleted in the ECa chromosome. From these results, we conclude that the Bombyx E complex consists of the Ubx, abd-A and possibly Abd-B genes, which may play similar roles to their homologues in the Drosophila bithorax complex.     

The localization and regulation of Antennapedia protein expression in Drosophila embryos

The homeotic Antennapedia (Antp) gene of Drosophila is required for the normal differentiation of the thoracic segments during embryonic development and metamorphosis. Antibodies to a recombinant Antp protein were used to localize the protein in whole mount embryos. Antp is expressed in the nuclei of cells of the thoracic embryonic epidermis and several segments of the ventral and peripheral nervous systems. Analysis of Antp expression in mutant embryos revealed three levels of Antp regulation by genes of the bithorax complex, pleiotropic homeotic loci, and Antp itself. The distributions of the Antp and the Ultrabithorax (Ubx) proteins in doubly-labeled embryos suggest that the Ubx protein may be one direct negative regulator of Antp gene expression.     

Embryonic development and evolutionary origin of the Orthopteran auditory organs

Two different types of ears characterize the order of Orthopteran insects. The auditory organs of grasshoppers and locusts (Caelifera) are located in the first abdominal segment, those of bushcrickets and crickets (Ensifera) are found in the tibiae of the prothoracic legs. Using neuron-specific antibody labelling, we describe the ontogenetic origin of these two types of auditory organs, use comparative developmental studies to identify their segmental homologs, and on the basis of homology postulate their evolutionary origin. In grasshoppers the auditory receptors develop by epithelial invagination of the body wall ectoderm in the first abdominal segment. Subsequently, at least a part of the receptor cells undergo active migration and project their out-growing axons onto the next anterior intersegmental nerve. During this time the receptor cells and their axons express the cell-cell adhesion molecule, Fasciclin I. Similar cellular and molecular differentiation processes in neighboring segments give rise to serially homologous sensory organs, the pleural chordotonal organs in the pregenital abdominal segments, and the wing-hinge chordotonal organs in the thoracic segments. In more primitive earless grasshoppers pleural chordotonal organs are found in place of auditory organs in the first abdominal segment. In bushcrickets the auditory receptors develop in association with the prothoracic subgenual organ from a common developmental precursor. The auditory receptor neurons in these insects are homologous to identified mechanoreceptors in the meso- and metathoracic legs. The established intra- and interspecies homologies provide insight into the evolution of the auditory organs of Orthopterans.