Isolation of an abdominal-A gene from the locust Schistocerca gregaria and its expression during early embryogenesis

Using sequence homology to Drosophila homeobox-containing genes, we have cloned a homologue of abdominal-A from the locust Schistocerca gregaria. The Schistocerca clone encodes a stretch of 78 amino acids including the homeodomain and its flanking regions identical to the corresponding region of abdominal-A. We have shown by in situ hybridization that this gene is transcribed and have used an antibody raised against its protein product to examine the expression of abdominal-A during early Schistocerca embryogenesis. Schistocerca is a short germ insect. Although the segmented body plan is very similar to that of Drosophila, the segments are generated sequentially by a process of growth, not simultaneously by subdivision of a syncytial blastoderm. In both organisms, abdominal-A is expressed throughout the abdomen from a sharp anterior boundary located within the first abdominal segment (A1). The initial activation of the genes in the two species differs. Schistocerca initiates expression in a small group of cells in the anterior of A2, shortly after this segment is defined by the appearance of engrailed protein. This contrasts with the appearance of abdominal-A expression in Drosophila, which appears simultaneously throughout the entire abdomen.     

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.     

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.     

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.     

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.     

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.     

Possible implication of Hox genes<Emphasis Type="Italic"> Abdominal-B</Emphasis> and<Emphasis Type="Italic"> abdominal-A</Emphasis> in the specification of genital and abdominal segments in cirripedes

The crustaceans cirripedes (barnacles) are characterised by the lack of fully developed abdominal segments at any stage of their life cycle. However, in nauplius larvae of the cirripede Sacculina carcini, we detected five small engrailed stripes in a postero-dorsal region behind the sixth thoracic segment, that we interpreted as a vestigial abdomen. Here, we present additional morphological and genetic data on Sacculina to further characterise this structure. Scanning electron microscopy analysis confirms the existence of a segmented region in this part of the naupliar body. However, at the late naupliar stage, this structure stops its development and degenerates. This region expresses the Hox gene Abdominal-B, which may indicate that it actually corresponds to the posterior-most part of the Sacculina trunk. In addition, Abdominal-B expression differentiates two types of larvae that probably correspond to male and female larvae, respectively. In contrast, no abdominal-A expression can be detected in the vestigial abdomen. We discuss the possible implication of the loss or divergence of the Abdominal-A protein in the impaired development of abdominal segments in cirripedes.     

Segmental determination in Drosophila central nervous system: analysis of the abdominal-A region of the bithorax complex.

The bithorax complex (BX-C) comprises several genes required for the diversification of posterior segments in Drosophila. The BX-C genes control segment differences not only in the epidermis but in other tissues as well, especially in the central nervous system. We have examined the control of one segment-specific neural structure: the lateral dots, a paired structure present in the first abdominal segment of the larval CNS and absent in all following abdominal segments. Our results show that the suppression of lateral dots in segments A3 and A4 requires the presence of two active copies of one of the BX-C genes, abdominal-A (abd-A). We also show that the adjacent BX-C regions, iab-3 and iab-4, can act in trans on abd-A not only when the two copies of BX-C are paired but also, at least to some extent, when pairing is disturbed.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.     

Segment polarity gene expression in a myriapod reveals conserved and diverged aspects of early head patterning in arthropods

Arthropods show two kinds of developmental mode. In the so-called long germ developmental mode (as exemplified by the fly Drosophila), all segments are formed almost simultaneously from a preexisting field of cells. In contrast, in the so-called short germ developmental mode (as exemplified by the vast majority of arthropods), only the anterior segments are patterned similarly as in Drosophila, and posterior segments are added in a single or double segmental periodicity from a posterior segment addition zone (SAZ). The addition of segments from the SAZ is controlled by dynamic waves of gene activity. Recent studies on a spider have revealed that a similar dynamic process, involving expression of the segment polarity gene (SPG) hedgehog (hh), is involved in the formation of the anterior head segments. The present study shows that in the myriapod Glomeris marginata the early expression of hh is also in a broad anterior domain, but this domain corresponds only to the ocular and antennal segment. It does not, like in spiders, represent expression in the posterior adjacent segment. In contrast, the anterior hh pattern is conserved in Glomeris and insects. All investigated myriapod SPGs and associated factors are expressed with delay in the premandibular (tritocerebral) segment. This delay is exclusively found in insects and myriapods, but not in chelicerates, crustaceans and onychophorans. Therefore, it may represent a synapomorphy uniting insects and myriapods (Atelocerata hypothesis), contradicting the leading opinion that suggests a sister relationship of crustaceans and insects (Pancrustacea hypothesis). In Glomeris embryos, the SPG engrailed is first expressed in the mandibular segment. This feature is conserved in representatives of all arthropod classes suggesting that the mandibular segment may have a special function in anterior patterning.     

Interactions of the Drosophila gap gene giant with maternal and zygotic pattern-forming genes.

The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.     

Scanning electron microscopy of Drosophila melanogaster embryogenesis: III. Formation of the head and caudal segments

The formation of both the anterior most and posterior most segments in higher dipteran embryos involves complex movements of primordia which can be best visualized with the scanning electron microscope. During head formation, the gnathocephalic segments partially involute through the stomodeum. The labial segment forms the floor of the mouth, and the fused maxillary and mandibular segments form the lateral sides of the mouth. The involuted clypeolabrum forms the roof of the mouth. Invaginations of cells for segmentally derived sense organs can be found prior to involution on all the gnathocephalic and thoracic segments as well as on the labrum. The antennal sense organ derives from the lateral surface of the procephalic lobe. Following involution of the mouth parts, the dorsal ridge, which arises just anterior to the first thoracic segment, is drawn over the dorsal procephalic lobe producing the deep dorsal sac. The optic lobes of the brain invaginate anterior to the dorsal ridge just prior to the covering over of the head. The formation of the anal segment is similarly complex. Two rudimentary segments are found posterior to the eighth abdominal segment. During shortening of the germ band, the posterior most segment is drawn around the posterior tip of the embryo to lie ventrally. Two large anal pads form lateral to the anus from this segment. The next segment, following dorsal closure, produces a pair of anal sense organs and a central tuft of setae. Finally, the eighth abdominal segment gives rise to the posterior spiracles. Following dorsal closure these three segments fuse to produce the terminal (anal) segment of the larva.     

hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo

The locus hunchback (hb) is a member of the gap class of segmentation genes of Drosophila. A number of X-ray-induced deletions locate the hb locus at the chromosomal site 85A3-B1, to the right of the pink locus, which maps in the same interval. A total of 14 EMS and 3 X-ray-induced hb alleles have been studied. Homozygous mutant embryos show deletions of segments in two separate regions. In the six strong alleles, the labium and all three thoracic segments are deleted anteriorly while posteriorly the 8th abdominal segment and adjacent parts of the 7th abdominal segment are lacking. The eight weak alleles show smaller deletions both in the thoracic and posterior abdominal region. In the weakest allele only part of the mesothorax is deleted. Three hb alleles produce a homoeotic transformation: superimposed on a strong or weak deletion phenotype, head or thoracic segments are transformed into abdominal segments, respectively. This suggests that hb might also be involved in the regulation of genes in the Bithorax complex (BX-C). Fate mapping of the normal-appearing segments in strong mutant embryos using the UV-laser beam ablation technique (Lohs-Schardin et al., 1979) shows that these segments arise from the normal blastoderm regions. The mutant phenotype can be recognized soon after the onset of gastrulation in a failure to fully extend the germ band. In 6-hr-old mutant embryos, two clusters of dead cells are observed in the thoracic and posterior abdominal region. These observations indicate region specific requirement of hb gene function. The analysis of germ line chimeras by transplantation of homozygous mutant pole cells shows that hb is already expressed during oogenesis. Homozygous mutant embryos derived from a homozygous mutant germ line have a novel phenotype. The anterior affected region is enlarged, including all three gnathal segments and the anterior three abdominal segments. In addition three abdominal segments with reversed polarity are formed between the remaining head structures and the posterior abdomen. Heterozygous mutant embryos derived from a homozygous mutant germ line develop normally, indicating that maternal gene expression is not required for normal development.     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.