Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant

 We have analyzed the Tribolium castaneum ortholog of the Drosophila homeotic gene Deformed (Dfd) and determined its expression pattern during embryogenesis in this beetle. Tc Deformed (Tc Dfd) is expressed in the blastoderm and the condensing germ rudiment in a region that gives rise to gnathal segments. During germ band extension Tc Dfd is expressed in the mandibular and maxillary segments, their appendages, and the dorsal ridge. Comparison of insect Dfd protein sequences reveals several highly conserved regions. To determine whether common molecular features reflect conserved regulatory functions we used the Gal4 system to express the Tribolium protein in Drosophila embryos. When Tc Dfd is expressed throughout embryonic ectoderm under the control of P69B, the beetle protein autoregulates the endogenous Dfd gene. In addition, the Drosophila proboscipedia gene (a normal target of Dfd) is ectopically activated in the antennal and thoracic segments. We also compared the ability of the beetle and fly proteins to rescue defects in Dfd ^– mutants by expressing each throughout the embryonic during embryogenesis. Both proteins rescued Dfd ^– defects to the same extent in that they each restore the development of mouth hooks and cirri, as well as cause gain-of-function abnormalities of posterior mouth parts. As before, pb was ectopically activated in the antennal segment. This is the first demonstration of the ability of a heterologous homeotic selector protein to directly regulate a target gene independent of an endogenous Drosophila autoregulatory loop. Received: 11 December 1998 / Accepted: 8 March 1999     

Hox genes in the honey bee Apis mellifera

Hox genes are known to control the identity of serially repeated structures in arthropods and vertebrates. We analyzed the expression pattern of the Hox genes Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), and Ultrabithorax/abdominal-A (Ubx/abd-A) from the honey bee Apis mellifera. We also cloned a cDNA with the complete coding region of the Antennapedia gene from Apis. Comparison with Antp proteins from other insect species revealed several regions of homology. The expression patterns of the isolated Hox genes from Apis showed that the original expression patterns of Dfd, Scr, and Antp appear between late blastoderm and early germ band stage in a temporal and spatial sequence. Each of them shows up as a belt, spanning approximately two segment anlagen, Dfd in the anterior gnathal region, Scr in the posterior gnathal and anterior thoracic region, and Antp in the thoracic region. Following expansion of the Antp domain in the abdomen as a gradient towards the posterior, Ubx/abd-A expression appears laterally in the abdomen. During gastrulation and in the germ band stage the domains of strong expression do not overlap any more, but touch each other. After gastrulation the borders of the expression domains partly correlate with parasegment and partly with segment boundaries. Laterally, gaps between the domain of each gene may show no expression of any of the genes examined. Received: 30 August 1999 / Accepted: 28 April 2000     

Pair-rule patterning in the honeybee Apis mellifera: Expression of even-skipped combines traits known from beetles and fruitfly

 We have studied the binding pattern of antibody mAB 2B8 directed against even-skipped orthologous proteins (EVE) in honeybee embryos. Primary and secondary EVE stripes form in roughly anterior-to-posterior succession; there are 8 primary and 16 secondary stripes. The most posterior primary stripes appear only after the onset of gastrulation. The secondary stripes form by a splitting of primary stripes; they demarcate the parasegmental pattern. While these findings resemble EVE expression in long-germ beetles, the honeybee differs from both beetles and dipterans by two transient pair-rule traits in the parasegmental EVE pattern: the secondary stripes in head and thorax alternate in strength, yet out of register with the Drosophila pattern, and over the whole pattern the odd-numbered stripes vanish earlier than their even-numbered counterparts. As in Drosophila, however, the strong EVE stripes coincide with the weak engrailed (EN) stripes. These findings are taken to indicate that (1) honeybee and beetles share a conserved mode of EVE stripe formation whilst Drosophila has diverged in this respect, (2) honeybee and Drosophila have diverged from the beetles in specific pair-rule traits during the parasegmental expression of both EVE and EN, and (3) some of these traits differ in the register of segment pairing and thus may reflect regulatory divergences at the pair-rule level between dipterans and the honeybee.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

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.     

Segmental organisation of the head in the embryo of Drosophila melanogaster

Summary Embryos of Drosophila melanogaster were irradiated in the presumptive head region with a UV-laser microbeam of 20 m diameter at two developmental stages, the cellular blastoderm and the extended germ band. The ensuing defects were scored in the cuticle pattern of the head of the first-instar larva, which is described in detail in this paper. The defects caused by irradiating germ band embryos when morphologically recognisable lobes appear in the head region were used to establish the segmental origin of various head structures. This information enabled us to translate the spatial distribution of blastoderm defects into a fate map of segment anlagen. The gnathal segments derive from a region of the blastoderm between 60% and 70% egg length (EL) dorsally and 60% and 80% ventrally. The area anterior to the mandibular anlage and posterior to the stomodaeum is occupied by the small anlagen of the intercalary and antennal segments ventrally and dorsally, respectively. The labrum, which originates from a paired anlage dorsally at 90% EL, is separated from the remaining head segments by an area for which we did not observe cuticle defects following blastoderm irradiation, presumably because those cells give rise to the brain. The dorsal and lateral parts of the cephalo-pharyngeal skeleton appear to be the only cuticle derivatives of the non-segmental acron. These structures derive from a dorso-lateral area just behind the putative brain anlage and may overlap the latter. In addition to the segment anlagen, the regions of the presumptive dorsal pouch, anterior lobe and post-oral epithelium, whose morphogenetic movements during head involution result in the characteristic acephalic appearance of the larva, have been projected onto the blastoderm fate map. The results suggest that initially the head of the Drosophila embryo does not differ substantially from the generalised insect head as judged by comparison of fate map and segmental organisation.     

Sites of Fgf signalling and perception during embryogenesis of the beetle Tribolium castaneum

The development of multicellular embryos depends on coordinated cell-to-cell signalling events. Among the numerous cell-signalling pathways, fibroblast growth factors (FGFs) are involved in important processes during embryogenesis, such as mesoderm formation during gastrulation and growth. In vertebrates, the Fgf superfamily consists of 22 family members, whereas only few FGFs are contained in the less complex genomes of insects and worms. In the recently sequenced genome of the beetle Tribolium, we identified four Fgf family members representing three subfamilies. Tribolium has Fgf1 genes that are absent in Drosophila but known from vertebrates. By phylogenetic analysis and microsynteny to Drosophila, we further classify Tc-fgf 8 as an ancestor of pyramus and thisbe, the fly Fgf8 genes. Tc-fgf8 expression in the growth zone suggests an involvement in mesoderm formation. In the embryonic head, expression of Tc-fgf8 subdivides the brain into a larger anterior and a smaller posterior region. The Fgf Tc-branchless is expressed in the embryonic tracheal placodes and in various gland-like structures. The expression patterns of the only Tribolium Fgf receptor and the adaptor molecule Downstream-of-Fgfr are largely congruent with Tc-Fgf8 and Tc-bnl. Thus, in contrast to Drosophila, only one Fgf receptor canalises Fgf signalling in different tissues in Tribolium. Our findings significantly advance our understanding of the evolution of Fgf signalling in insects.     

Cell-Autonomous and Non-cell-autonomous Function of Hox Genes Specify Segmental Neuroblast Identity in the Gnathal Region of the Embryonic CNS in Drosophila

During central nervous system (CNS) development neural stem cells (Neuroblasts, NBs) have to acquire an identity appropriate to their location. In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of NBs prior to their delamination from the neuroectoderm. Compared to the thoracic, ground state segmental units in the head region are derived to different degrees, and the precise mechanism of segmental specification of NBs in this region is still unclear. We identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. We show that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. We identify the secreted molecule Amalgam (Ama) as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, we provide genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental patterning in the developing CNS.     

Subdivision and developmental fate of the head mesoderm in Drosophila melanogaster

In this paper, we define temporal and spatial subdivisions of the embryonic head mesoderm and describe the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the “T-bar” of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. We distinguish between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that we interpret as a rudimentary cephalic aorta (“cephalic vascular rudiment”). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells.     

The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera

Members of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate neurotransmission in insects and are targets of successful insecticides. We have described the cys-loop LGIC superfamily of the honeybee, Apis mellifera, which is an important crop pollinator and a key model for social interaction. The honeybee superfamily consists of 21 genes, which is slightly smaller than that of Drosophila melanogaster comprising 23 genes. As with Drosophila, the honeybee possesses ion channels gated by acetylcholine, γ-amino butyric acid, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel (pHCl), CG8916, CG12344 and CG6927. Similar to Drosophila, honeybee cys-loop LGIC diversity is broadened by differential splicing which may also serve to generate species-specific receptor isoforms. These findings on Apis mellifera enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. DQ667181–DQ667195.     

Changes in the Pattern of Twisted Gastrulation Gene Expression Among Drosophila Species

A long-standing hypothesis posits that morphological changes may be more likely to result from changes in regulation of gene expression than from changes in the protein coding sequences of genes. We have compared the expression pattern of the twisted gastrulation (tsg) gene among five Drosophila species: D. melanogaster, D. simulans, D. subobscura, D. mojavensis, and D. virilis. The tsg gene encodes a secreted protein that is required for the specification of dorsal midline fates in the Drosophila early embryo. TSG is unlike other secreted growth and differentiation factors in Drosophila in that its expression pattern can be experimentally varied and still result in normal development. Because of this, its regulatory region may be freer to diverge than that of other developmental genes whose misexpression may lead to lethal defects. Thus, the tsg gene may be a good indicator of the frequency and nature of evolutionary changes affecting patterns of gene expression. Over ∼60 million years (Myr), the tsg gene has retained a dorsal-on/ventral-off pattern and a middorsal region of expression; but there have been marked changes in the middorsal domain of expression as well as the appearance/loss of other domains of expression along the anterior/posterior axis. Changes between closely related species (∼2–5 Myr since divergence) that are not reflected among more distantly related species suggest frequent changes in gene expression over evolutionary time. These changes in gene expression may serve as the raw material for eventual evolutionary changes in morphology. Received: 24 March 1997 / Accepted: 20 June 1997     

Homeobox gene expression in Brachiopoda: the role of Not and Cdx in bodyplan patterning, neurogenesis, and germ layer specification

The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa. Not is a homeobox containing gene that regulates the formation of the notochord in chordates, while Cdx (caudal) is a ParaHox gene involved in the formation of posterior tissues of various animal phyla. The T. transversa homolog, TtrNot, is expressed in the ectoderm from the beginning of gastrulation until completion of larval development, which is marked by a three-lobed body with larval setae. Expression starts at gastrulation in two areas lateral to the blastopore and subsequently extends over the animal pole of the gastrula. With elongation of the gastrula, expression at the animal pole narrows to a small band, whereas the areas lateral to the blastopore shift slightly towards the future anterior region of the larva. Upon formation of the three larval body lobes, TtrNot expressing cells are present only in the posterior part of the apical lobe. Expression ceases entirely at the onset of larval setae formation. TtrNot expression is absent in unfertilized eggs, in embryos prior to gastrulation, and in settled individuals during and after metamorphosis. Comparison with the expression patterns of Not genes in other metazoan phyla suggests an ancestral role for this gene in gastrulation and germ layer (ectoderm) specification with co-opted functions in notochord formation in chordates and left/right determination in ambulacrarians and vertebrates. The caudal ortholog, TtrCdx, is first expressed in the ectoderm of the gastrulating embryo in the posterior region of the blastopore. Its expression stays stable in that domain until the blastopore is closed. Thereafter, the expression is confined to the ventral portion of the mantle lobe in the fully developed larva. No TtrCdx expression is detectable in the juvenile after metamorphosis. This expression of TtrCdx is congruent with findings in other metazoans, where genes belonging to the Cdx/caudal family are predominantly localized in posterior domains during gastrulation. Later in development this gene will play a fundamental role in the formation of posterior tissues.     

<Emphasis Type="Italic">Drosophila</Emphasis> Bys is nuclear and shows dynamic tissue-specific expression during development

Although the bys-like family of genes has been conserved from yeast to humans, it is not apparent to what extent the function of Bys-like proteins has been conserved across phylogenetic groups. Human Bystin is thought to function in a novel cell adhesion complex involved in embryo implantation. The product of the yeast bys-like gene, Enp1, is nuclear and has a role in pre-ribosomal RNA (pre-rRNA) splicing and ribosome biogenesis. To gain insight into the function of the Drosophila melanogaster bys-like family member, termed bys, we examined bys mRNA expression and the localization of Bys protein. In embryos, bys mRNA is expressed in a tissue-specific pattern during gastrulation. In the larval wing imaginal disc, bys mRNA is expressed in the ventral and dorsal regions of the wing pouch, regions that give rise to epithelia that adhere to one another after the wing disc everts. The bys mRNA expression patterns could be interpreted as being consistent with a role for Bys in events requiring cell-cell interactions. However, embryonic bys mRNA expression patterns mirror those of genes that are potential targets of the growth regulator Myc and encode nucleolar proteins implicated in cell growth. Additionally, in Schneider line 2 (S2) cells, an epitope-tagged Bys protein is localized to the nucleus, suggesting that Drosophila Bys function may be conserved with that of yeast Enp1.Edited by D.A. Weisblat     

<Emphasis Type="Italic">snail</Emphasis> expression during embryonic development of the coral<Emphasis Type="Italic"> Acropora</Emphasis>: blurring the diploblast/triploblast divide?

Although corals are nominally diploblastic, the early development of Acropora millepora involves a process that clearly resembles gastrulation in higher metazoans. This similarity at the morphological level led us to search for the Acropora equivalents of genes whose key roles in gastrulation are conserved across the higher Metazoa. We here report the characterisation of one such gene, snail, which in both Drosophila and the mouse is expressed in cells undergoing an epithelial-mesenchyme transition and/or morphogenetic movements. In addition to an N-terminal SNAG domain, the Acropora snail protein contains four zinc fingers with sequences diagnostic for members of the snail protein subfamily. In situ hybridisation reveals expression in epithelial tissue in the central portion of one side of the flattened pre-gastrulation embryo, which continues to express snail as it is engulfed by its opposite layer. Comparison to snail expression during gastrulation in bilaterians such as Drosophila reveals striking similarities and suggests mechanistic, and possibly evolutionary, links between the processes of mesoderm formation in bilaterians and endoderm formation in the Cnidaria.Edited by P. Simpson     

Functional stability of the aristaless gene in appendage tip formation during evolution

The appendages of an insect are subdivided into distinct segments or podomeres. Many genes responsible for the regionalization of the growing limb into subdomains have been isolated from Drosophila. So far, only one gene is known in the leg that is solely required for specifying the distal-most pattern element—the pretarsal claw. In Drosophila, the gene aristaless is expressed in the centre of the antennal and leg imaginal disc that represents the most distal position of appendages, and in a proximal region. When Drosophila aristaless function is impaired, antennae and legs develop without their distal-most structures—the arista and the claw. We describe here the analysis of aristaless in the beetle Tribolium—an insect that shows a different, more ancestral mode of appendage formation than Drosophila. In Tribolium, appendages grow out continuously during embryogenesis, and no imaginal discs are formed. Tribolium aristaless (Tc-al) expression starts midway during appendage elongation, and is seen in a distal and a proximal position of head and trunk appendages. At the end of embryogenesis, Tc-al is seen in four expression domains in the leg, in the dorsal epidermis, and ventrally in every segment in lateral groups of cells, presumably the histoblasts. Like in the Drosophila adult, Tc-al is required in the larva for the formation of the most distal structures of the leg and the antenna as revealed by RNAi experiments. We conclude that aristaless is evolutionarily robust, meaning that it has retained its expressional and functional characteristics, although a heterochronic change of the process of appendage elongation took place towards the evolution of the highly derived diptera.Edited by D. Tautz     

Lox6, a leech Dfd ortholog, is expressed in the central nervous system and in peripheral sensory structures

 Sequence analysis of a newly isolated Hirudo medicinalis cDNA containing an Antennapedia (Antp)-class homeobox suggests that the corresponding gene, Lox6, is an ortholog of the Drosophila Deformed (Dfd) gene. In situ hybridization of whole-mounted preparations shows that the major sites of Lox6 expression during embryogenesis are the central nervous system (CNS) and the peripheral sensory system. Lox6 mRNA can be detected in a subset of neurons in each ganglion from the subesophageal ganglion (RG2) to the most posterior ganglion, with the highest level of expression seen in RG3. Peripherally, Lox6 is expressed principally in the primordia of the sensillae and in the eyes. This pattern of expression of Lox6 suggests that one of its functions may be to contribute to the diversification of neuronal phenotypes. Received: 16 August 1997/Accepted: 20 December 1997     

An amphioxus Msx gene expressed predominantly in the dorsal neural tube

 Genomic and cDNA clones of an Msx class homeobox gene were isolated from amphioxus (Branchiostoma floridae). The gene, AmphiMsx, is expressed in the neural plate from late gastrulation; in later embryos it is expressed in dorsal cells of the neural tube, excluding anterior and posterior regions, in an irregular reiterated pattern. There is transient expression in dorsal cells within somites, reminiscent of migrating neural crest cells of vertebrates. In larvae, mRNA is detected in two patches of anterior ectoderm proposed to be placodes. Evolutionary analyses show there is little phylogenetic information in Msx protein sequences; however, it is likely that duplication of Msx genes occurred in the vertebrate lineage. Received: 12 October 1998 / Accepted: 26 December 1998