Trunk-specific modulation of wingless signalling in Drosophila by teashirt binding to armadillo.

BACKGROUND: One function of the Wingless signal cascade is to determine the 'naked' cuticle cell-fate choice instead of the denticled one in Drosophila larvae. Wingless stabilises cytoplasmic Armadillo, which may act in a transcriptional activator complex with the DNA-binding protein T-cell factor (also known as Pangolin). As these components are critical for all Wingless-dependent patterning events, the problem arises as to how specific outputs are achieved. RESULTS: The Teashirt zinc finger protein was found to be necessary for a subset of late Wingless-dependent functions in the embryonic trunk segments where the teashirt gene is expressed. Teashirt was found to be required for the maintenance of the late Wingless signalling target gene wingless but not for an earlier one, engrailed. Armadillo and Teashirt proteins showed similar Wingless-dependent modulation patterns in homologous parts of each trunk segment in embryos, with high levels of nuclear Teashirt and intracellular Armadillo within cells destined to form naked cuticle. We found that Teashirt associates with, and requires, Armadillo in a complex for its function. CONCLUSIONS: Teashirt binds to, and requires, Armadillo for the naked cell-fate choice in the larval trunk. Teashirt is required for trunk segment identity, suggesting that Teashirt provides a region-specific output to Armadillo activity. Further modulation of Wingless is achieved in homologous parts of each trunk segment where Wingless and Teashirt are especially active. Our results provide a novel, cell-intrinsic mechanism to explain the modulation of the activity of the Wingless signalling pathway.

Morphogen gradients. A question of time or concentration?

Morphogens are secreted proteins that organize surrounding tissues into distinct territories and are thought to act as a function of a threshold of their concentration. Although it has been demonstrated that morphogens act directly on the cells and do not rely on secondary signalling relays, intracellular sequential induction mechanisms, which are dependent on a simple signalling instruction, have not been excluded. Here, we present an alternative model to account for the organizing properties of morphogens, and propose that initial exposure to signalling changes cell context, which in combination with continuing morphogen activity, results in the expression of novel targets.

The levels of the bancal product, a Drosophila homologue of vertebrate hnRNP K protein, affect cell proliferation and apoptosis in imaginal disc cells.

We have characterized the Drosophila bancal gene, which encodes a Drosophila homologue of the vertebrate hnRNP K protein. The bancal gene is essential for the correct size of adult appendages. Reduction of appendage size in bancal mutant flies appears to be due mainly to a reduction in the number of cell divisions in the imaginal discs. Transgenes expressing Drosophila or human hnRNP K are able to rescue weak bancal phenotype, showing the functional similarity of these proteins in vivo. High levels of either human or Drosophila hnRNP K protein in imaginal discs induces programmed cell death. Expression of the antiapoptotic P35 protein suppresses this phenotype in the eye, suggesting that apoptosis is the major cellular defect caused by overexpression of K protein. Finally, the human K protein acts as a negative regulator of bancal gene expression. We propose that negative autoregulation limits the level of Bancal protein produced in vivo.

Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt.

In Drosophila the teashirt gene, coding for a zinc finger protein, is active in specific body parts for patterning. For example, Teashirt is required in the trunk (thorax and abdomen) tagmata of the embryo, parts of the intestine and the proximal parts of appendages. Here we report the isolation of vertebrate cDNAs related to teashirt. As in Drosophila, human and murine proteins possess three widely spaced zinc finger motifs. Additionally, we describe the expression patterns of the two murine genes. Both genes show regionalized patterns of expression, in the trunk, in the developing limbs and the gut.

Transcriptional regulation of the Drosophila homeotic gene teashirt by the homeodomain protein Fushi tarazu.

The Drosophila melanogaster gene teashirt (tsh) is essential for segment identity of the embryonic thorax and abdomen. A deletion 3' to the tsh transcription unit causes the loss of tsh early expression in the even-numbered parasegments, and the corresponding larval cuticular patterns are disrupted. tsh function in the odd-numbered parasegments in these mutants is normal by both criteria. The in vivo activities of genomic fragments from the deleted region were tested in transgenic embryos. A 2.0 kb enhancer from the 3' region acts mainly in the even-numbered parasegments and is dependent on fushi tarazu (ftz) activity, which encodes a homeodomain protein required for the development of even-numbered parasegments. Ftz protein binds in vitro to four distinct sequences in a 220 bp sub-fragment; these and neighboring sequences are conserved in the equivalent enhancer isolated from Drosophila virilis. Tsh protein produced under the control of the 220 bp enhancer partially rescues a null tsh mutation, with its strongest effect in the even-numbered parasegments. Mutation of the Ftz binding sites partially abrogates the capacity for rescue. These results suggest a composite mechanism for regulation of tsh, with different activators such as ftz contributing to the overall pattern of expression of this key regulator.

Identification of a regulatory allele of teashirt (tsh) in Drosophila melanogaster that affects wing hinge development. An adult-specific tsh enhancer in Drosophila.

A cis-acting regulatory element defined herein is required to drive teashirt (tsh) expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. This represents the first description of a viable adult-specific regulatory allele of tsh with a visible phenotype, and it enlarges our understanding of the expression of tsh and its function during the development of the adult.

The Drosophila teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modifier of variegation, is a likely candidate for being a direct target gene.

The Drosophila teashirt (tsh) gene has an homeotic function which, in combination with HOM-C genes, determines thoracic and abdominal (trunk) identities. Analysis of TSH protein distribution during embryogenesis using a specific polyclonal antibody shows that it is nuclear. The protein is present with regional modulation in several tissues within the trunk, suggesting additional tsh functions to those already studied. We identified a candidate tsh target shared with some HOM-C genes, the modifier of variegation gene modulo (mod). The TSH zinc-finger protein recognizes in vitro two specific sites within a 5' control element of the mod gene which responds in vivo to tsh activity. TSH is therefore a DNA binding protein and might directly control mod expression.

A 1-year follow-on study from a randomised, head-to-head, multicentre, open-label study of two pandemic influenza vaccines in children.

INTRODUCTION: Pandemic influenza A H1N1 infections occurred worldwide from 2009. Children were particularly vulnerable. Novel vaccines were used during the pandemic. OBJECTIVE: To assess the persistence of antibody to H1N1 influenza 1 year after children aged 6 months to 12 years had been immunised with two doses of either a non-adjuvanted whole-virion H1N1 influenza vaccine or an AS03B-adjuvanted split-virion H1N1 influenza vaccine; and also to assess the immunogenicity and reactogenicity in this population of a single dose of 2010-11 trivalent seasonal influenza vaccine. DESIGN: Multicentre, open-label, follow-on from randomised, head-to-head trial. SETTING: Five UK sites (Southampton, Oxford, Bristol, London and Exeter). PARTICIPANTS: Children who completed last year's head-to-head randomised study were invited to participate. Children who had subsequently received a further dose of H1N1 vaccine, or who had already received a dose of 2010-11 trivalent seasonal influenza vaccine, were excluded. INTERVENTIONS: In the previous study, children were randomised (in a 1 : 1 ratio) to receive two doses, 21 days apart, of either a non-adjuvanted whole-virion H1N1 influenza vaccine or an AS03B-adjuvanted split-virion H1N1 influenza vaccine. In this follow-on study, a blood sample was taken to assess the persistence of antibody 1 year later, followed by administration of one 0.5 ml-dose of trivalent seasonal influenza vaccine. A second blood sample was taken 3 weeks later. MAIN OUTCOME MEASURES: Comparison between vaccines of the percentage of participants with a microneutralisation (MN) titre ≥ 1 : 40 and a haemagglutination titre ≥ 1 : 32, 1 year after vaccination. Immunogenicity of the trivalent seasonal influenza vaccine was assessed 3 weeks after vaccination by both the MN and the haemagglutination inhibition (HI) titres. Reactogenicity data were recorded for 7 days after vaccination. RESULTS: A total of 323 children were enrolled and 318 were included in the analysis of the persistence of antibody. One year after receipt of whole-virion vaccine, the MN titre was ≥ 1 : 40 in 32.4% of those vaccinated when < 3 years old and in 65.9% of those vaccinated when ≥ 3 years old; the HI titre was ≥ 1 : 32 in 63.2% and 79.1% of children in the respective age groups. One year after receipt of the adjuvanted vaccine, the MN titre was ≥ 1 : 40 in 100% of those vaccinated when < 3 years old and in 96.9% of those vaccinated when ≥ 3 years old; the HI titre was ≥ 1 : 32 in 98.4% and 96.9% of children in the respective age groups. Three hundred and two children were given trivalent seasonal influenza vaccination. Three weeks later, sera were obtained from 282 children; 100% had an MN titre ≥ 1 : 40 and HI titre ≥ 1 : 32. Trivalent seasonal influenza vaccine was well tolerated, although in children < 5 years old, fever ≥ 38 °C was reported in 13.6% of those who had previously received whole-virion vaccine, and in 18.3% of those who had received adjuvanted vaccine. CONCLUSIONS: Nearly all children who received two doses of AS03B-adjuvanted split-virion pandemic H1N1 influenza vaccine had titres of antibody deemed protective (HI titre ≥ 1 : 32, MN titre ≥ 1 : 40) 1 year later. Children who received two doses of whole-virion vaccine had lower titres, although many were above the putative protective thresholds. One year after either pandemic vaccine, the 2010-11 trivalent seasonal influenza vaccine produced a marked serological response to the H1N1 component of the vaccine and was well tolerated. We propose to investigate whether or not previous receipt of monovalent influenza vaccines affected serological response to the H3N2 and B components of the 2010-11 seasonal influenza vaccine, using stored sera. TRIAL REGISTRATION: ClinicalTrials.gov NCT01239537. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Distal into proximal (Dipr): a homoeotic mutation of Drosophila melanogaster.

The morphology and genetical characteristics of a new dominant homoeotic mutation, called Distal into proximal (Dipr), are described. Dipr causes two main abnormalities, both of which are specific to distal regions of the adult appendages (i.e. the wing, haltere, legs, antenna, and proboscis); first that distal parts are reduced in size and second that the patterns found distally resemble those normally localised in more proximal parts. The mutation maps to the right arm of chromosome 3 and is associated with an inversion with breakpoints in 84D and 84F. Analysis of revertants of Dipr show that the right breakpoint of In(3R)Dipr is the one responsible for the mutant phenotype. Complementation analyses of Dipr revertants and dosage studies of Dipr with different doses of Dipr+ indicate that the mutant is a hypermorph affecting the normal expression of a gene localised in 84F. The developmental significance of the mutation is discussed.

Monitoring positional information during oogenesis in adult Drosophila.

About 184P[lac, ry+]A insertions (O'Kane & Gehring, 1987) have been incorporated into the genome via P element-mediated transformation. The temporal-spatial localization of beta-galactosidase, synthesized by these insertions during oogenesis, is described. 32% present control levels of endogenous beta-galactosidase expression and 68% show novel patterns. 13% of the insertions are germline-specific; 33%, follicle-cell-specific; 20% are expressed in both germ line and follicle cells; and 2%, specific to the germarium. Several lines exhibit strict temporal-spatial localizations of beta-galactosidase; notably those expressed in specific populations of follicle cells. The results are discussed with respect to some of the positional information encoded in the genome to which the insertions respond, the use of the insertions as markers for cell differentiation and the potential of the technique for isolating new genes involved in egg production.

The role of position in determining homoeotic gene function in Drosophila.

Homoeotic mutations of Drosophila lead to the replacement of one structure by another, for example, Antennapedia replaces the antenna with a mesothoracic leg and bithorax produces an anterior wing instead of the anterior haltere. The transformed structures differentiated by the homoeotic mutants are essentially normal--only the position in which they appear is abnormal. The mutant phenotypes suggest that in normal development homoeotic genes are involved in developmental alternatives and contribute to a genetic address that defines the particular developmental pathway taken by a primordial group of cells. Thus, in the absence of homoeotic gene function, primordia in different positions should follow the same basic developmental pathway. This is indeed the case for embryos that show no activity of bithorax genes; thoracic and abdominal segments develop as mesothorax. The simplest view on homoeotic gene function is that the genes act selectively on primordia depending on their position in the embryo. To test this hypothesis, we used a mutation at the Antennapedia locus, Antp, which transforms the antenna into a mesothoracic leg, and we observed the function of the homoeotic genes engrailed and Ultrabithorax in two apparently morphologically identical appendages which develop from primordia in different positions. Our results indicate that position is the relevant factor in the function of these two homoeotic genes.

Sequential functions of the bithorax complex of Drosophila.

The bithorax genes are a group of homoeotic genes of Drosophila whose function is related to the control of segment development. They form a gene complex in which at least seven different loci have been identified. Mutations at each of these loci produce a specific homoeotic transformation whereby a segment is transformed either in part or completely into another. In embryos completely deficient for the bithorax genes, all the thoracic and abdominal segments resemble mesothoracic segments. This observation, together with the segmental specificity of the mutant phenotypes, led Lewis to suggest a model of genetic control in which the type of development in each segment is specified by the activation of a fraction of the total number of bithorax genes. Thus, lack of activity of the bithorax genes results in mesothoracic development, activation of the bithorax (bx+) and postbithorax (pbx+) genes produces metathorax (anterior and posterior respectively), and activation of these two genes with bithoraxoid (bxd+) produces the first abdominal segment and so on. Although this model explains the genetic and developmental data, it leaves unexplained formation of the prothoracic segment. We now describe two unexpected results: deficiencies for bithorax genes can lead to segments being transformed into prothorax, and there is a temporal sequence in the function of the bithorax system.

The role of the teashirt gene in trunk segmental identity in Drosophila.

The phenotypes of different mutant combinations of teashirt (tsh) and homeotic genes together with their regulatory interactions are described in order to gain insight into tsh gene function. We show that when tsh, Scr, Antp and BX-C genes are missing, the ventral part of the trunk (or thorax and abdomen) is transformed to anterior head identity showing that tsh is a homeotic gene. These genes act synergistically to suppress the expression of the procephalic gene labial (lab) in subsets of cells in each segment of the trunk. Transcripts from the tsh gene always accumulate in segments destined to acquire trunk identities. tsh gene activity is required for the normal function of the Antp and BX-C genes, which modulate in part the expression of tsh. As a whole, our results suggest that tsh plays an essential dual role, during embryogenesis, for determining segmental identity of the trunk. First, tsh is required critically for the identity of the anterior prothorax. Second, tsh is required globally for segmental identity throughout the entire trunk whereas the "classical" homeotic genes have more specific roles. Our results are consistent with the idea that tsh is defining the ground state of the Drosophila trunk region seen in the absence of the Antp and BX-C genes.

Developmental analysis of the homoeotic mutation bithoraxoid of Drosophila melanogaster.

The homoeotic transformations caused by bxd are described in detail. The anterior histoblast nests of the first abdominal segment are missing, and are replaced by one or two leg discs ventrally. Mainly anterior compartment patterns are found in the ectopic, abdominal legs of adult flies. However, cell lineage analyses show that both anterior and posterior polyclones are established early in the development of these ectopic legs, but the posterior polyclone is smaller. Cells of the anterior polyclone may regulate later in development to adjust for this and form pattern elements normally derived from the posterior polyclone. In addition, experiments show that bxd+ is required by the second larval instar stage, and possibly as early as the blastoderm stage.

Cubitus interruptus-independent transduction of the Hedgehog signal in Drosophila.

The Hedgehog (Hh) family of secreted proteins are key factors that control pattern formation in invertebrates and vertebrates. The manner in which Hh molecules regulate a target cell remains poorly understood. In the Drosophila embryo, Hh is produced in identical stripes of cells in the posterior compartment of each segment. From these cells a Hh signal acts in both anterior and posterior directions. In the anterior cells, the target genes wingless and patched are activated whereas posterior cells respond to Hh by expressing rhomboid and patched. Here, we have examined the role of the transcription factor Cubitus interruptus (Ci) in this process. So far, Ci has been thought to be the most downstream component of the Hh pathway capable of activating all Hh functions. However, our current study of a null ci allele, indicates that it is actually not required for all Hh functions. Whereas Hh and Ci are both required for patched expression, the target genes wingless and rhomboid have unequal requirements for Hh and Ci activity. Hh is required for the maintenance of wingless expression before embryonic stage 11 whereas Ci is necessary only later during stage 11. For rhomboid expression Hh is required positively whereas Ci exhibits negative input. These results indicate that factors other than Ci are necessary for Hh target gene regulation. We present evidence that the zinc-finger protein Teashirt is one candidate for this activity. We show that it is required positively for rhomboid expression and that Teashirt and Ci act in a partially redundant manner before stage 11 to maintain wingless expression in the trunk.

Role of the teashirt gene in Drosophila midgut morphogenesis: secreted proteins mediate the action of homeotic genes.

Homeotic genes control the development of embryonic structure by coordinating the activities of downstream 'target' genes. The identities and functions of target genes must be understood in order to learn how homeotic genes control morphogenesis. Drosophila midgut development is regulated by homeotic genes expressed in the visceral mesoderm, where two of their target genes have been identified. Both encode secreted proteins. The Ultrabithorax (Ubx) homeotic gene activates transcription of the decapentaplegic (dpp) gene, which encodes a TGF beta class protein, while in adjacent mesoderm cells the abdominal-A (abd-A) homeotic gene activates transcription of the wingless (wg) gene, which encodes a Wnt class protein. The homeotic genes Antennapedia (Antp) and Sex combs reduced (Scr) act in more anterior midgut regions. Here we report the identification of another homeotic gene target in the midgut mesoderm, the teashirt (tsh) gene, which encodes a protein with zinc finger motifs. tsh is necessary for proper formation of anterior and central midgut structures. Antp activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx, abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the tsh transcription factor is produced in a spatial pattern distinct from any of the homeotic genes.

Homeotic complex and teashirt genes co-operate to establish trunk segmental identities in Drosophila.

Homeotic genes determine the identities of metameres in Drosophila. We have examined functional aspects of the homeotic gene teashirt by ectopically expressing its product under the control of a heat-shock promoter during embryogenesis. Our results confirm that the gene is critical for segmental identity of the larva. Under mild heat-shock conditions, the Teashirt protein induces an almost complete transformation of the labial to prothoracic segmental identity, when expressed before 8 hours of development. Positive autoregulation of the endogenous teashirt gene and the presence of Sex combs reduced protein in the labium explain this homeosis. Patterns in the maxillary and a more anterior head segment are partly replaced with trunk ones. Additional Teashirt protein has no effect on the identity of the trunk segments where the gene is normally expressed; teashirt function is overridden by some homeotic complex acting in the posterior trunk. Strong heat-shock regimes provoke novel defects: ectopic sense organs differentiate in posterior abdominal segments and trunk pattern elements differentiate in the ninth abdominal segment. Teashirt acts in a partially redundant way with certain homeotic complex proteins but co-operates with them for the establishment of specific segment types. We suggest that Teashirt and HOM-C proteins regulate common sets of downstream target genes.

Homeotic response elements are tightly linked to tissue-specific elements in a transcriptional enhancer of the teashirt gene.

Along the anterior-posterior axis of animal embryos, the choice of cell fates, and the organization of morphogenesis, is regulated by transcription factors encoded by clustered homeotic or 'Hox' genes. Hox genes function in both epidermis and internal tissues by regulating the transcription of target genes in a position- and tissue-specific manner. Hox proteins can have distinct targets in different tissues; the mechanisms underlying tissue and homeotic protein specificity are unknown. Light may be shed by studying the organization of target gene enhancers. In flies, one of the target genes is teashirt (tsh), which encodes a zinc finger protein. tsh itself is a homeotic gene that controls trunk versus head development. We identified a tsh gene enhancer that is differentially activated by Hox proteins in epidermis and mesoderm. Sites where Antennapedia (Antp) and Ultrabithorax (Ubx) proteins bind in vitro were mapped within evolutionarily conserved sequences. Although Antp and Ubx bind to identical sites in vitro, Antp activates the tsh enhancer only in epidermis while Ubx activates the tsh enhancer in both epidermis and in somatic mesoderm. We show that the DNA elements driving tissue-specific transcriptional activation by Antp and Ubx are separable. Next to the homeotic protein-binding sites are extensive conserved sequences likely to control tissue activation by different homeodomain proteins. We propose that local interactions between homeotic proteins and other factors effect activation of targets in proper cell types.

Two transcripts from the rotund region of Drosophila show similar positional specificities in imaginal disc tissues.

The rotund (rn) mutation in Drosophila is unique in that its phenotype is limited to the deletion of specific distal parts, though not the extremities, of all adult appendages. We have cloned the rn gene, located at cytogenetic position 84D3,4, by chromosomal walking. The functional rn unit, defined genetically by the localization of 13 noncomplementing rn alleles, covers approximately 50 kb of DNA. Despite different developmental profiles, two transcript size classes (1.7 and 5.3 kb) from this region show an indistinguishable pattern of spatial expression in the imaginal discs at the white pupa stage. There is a high correlation between the specificity of the mutant phenotype and the accumulation of transcripts in the presumptive distal regions of the cuticle-forming epithelial cells of the affected discs; it is, in fact, the first gene reported whose expression is localized with respect to the proximo distal-forming axis. For both transcripts, we have also found, uniquely in the wing disc, expression limited to the anterior region of the mesodermally derived epithelial cells, which contribute to the muscles of the thorax.