WRS-85D: A tryptophanyl-tRNA synthetase expressed to high levels in the developing Drosophila salivary gland

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.     

Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Here, we analyze appendage genes in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic (dpp) expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax (hth) and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role of this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. We comment on the significance of this finding for attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, our results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies.     

Patterning function of homothorax/extradenticle in the thorax of Drosophila

In Drosophila, the morphological diversity is generated by the activation of different sets of active developmental regulatory genes in the different body subdomains. Here, we have investigated the role of the homothorax/extradenticle (hth/exd) gene pair in the elaboration of the pattern of the anterior mesothorax (notum). These two genes are active in the same regions and behave as a single functional unit. We find that their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and betasubdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. We also present evidence for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain.     

Organogenesis in Drosophila melanogaster: embryonic salivary gland determination is controlled by homeotic and dorsoventral patterning genes.

We have investigated Drosophila salivary gland determination by examining the effects of mutations in pattern forming genes on the salivary gland primordium. We find that the anterior-posterior extent of the primordium, a placode of columnar epithelial cells derived from parasegment 2, is established by the positive action of the homeotic gene Sex combs reduced (Scr). Embryos mutant for Scr lack a detectable placode, while ectopic Scr expression leads to the formation of ectopic salivary glands. In contrast, the dorsal-ventral extent of the placode is regulated negatively. Functions dependent on the decapentaplegic product place a dorsal limit on the placode, while dorsal-dependent genes act to limit the placode ventrally. We propose a model in which these pattern forming genes act early to determine the salivary gland anlage by regulating the expression of salivary gland determining genes, which in turn control genes that are involved in salivary gland morphogenesis.     

Coordinate regulation of downstream genes by extradenticle and the homeotic selector proteins.

Mutations in the Drosophila gene extradenticle (exd), a homologue of the human proto-oncogene pbx1, cause homeotic transformations by altering the morphological consequences of homeotic selector gene activity. exd has been proposed to act by contributing to the specificity of selector homeodomain proteins for their downstream targets. Here we show that exd is indeed required for the appropriate regulation of at least some of these target genes. Expression patterns of wingless, teashirt and decapentaplegic (dpp) are altered in the embryonic midgut of embryos lacking exd, while the expression of their respective regulators (abd-A, Antp and Ubx) remains normal. Co-regulation of dpp by exd and Ubx was investigated in greater detail by examining the expression of reporter constructs in exd embryos. These experiments not only define dpp regulatory regions responsive to exd, but also distinguish two functions of exd in the regulation of dpp. exd acts with Ubx to activate dpp expression in parasegment 7 (PS7), via a minimal visceral mesoderm enhancer, and exd represses dpp expression anterior to PS7. We show that even when Ubx is ubiquitously expressed at high levels in exd embryos, Ubx is incapable of activating dpp enhancer expression. Thus, exd is an indispensable component in target gene regulation by the homeotic selector proteins.     

Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila

The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium of the eye-antennal disc by a 3.5kb enhancer in the 5' end of the gene. We recovered a 15bp deletion mutation within this enhancer that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head.     

Patterning defects in the primary axonal scaffolds caused by the mutations of the extradenticle and homothorax genes in the embryonic Drosophila brain

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent. Received: 4 October 1999 / Accepted: 10 January 2000     

A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis

The decapentaplegic (dpp) gene product, a member of the transforming growth factor-beta family, is required in Drosophila embryos for normal gastrulation and the establishment of dorsal-ventral polarity in the embryo. dpp is also expressed at specific positions in the visceral mesoderm along the developing midgut. We find that mutations that eliminate the visceral mesoderm expression of dpp lead to defects in midgut morphogenesis and alter the spatially localized expression of the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Antennapedia (Antp) in the visceral mesoderm. The extracellular dpp protein migrates from the visceral mesoderm across the apposing endodermal cell layer in a region of the endoderm that expresses the homeotic gene labial (lab). Mesodermal expression of dpp is required for the expression of lab in these endodermal cells indicating that dpp mediates an inductive interaction between the two germ layers. We propose that extracellular dpp protein regulates gut morphogenesis, in part, by regulating homeotic gene expression in the visceral mesoderm and endoderm of the developing midgut.     

Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

BACKGROUND: Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. RESULTS: Here, we show that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam was able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. CONCLUSIONS: Although we have previously shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. Our data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner.