Travis K. Johnson

A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila

Body size often shows adaptive clines in many ectotherms across altitude and latitude, but little is known about the genetic basis of these adaptive clines. Here we identify a polymorphism in the Dca (Drosophila cold acclimation) gene in Drosophila melanogaster that influences wing size, affects wing:thorax allometry and also controls a substantial proportion of the clinal wing‐size variation. A polymorphism in the promoter region of Dca had two common alleles showing strong reciprocal clinal variation in frequency with latitude along the east coast of Australia. The Dca‐237 allele increased towards the tropics where wing size is smaller. A within‐population association study highlighted that an increase in the frequency of this allele decreased wing size but did not influence thorax size. A manipulated increase in the level of expression of Dca achieved through UAS‐GAL4 was associated with a decrease in wing size but had no effect on thorax size. This was consistent with higher Dca expression levels in family lines with higher frequency of the Dca‐237 allele. Genetic variation in the promoter region of the Dca gene appears to influence adaptive size variation in the eastern Australian cline of Drosophila melanogaster and accounts for more than 10% of the genetic variation in size within and between populations.

The Toll and Imd pathways are not required for Wolbachia mediated dengue virus interference.

ABSTRACT Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virus-blocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system.

Torso-like functions independently of Torso to regulate Drosophila growth and developmental timing

Significance Torso-like (Tsl) is the sole Drosophila member of the membrane attack complex/perforin-like protein superfamily, generally known for pore-forming function and immune defence roles. Tsl, however, has a well-characterized developmental role in controlling activation of the receptor tyrosine kinase Torso (Tor) to achieve patterning of the termini of the early embryo. Here we report that the second known role of Tor, as the receptor for the hormone that induces metamorphosis, does not require Tsl. Instead, we find that Tsl controls developmental timing and growth independently of Tor. We conclude that Tsl plays a broader than expected role during development and is not merely a specialized cue for Tor signaling. Activation of the Drosophila receptor tyrosine kinase Torso (Tor) only at the termini of the embryo is achieved by the localized expression of the maternal gene Torso-like (Tsl). Tor has a second function in the prothoracic gland as the receptor for prothoracicotropic hormone (PTTH) that initiates metamorphosis. Consistent with the function of Tor in this tissue, Tsl also localizes to the prothoracic gland and influences developmental timing. Despite these commonalities, in our studies of Tsl we unexpectedly found that tsl and tor have opposing effects on body size; tsl null mutants are smaller than normal, rather than larger as would be expected if the PTTH/Tor pathway was disrupted. We further found that whereas both genes regulate developmental timing, tsl does so independently of tor. Although tsl null mutants exhibit a similar length delay in time to pupariation to tor mutants, in tsl:tor double mutants this delay is strikingly enhanced. Thus, loss of tsl is additive rather than epistatic to loss of tor. We also find that phenotypes generated by ectopic PTTH expression are independent of tsl. Finally, we show that a modified form of tsl that can rescue developmental timing cannot rescue terminal patterning, indicating that Tsl can function via distinct mechanisms in different contexts. We conclude that Tsl is not just a specialized cue for Torso signaling but also acts independently of PTTH/Tor in the control of body size and the timing of developmental progression. These data highlight surprisingly diverse developmental functions for this sole Drosophila member of the perforin-like superfamily.

Latitudinal and cold-tolerance variation associate with DNA repeat-number variation in the hsr-omega RNA gene of Drosophila melanogaster

An 8-bp deletion in the hsr-omega heat-stress gene of Drosophila melanogaster has previously been associated with latitude, and with heat tolerance that decreases with latitude. Here we report a second polymorphic site, at the 3′-end of hsr-omega, at which multiple alleles segregate in natural populations for copy number of a ∼280 bp tandem repeat. On each of 3 consecutive years (2000, 2001 and 2002) among populations sampled along the Australian eastern coast, repeat number was negatively associated with latitude. Neither altitudinal association was detected in 2002 when five high-altitude sites were included, nor was a robust association detected with local temperature or rainfall measures. Although in a large number of family lines, derived from a population located centrally in the latitudinal transect, no association between hsr-omega repeat number and heat tolerance occurred, a negative association of repeat number with cold tolerance was detected. As cold tolerance also exhibits latitudinal clines we examined a set of cold-tolerant populations derived by selection and found both reduced repeat number and low constitutive levels of the omega-n repeat-bearing transcript. In a sample from the central population, linkage disequilibrium was measured between repeat number and linked markers that also cline latitudinally. However, such disequilibrium could not account for the cline in repeat number or tolerance associations. Finally, during adult recovery from cold exposure a large increase occurred in tissue levels of the omega-c transcript. Together these data suggest that a latitudinal cline in hsr-omega repeat number influences cold-tolerance variation in this species.

Torso-like mediates extracellular accumulation of Furin-cleaved Trunk to pattern the Drosophila embryo termini.

Patterning of the Drosophila embryonic termini is achieved by localized activation of the Torso receptor by the growth factor Trunk. Governing this event is the perforin-like protein Torso-like, which is localized to the extracellular space at the embryo poles and has long been proposed to control localized proteolytic activation of Trunk. However, a protease involved in terminal patterning remains to be identified, and the role of Torso-like remains unknown. Here we find that Trunk is cleaved intracellularly by Furin proteases. We further show that Trunk is secreted, and that levels of extracellular Trunk are greatly reduced in torso-like null mutants. On the basis of these and previous findings, we suggest that Torso-like functions to mediate secretion of Trunk, thus providing the mechanism for spatially restricted activation of Torso. Our data represent an alternative mechanism for the spatial control of receptor signalling, and define a different role for perforin-like proteins in eukaryotes.

Trunk cleavage is essential for Drosophila terminal patterning and can occur independently of Torso-like

Terminal patterning in Drosophila is governed by a localized interaction between the Torso kinase (Tor) and its ligand Trunk (Trk). Currently, it is proposed that Trk must be cleaved in order to bind Tor, and that these proteolytic events are controlled by secretion of Torso-like (Tsl) only at the embryo poles. However, controversy surrounds these ideas since neither cleaved Trk nor a protease that functions in terminal patterning have been identified. Here we show that Trk is cleaved multiple times in vivo and that these proteolytic events are essential for its function. Unexpectedly, however, the Trk cleavage patterns we observe are unaltered in tsl-null mutants. One explanation for these data is that the influence of Tsl on localized Trk cleavage at the embryo poles is subtle and cannot be readily detected. Alternatively, we favour a scenario where Tsl functions post proteolytic processing of Trk to control localized terminal patterning.

Transcripts from the Drosophila heat-shock gene hsr-omega influence rates of protein synthesis but hardly affect resistance to heat knockdown.

While hundreds of genes have recently been implicated in an organism’s response to thermal stress, our insight into the cellular and physiological mechanisms affected by these genes has advanced to a lesser extent. We focus on an enigmatic Drosophila heat stress RNA gene, hsr-omega, which encodes two RNA transcripts that are constitutively expressed in almost all developing and adult tissues, omega-n in the nucleus and omega-c in the cytoplasm; both being readily induced to high levels by mild heat stress. We derived three hsr-omega mutant lines via imprecise P-element excision and characterised them for changes in expression, in both the presence and absence of heat stress. Viability estimates indicate that a low level of omega-n is required for normal development. Consistent with the model of omega-n as a negative regulator of intron-processed mRNA levels the mutants displayed a 1.5-fold increase in rates of protein synthesis measured in ovarian tissue in the absence of heat stress, a result suggesting that an important function of hsr-omega is the modulation of general protein synthesis. The mutants had little effect on two measures commonly used to assess heat tolerance, heat-knockdown time and heat hardening ability, suggesting that more subtle heat-related fitness components need to be examined for effects of these mutations.

The Nucleus- and Endoplasmic Reticulum-Targeted Forms of Protein Tyrosine Phosphatase 61F Regulate Drosophila Growth, Life Span, and Fecundity

ABSTRACT The protein tyrosine phosphatases (PTPs) T cell PTP (TCPTP) and PTP1B share a high level of catalytic domain sequence and structural similarity yet display distinct differences in substrate recognition and function. Their noncatalytic domains contribute to substrate selectivity and function by regulating TCPTP nucleocytoplasmic shuttling and targeting PTP1B to the endoplasmic reticulum (ER). The Drosophila TCPTP/PTP1B orthologue PTP61F has two variants with identical catalytic domains that are differentially targeted to the ER and nucleus. Here we demonstrate that the PTP61F variants differ in their ability to negatively regulate insulin signaling in vivo, with the nucleus-localized form (PTP61Fn) being more effective than the ER-localized form (PTP61Fm). We report that PTP61Fm is reliant on the adaptor protein Dock to attenuate insulin signaling in vivo. Also, we show that the PTP61F variants differ in their capacities to regulate growth, with PTP61Fn but not PTP61Fm attenuating cellular proliferation. Furthermore, we generate a mutant lacking both PTP61F variants, which displays a reduction in median life span and a decrease in female fecundity, and show that both variants are required to rescue these mutant phenotypes. Our findings define the role of PTP61F in life span and fecundity and reinforce the importance of subcellular localization in mediating PTP function in vivo.

Spatial analysis of gene regulation reveals new insights into the molecular basis of upper thermal limits

The cellular stress response has long been the primary model for studying the molecular basis of thermal adaptation, yet the link between gene expression, RNA metabolism and physiological responses to thermal stress remains largely unexplored. We address this by comparing the transcriptional and physiological responses of three geographically distinct populations of Drosophila melanogaster from eastern Australia in response to, and recovery from, a severe heat stress with and without a prestress hardening treatment. We focus on starvin (stv), recently identified as an important thermally responsive gene. Intriguingly, stv encodes seven transcripts from alternative transcription sites and alternative splicing, yet appears to be rapidly heat inducible. First, we show genetic differences in upper thermal limits of the populations tested. We then demonstrate that the stv locus does not ubiquitously respond to thermal stress but is expressed as three distinct thermal and temporal RNA phenotypes (isoforms). The shorter transcript isoforms are rapidly upregulated under stress in all populations and show similar molecular signatures to heat‐shock proteins. Multiple stress exposures seem to generate a reserve of pre‐mRNAs, effectively ‘priming’ the cells for subsequent stress. Remarkably, we demonstrate a bypass in the splicing blockade in these isoforms, suggesting an essential role for these transcripts under heat stress. Temporal profiles for the weakly heat responsive stv isoform subset show opposing patterns in the two most divergent populations. Innate and induced transcriptome responses to hyperthermia are complex, and warrant moving beyond gene‐level analyses.

New Levels of Transcriptome Complexity at Upper Thermal Limits in Wild Drosophila Revealed by Exon Expression Analysis

While the cellular heat-shock response has been a paradigm for studying the impact of thermal stress on RNA metabolism and gene expression, the genome-wide response to thermal stress and its connection to physiological stress resistance remain largely unexplored. Here, we address this issue using an array-based exon expression analysis to interrogate the transcriptome in recently established Drosophila melanogaster stocks during severe thermal stress and recovery. We first demonstrated the efficacy of exon-level analyses to reveal a level of thermally induced transcriptome complexity extending well beyond gene-level analyses. Next, we showed that the upper range of both the cellular and physiological thermal stress response profoundly affected message expression and processing in D. melanogaster, limiting expression to a small subset of transcripts, many that share features of known rapidly responding stress genes. As predicted from cellular heat-shock research, constitutive splicing was blocked in a set of novel genes; we did not detect changes to alternative splicing during heat stress, but rather induction of intronless isoforms of known heat-responsive genes. We observed transcriptome plasticity in the form of differential isoform expression during recovery from heat shock, mediated by multiple mechanisms including alternative transcription and alternative splicing. This affected genes involved in DNA regulation, immune response, and thermotolerance. These patterns highlight the complex nature of innate transcriptome responses under stress and potential for adaptive shifts through plasticity and evolved genetic responses at different hierarchical levels.