Drosophila Insulin receptor regulates the persistence of injury-induced nociceptive sensitization

Diabetes-associated nociceptive hypersensitivity affects diabetic patients with hard-to-treat chronic pain. Because multiple tissues are affected by systemic alterations in insulin signaling, the functional locus of insulin signaling in diabetes-associated hypersensitivity remains obscure. Here, we used Drosophila nociception/nociceptive sensitization assays to investigate the role of Insulin receptor (Insulin-like receptor, InR) in nociceptive hypersensitivity. InR mutant larvae exhibited mostly normal baseline thermal nociception (absence of injury) and normal acute thermal hypersensitivity following UV-induced injury. However, their acute thermal hypersensitivity persists and fails to return to baseline, unlike in controls. Remarkably, injury-induced persistent hypersensitivity is also observed in larvae that exhibit either type 1 or type 2 diabetes. Cell type-specific genetic analysis indicates that InR function is required in multidendritic sensory neurons including nociceptive class IV neurons. In these same nociceptive sensory neurons, only modest changes in dendritic morphology were observed in the InRRNAi-expressing and diabetic larvae. At the cellular level, InR-deficient nociceptive sensory neurons show elevated calcium responses after injury. Sensory neuron-specific expression of InR rescues the persistent thermal hypersensitivity of InR mutants and constitutive activation of InR in sensory neurons ameliorates the hypersensitivity observed with a type 2-like diabetic state. Our results suggest that a sensory neuron-specific function of InR regulates the persistence of injury-associated hypersensitivity. It is likely that this new system will be an informative genetically tractable model of diabetes-associated hypersensitivity

Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila

Pain signaling in vertebrates is modulated by neuropeptides like Substance P (SP). To determine whether such modulation is conserved and potentially uncover novel interactions between nociceptive signaling pathways we examined SP/Tachykinin signaling in a Drosophila model of tissue damage-induced nociceptive hypersensitivity. Tissue-specific knockdowns and genetic mutant analyses revealed that both Tachykinin and Tachykinin-like receptor (DTKR99D) are required for damage-induced thermal nociceptive sensitization. Electrophysiological recording showed that DTKR99D is required in nociceptive sensory neurons for temperature-dependent increases in firing frequency upon tissue damage. DTKR overexpression caused both behavioral and electrophysiological thermal nociceptive hypersensitivity. Hedgehog, another key regulator of nociceptive sensitization, was produced by nociceptive sensory neurons following tissue damage. Surprisingly, genetic epistasis analysis revealed that DTKR function was upstream of Hedgehog-dependent sensitization in nociceptive sensory neurons. Our results highlight a conserved role for Tachykinin signaling in regulating nociception and the power of Drosophila for genetic dissection of nociception

Prevalence and detection of low-allele-fraction variants in clinical cancer samples

Accurate detection of genomic alterations using high-throughput sequencing is an essential component of precision cancer medicine. We characterize the variant allele fractions (VAFs) of somatic single nucleotide variants and indels across 5095 clinical samples profiled using a custom panel, CancerSCAN. Our results demonstrate that a significant fraction of clinically actionable variants have low VAFs, often due to low tumor purity and treatment-induced mutations. The percentages of mutations under 5% VAF across hotspots in EGFR, KRAS, PIK3CA, and BRAF are 16%, 11%, 12%, and 10%, respectively, with 24% for EGFR T790M and 17% for PIK3CA E545. For clinical relevance, we describe two patients for whom targeted therapy achieved remission despite low VAF mutations. We also characterize the read depths necessary to achieve sensitivity and specificity comparable to current laboratory assays. These results show that capturing low VAF mutations at hotspots by sufficient sequencing coverage and carefully tuned algorithms is imperative for a clinical assay

The convergence of PDF signaling and CRYPTOCHROME in the neuronal network underlying Drosophila circadian behavior

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest the network is composed of heterogeneous oscillators which normally interact and synchronize. Sixteen oscillator neurons (the small and large LNvs) express a neuropeptide called pigment dispersing factor (PDF) whose signaling is required for the synchrony between oscillators. Given the significance of PDF signaling for numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. In this study, I pursued the expression patterns of PDFR via two independent approaches. I utilized GAL4 mediated rescue of PDFR, and also generated a large transgene of PDFR through recombineering methodology. I found that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, a ~70 kB PDF receptor (pdfr) transgene does rescue the entire pdfr circadian behavioral phenotype. The transgene (pdfr-myc) is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. My results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of pdfr-myc confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a subset of glia that resides at the base of the eye. Next, using this authentic pdfr reporter, the 70 kB pdfr-myc, I observed precise coincidence of PDFR expression and high CRYPTOCHROME (CRY) expression, the circadian photoreceptor, within the circadian clock neurons. PDFR and CRY are highly co-expressed in the 5th small LNv, three LNd, and six to seven DN1s. This observation of precise co-expression of these two genes prompted me to test potential genetic interactions of pdf signaling and cry. Flies doubly mutant for pdfr and cry display severely disrupted locomotor rhythms with an absence of both morning and evening anticipatory peaks. Moreover, these double mutant flies were also arrhythmic under constant dark or light conditions, further suggesting the lack of a functional clock. I also observed an unexpected genetic interaction of PDF and CRY signaling in over-expression conditions. An extra copy of pdfr gene in a cryb background causes long period phenotype, whereas an extra copy of pdfr in a wild-type background does not change any aspect of circadian behavior. This suggests that these two signaling pathways are not simply redundancy since over-expression of one signaling cannot replace the lack of the other: instead of restoring normal behavior that genetic combination generates abnormalities in behavior. Finally, I tested how these behavioral phenotypes can be related to the changes in the cellular properties of the clock network. In pdfr;; cryb double mutants, which display arrhythmic behavior under LD and constant conditions, molecular rhythms were abnormal in a critical subset of clock neurons (the 5th small LNv and all LNd). In these pacemakers, I observed sustained levels of high nuclear PERIOD. I conclude that this arrested PER rhythms in the subset of clock neurons is the principle basis for the lack of circadian coordination of behavior in the double mutant flies. In the gain-of-function of pdfr flies, I observed cell fate changes from PDF-expressing oscillators to PDFR-expressing slow-clock oscillators. When this same phenomenon was studied in a cryb mutant background, I saw clear period extension in the flies\u27 circadian behavior. This is a new finding in the field: that the balance of different clock cell types may be a point of physiological regulation and may affect the period of circadian rhythms. Overall, this thesis work reveals an unexpected convergence of Cry-mediated and PDF signaling pathways. This convergence is required to support circadian behavioral rhythms, and initiates a possible link between the balance of cell types and behavioral phenotypes

PDFR-MYC and CRYPTOCHROME are precisely co-expressed in the same subsets of clock neurons.

<p>PDFR-MYC fly brains were triple-stained with anti-MYC (green), anti-PER (magenta), and anti-PDF (blue) antibodies (A, C, E), or double-stained with anti-MYC (green) and anti-CRY (blue) antibodies (B, D, F). (A) Three LNds of six show strong PDFR-MYC staining (white arrowheads), whereas the others show no PDFR-MYC (magenta arrowheads). (B) Three LNds express both PDFR-MYC and CRY (arrowheads). (C) The 5^th s-LNv showed strong staining of PDFR-MYC (arrow). (D) Nine LNv stained with anti-CRY antibody, three of these were also stained with anti-MYC. By reference to results shown in panel C, we assigned the strongest MYC expressing neuron to the 5^th s-LNv (arrow). The two remaining MYC(+) neurons are marked with white arrowheads: By size, we speculate these are l-LNv. Two CRY(+) l-LNvs (by size) were not detected with anti-MYC antibody (magenta arrowheads). (E) Six of the 17 DN1s show PDFR-MYC staining at strong levels (white arrowheads), whereas the remaining ones show little or no MYC staining. (F) Six of 15 DN1ps express both PDFR-MYC and CRY (arrowheads). Asterisks (in A, B, D, F) - non-specific staining by either anti-MYC or anti-CRY rabbit antibodies. Scale bars, 10 µm. (G) A summary diagram of the precise PDFR and CRY co-expression in discreet subsets of the three major pacemaker cell groups.</p

Lack of PDF and CRY signaling causes weak, short behavioral rhythms under LL.

<p>Group-averaged actograms of each genotype. (A) <i>w¹¹¹⁸; ; cry^b/01</i> single mutant flies; (B) <i>pdfr⁵³⁰⁴</i> single mutants; (C) <i>pdfr⁵³⁰⁴; ; cry^b/01</i> double mutant flies; (D) <i>cry-G4⁽¹⁹⁾</i>; <i>cry^b ss Pdf⁰¹</i> double mutant flies.</p

Daily locomotor activities under LD cycles reveal genetic interactions between PDF and CRY signaling pathways.

<p>Averaged activity of various genotype flies for a six-day-period under 8∶16 LD (A–D), 12∶12 LD (E–H), and 16∶8 LD (I–L) entrainment conditions. (A, E, I) <i>w¹¹¹⁸</i> control flies; (B, F, J) <i>pdfr⁵³⁰⁴</i> single mutant flies; (C, G, K) <i>pdfr⁵³⁰⁴; ; cry^b/01</i> double mutant flies; (D, H, L) <i>cry-G4⁽¹⁹⁾</i>; <i>cry^b ss Pdf⁰¹</i> double mutant flies. Both double mutant flies display lack of anticipatory peaks under LD cycles. Note that, in <i>pdfr</i> single mutants, the longer the day length becomes the more pronounced the advanced evening phenotype. For the experiment shown, the numbers of animals averaged are 32 (A), 31 (B), 32 (C), 32 (D), 30 (E), 14 (F), 15 (G), 32 (H), 32 (I), 31 (J), 31 (K), and 32 (L).</p

LD Molecular rhythms in the 5^th s-LNv and LNd are deranged in the double mutants.

<p>At various time-points, PER levels were monitored in the nucleus (filled histograms) and cytoplasm (open histograms) of the 5^th s-LNv (A, B) and the ITP(+) LNd (C, D). (A) In the 5^th s-LNv of <i>w¹¹¹⁸; ; cry^b/01</i>, PER levels in the nucleus and cytoplasm are robustly cycling: nuclear amplitude rhythm – 19.4-fold; cytoplasmic amplitude rhythm – 9.0-fold. ANOVA test revealed that the differences in nuclear staining levels are significant (P<0.0001). (B) In the 5^th s-LNv of <i>pdfr⁵³⁰⁴; ; cry^b/01</i>, PER staining is always found in the nucleus with very low amplitude rhythms and no phase difference between nucleus and cytoplasm: nuclear amplitude rhythm –2.4-fold; cytoplasm amplitude rhythm – 3.0-fold. ANOVA test revealed that the difference in this group is significant (P = 0.03). (C) In the ITP(+) LNd of <i>w¹¹¹⁸; ; cry^b/01</i>, PER levels in the nucleus and cytoplasm are robustly cycling, nuclear amplitude rhythm – 19.0-fold; cytoplasmic amplitude rhythm – 11.4-fold. ANOVA test revealed that the difference in this group is significant (P<0.0001). (D) In the ITP(+) LNd of <i>pdfr⁵³⁰⁴; ; cry^b/01</i>, PER staining is always found in the nucleus with very low amplitude rhythms and no phase difference between nucleus and cytoplasm: nuclear amplitude rhythm – 3.8-fold; cytoplasmic amplitude rhythm – 2.8-fold. ANOVA test revealed that the difference in this group is significant (P<0.0001). Results from post-hoc statistical tests are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018974#pone-0018974-t002" target="_blank">Table 2</a>.</p