The Receptor Tyrosine Kinase Alk Controls Neurofibromin Functions in Drosophila Growth and Learning

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1

An LQG approach to self-tuning control with application to robotics

Self-tuning control has been recognised as an effective approach for mechanical manipulator control design due to its ability to cope with the presence of nonlinearities and uncertainties in robot dynamic models. The vast majority of existing self-tuning controllers are based on a linear plant description, the fact that most industrial processes are nonlinear is taken into account by regarding the plant as a sequence of pseudolinear descriptions. Therefore, as the plant operating point changes, the nonlinear plant dynamics are reflected as time varying parameters in the linear plant description. The applicability of this approach has been demonstrated by Koivo and Guo [1] where manipulator joint angular position is the controlled variable. This work is extended in Koivo et al [2] to the case in which the manipulator is controlled directly in the cartesian coordinate system. It is found that convergence of the parameter estimates may not be achieved during the finite time over which the· motion takes place. Therefore this approach is particularly suited to repetitive tasks where the last estimates from the previous run can be used as the initial estimates. Lelic and Wellstead [3] have successfully applied generalised pole placement to the control of a 5 axis electrically actuated robot-manipulator

Deficiency screen summary.

<p>Indicated are the number of chromosome 1, 2L and 2R deficiencies screened, the fraction of genes uncovered (based on the FB2013_03 FlyBase release), the number of <i>dNf1</i> modifying deficiencies and loci identified, and the number of non-specific modifiers.</p

Validation of <i>dNf1</i> modifiers with neuronal functions.

<p>(A) <i>Ras2-Gal4</i> or <i>C23-Gal4</i> driven neuronal RNAi knockdown of <i>CCKLR-17D1</i> but not <i>CCKLR-17D3</i> suppressed the <i>dNf1</i> pupal size defect. (B) Identification of dynamin-associated protein 160 (Dap160) as a suppressor of <i>dNf1</i> growth. Neuronal RNAi targeting of <i>Dap160</i> increased <i>dNf1</i> pupal size as did two <i>Dap160</i> loss-of-function alleles. (C) Two <i>elav</i> alleles dominantly suppress the <i>dNf1</i> size defect. (D) Neuronal expression of a Rab9 RNAi transgene or of a dominant negative Rab9 mutant suppresses the <i>dNf1</i> size defect.</p

<i>dNf1</i> systemic growth related RAS/ERK and cAMP/PKA signals appear functionally and topographically distinct.

<p>(A) The elevated larval CNS pERK level of <i>dNf1</i> mutants is reduced by neuronal expression of <i>dNf1</i>, but not by neuronal or heat-shock induced ubiquitous expression of PKA*. Western blot of pERK levels in larval CNS of the indicated genotypes. In lane 6, larvae received a daily 20 min 37°C heat shock throughout development, a protocol that suppresses the <i>dNf1</i> growth defect <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003958#pgen.1003958-The1" target="_blank">[4]</a>. (B) Structure of <i>UAS-PKA*</i> transgenes with 1 to 5 UAS elements. The lethality of these transgenes when driven with either <i>Ac5C-Gal4</i> or <i>elav-Gal4</i> is indicated by † whereas (−) indicates viable offspring. (C) Western blot of adult head lysates showing relative expression of <i>GMR-Gal4</i>-driven transgenic PKA*. Tubulin is used as a loading control. (D) Expression of PKA* or knockdown of <i>dnc</i> by shRNAi in the ring gland rescues the <i>dNf1</i> pupal size defect. In contrast, <i>UAS-dNf1</i> expression with the same ring gland drivers fails to restore systemic growth. (E–H) Expression pattern of <i>Akh-Gal4</i> driving <i>UAS-GFP</i>, co-stained with DAPI and anti-dNF1. GFP expression in the corpora cardiaca (CC) is indicated. Scale bar = 50 µm. As previously noted <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003958#pgen.1003958-Walker2" target="_blank">[74]</a>, anti-dNf1 staining is strong in the CNS, whereas staining in the ring gland is close to background.</p

Deficiency screen for dominant modifiers of the <i>dNf1</i> growth defect.

<p>Isogenic 1^st and 2^nd chromosomes deficiencies from the Exelixis, DrosDel and Bloomington Stock Center collections were tested for their ability to alter <i>dNf1</i> female pupal size. Crossing schemes to generate <i>Df(1)/+; dNf1^E2</i> (A) and <i>Df(2)/CyO; dNf1^E2</i> (B) screening stocks. The <i>tubby</i>-marked <i>TM6B</i> 3^rd chromosome balancer allowed the selection of <i>dNf1^E2</i> homozygotes for measurements. (C) Examples of deficiencies that suppress or enhance the <i>dNf1</i> size defect. Scale bar = 1 mm.</p

Restoration of systemic growth by <i>dNf1</i> and cAMP/PKA involves different tissues.

<p><i>Act5C</i>-<i>Gal4</i> driven ubiquitous <i>dNf1</i> re-expression, or <i>elav</i>-<i>Gal4</i> and <i>Ras2</i>-<i>Gal4</i> driven neuronal re-expression rescues the <i>dNf1</i> pupal size defect, whereas <i>dnc</i> RNAi or UAS-PKA* expression controlled by the same drivers is ineffective. By contrast, expressing <i>dNf1</i> in specific parts of the neuroendocrine ring gland with the <i>Akh</i>-<i>Gal4</i>, Feb36-<i>Gal4</i> or Aug21-<i>Gal4</i> drivers fails to rescue, whereas using the same drivers to express <i>dnc</i> RNAi or attenuated UAS-PKA* transgenes does increase <i>dNf1</i> pupal size. All crosses produced viable adults unless otherwise indicated.</p>†<p>denotes lethality, SV sub-viable, n/a not applicable, NR non-rescue.</p><p>The data shown summarize results of a larger effort to identify the tissues in which <i>dNf1</i> and cAMP/PKA affect systemic growth. Full results are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003958#pgen.1003958.s013" target="_blank">Table S5</a>.</p

Modifying deficiencies and identification of responsible genes.

<p>Modifying deficiencies for which the responsible <i>dNf1</i> interacting gene has been identified. The cytological location, and the dominant effect on <i>dNf1</i> pupal size (SUP - suppressor, ENH – enhancer) of each deficiency is given. The responsible genes for each modifying deficiency are shown with the mutant alleles, VDRC and TRiP RNAi lines used in their identification. Expression of RNAi transgenes was induced with the <i>Ras2</i>-<i>Gal4</i>, <i>elav</i>-<i>Gal4</i>, <i>n-syb</i>-<i>Gal4</i> and/or C23-<i>Gal4</i> drivers. Abbreviations: hypo: hypomorphic; leth: lethal; lof: loss-of-function; amorph: amorphic; Δ: deletion; via: viable.</p

Loss of <i>dNf1</i> does not phenocopy starvation or alter developmental timing.

<p>(A) Wild-type (<i>w¹¹¹⁸</i>) and <i>dNf1</i> larvae ingest similar amounts of food. Larvae at different stages of development were photographed after 25 minutes of feeding on dye-colored yeast paste. (B) As opposed to <i>ppl</i> mutants, wild-type and <i>dNf1</i> larvae do not wander from a food source (fraction of wandering larvae: WT 3.5% (SD 0.007), <i>dNf1</i> 2.5% (SD 0.007) and <i>ppl</i> 65% (SD 0.057)). In a similar assay, <i>dNf1</i> larvae also showed no abnormality in moving towards a food source (not shown). (C) RNA blot analysis of the starvation-sensitive genes, PEPCK and <i>Lip3</i> shows that <i>dNf1</i> larvae do not show elevated levels of either mRNA under normal feeding conditions. (D) Wild-type and <i>dNf1</i> larvae show no significant differences in developmental timing, as assessed by time of pupariation after egg deposition (AED). (E) The <i>dNf1</i> growth rate, as assessed by larval weight, is reduced throughout larval development when compared to wild-type or a <i>Ras2>UAS-dNf1</i> control. (F) Two hypomorphic insulin receptor alleles, <i>InR⁰⁵⁵⁴⁵</i> and <i>InR³²⁷</i>, do not modify <i>dNf1</i> pupal size. (G) ILP mRNA expression is not obviously reduced in <i>dNf1</i> larvae. H) <i>dNf1</i> adult flies show no altered longevity compared to wild-type controls. (I) Over-expression of <i>Ilp2</i> from a <i>hs-Ilp2</i> transgene in <i>dNf1</i> larvae results in a similar increase in size as in wild-type flies.</p