- Title
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A Genome-wide Screen Identifies PAPP-AA-Mediated IGFR Signaling as a Novel Regulator of Habituation Learning
- Authors
- Wolman, M.A., Jain, R.A., Marsden, K.C., Bell, H., Skinner, J., Hayer, K.E., Hogenesch, J.B., Granato, M.
- Source
- Full text @ Neuron
Genetic Screen Identifies Mutations Affecting Acoustic Startle Habituation (A) Schematic of acoustic startle habituation assay. Larvae are exposed to 10 non-habituating acoustic stimuli, delivered at 20 s interstimulus intervals (ISI), and then 30 habituating stimuli at a 1 s ISI. (B) Mean acoustic startle habituation percentage calculated by comparing the average frequency of startle responsiveness of an individual to stimuli 1-10 and stimuli 31-40 (Wolman et al., 2011). Behaviorally defined wild-type siblings shown in white bars, mutants in gray bars. (C) Estimated truncated PCXAp171 protein in information overloadp171 mutants due to Y1163X mutation. BC, biotin carboxylase; CT, carboxyl transferase; PT, pyruvate carboxylase tetramerization; BCCP, biotin-carboxy carrier protein. (D) Estimated truncated PAPP-AAp170 protein product in unfilteredp170 mutants due to Q322X mutation. LG, laminin G-like module; LNR, Lin-12/Notch repeats, MPD, metzincin proteolytic domain containing zinc-binding consensus sequence (Zn) and Met-turn motif (M); CCP, complement control protein modules 1-5. E) pappaap170 larvae injected with increasing doses of wild-type pappaa mRNA show improved habituation at 5 dpf. *p < 0.01, **p < 0.001, ANOVA with Bonferonni correction versus wild-type sibling (B) or uninjected pappaap170 (E) larvae. N = number of larvae shown within or below each bar. Error bars indicate SEM. |
pappaa Expression in Neurons of the Acoustic Startle Circuit (A and A′) Schematic representation of acoustic startle circuit at larval stage from a lateral (A) and dorsal (A′) perspective. The acoustic startle circuit includes cranial ganglion (blue), Mauthner neuron and homologs (green), spiral fiber neurons (orange), passive hyperpolarizing (PHP) neurons (red), and feedback inhibitory neurons (purple). (B and C) In situ hybridization for pappaa at 48 hpf (B, purple) and 5 dpf (C, red). Brackets (A and B) mark site of hindbrain neurons controlling startle behavior. (C) Dorsal view, anterior to the top. pappaa mRNA in red, Mauthner neuron (M) in green. Arrowheads mark site of spiral fiber neurons, asterisks mark position of PHP neurons, and arrows indicate location of feedback inhibitory neurons. SAG, statoacoustic ganglion; aLL, anterior lateral line ganglion; pLL, posterior lateral line ganglion; M, Mauthner. Scale bars = 50 µm (B) and 10 µm (C). |
Acoustic Startle Circuit Appears Intact in pappaap170 Larvae (A-C) DASPEI labeling (A and B) and mean number (C) of sensory neuromasts. (D-I and K-L) Projections of confocal stacks acquired at 5 dpf. Lateral views, anterior to the left in (A), (B), (D), and (E). Dorsal views, anterior to the top in (F)-(I) and (K)-(L). (D and E) Arrowheads mark projections (anti-neurofilament, red) from cranial ganglia (anti-HuC, green) to hindbrain. (F-I, K, L) Green label marks hspGFF130DMCA:UAS-gap43-citrine expression in Mauthner and Mid2cm Mauthner homologs. (F and G) Open arrows mark Mauthner (M) axon cap. Open arrowhead marks spiral fiber neuron projection and closed arrows mark contralateral passive hyperpolarizing (PHP) projection. (H and I) Brackets mark lateral dendrite of Mauthner with Cx35-positive club endings. Asterisk marks blood cell. (J) Mean number of club endings. (K and L) Glycineric receptors on Mauthner soma. SAG, statoacoustic ganglion; aLL, anterior lateral line ganglion; pLL, posterior lateral line ganglion. N, larvae shown within bars. Error bars indicate SEM. Scale bars = 1 mm (B), 50 µm (E), and 10 µm (G, I, and L). EXPRESSION / LABELING:
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pappaap170 Mutants Show Reduced Swim Bladder Inflation, Habituation, and Survival (A) pappaap170 mutants appear grossly normal at 5 dpf, with exception of some showing an uninflated swim bladder. (B) pappaap170 larvae show startle habituation deficit at 5 and 12 dpf. (C) Kaplan-Meier survival curve shows reduced viability in pappaap170 mutants. *p < 0.001, one-way ANOVA (with Bonferonni correction) versus pappaa+/+, **p < 0.0001 Mantel-Cox test versus wild-type siblings. N = number of larvae shown within bars (B) or in legend (C). Error bars indicate SEM. PHENOTYPE:
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PAPP-AA Metalloprotease Activity Is Required for Startle Habituation Mean habituation percentage after injection of human wild-type pappa mRNA or proteolytically inactive pappaE483AmRNA. p < 0.001, ANOVA with Bonferonni correction versus DMSO treated larvae of the same genotype. N, larvae shown within bars. Error bars indicate SEM. PHENOTYPE:
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PAPP-AA Mediates Habituation through Acute Regulation of IGF Signaling (A) Mean acoustic startle habituation percentage at 5 dpf of larvae treated with DMSO or 1 µM SC79. Treatment period (dpf) indicated below bars. (B) Mean acoustic startle habituation percentage of 5 dpf larvae treated with 740 Y-P for 30 min prior to and during habituation assay. (C and D) Mean acoustic startle habituation percentage after 30 min treatment with BMS-754807 or DMSO at 5 dpf (C) or 12 dpf (D). p < 0.01, p < 0.001, ANOVA with Bonferonni correction versus DMSO-treated larvae of the same genotype. N = number of larvae shown within bars. Error bars indicate SEM. PHENOTYPE:
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Mauthner neuron morphology (related to Figure 3). (A) 3D reconstruction of Mauthner neuron of pappaa+/+ larva showing soma, lateral and ventral dendrites, and initial axon segment. Scale bar = 10µm. Quantification of Mauthner neuron morphology includes: mean surface area of soma (B), lateral dendrite (D), ventral dendrite (F), and initial axon segment (H) and mean volume of soma (C), lateral dendrite (E), ventral dendrite (G), and initial axon segment (I). Each mark indicates a Mauthner neuron from a single larva. Error bars denote SEM. No significant differences between pappaa+/+ and pappaap170 larvae.PHENOTYPE:
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