Fig. 3

Ca²⁺ Signaling Prefigures and Guides Microglial Migration(A) Microglial tracking (pU1::Gal4-UAS-TagRFP) upon control injection of water in a 3 dpf larval brain. The starting point of individual tracks is marked with an X and the end point with a dot.(B) Injection of EGTA blocks the formation of Ca²⁺ waves (beta-actin::GCAMP3.1) upon laser injury (Movie S3, middle). The site of injury is marked with a white arrowhead.(C₁ and C₂) Control injury in the trunk of the same embryo as in (B). Ca²⁺ imaging (beta-actin::GCAMP3.1) before (C₁) and after (C₂) injury (Movie S3, right).(D) Cell tracking of microglia (pU1::Gal4-UAS-TagRFP) upon central brain injury in a control embryo. The white arrowhead marks the site of injury. The starting point of individual tracks is marked with an X and the end point with a dot.(E) Cell tracking of microglia (pU1::Gal4-UAS-TagRFP) upon central brain injury in a EGTA injected embryo. The site of injury is marked with a white arrowhead. The starting point of individual tracks is marked with an X and the end point with a dot.(F) Quantification of microglia movement to an injury site (percentage of microglia in the optic tectum responding to injury) in control (left, n = 5) and EGTA-injected brains (right, n = 5). Error bars represent standard deviations.(G) Single-hemisphere injections of caged IP3 (experiment). Single-neuron uncaging leads to Ca²⁺ signaling (beta-actin::GCaMP3.1) in the injected, but not in the control, side. Laser uncaging sites before (G₁) and after (G₂) uncaging are marked by white arrowheads.(H) Rose plot of microglial response to IP3 uncaging, showing microglia positions before (white) and 12 min after (red) uncaging.Scale bars for all images: 20 μm. Images in (B)–(F) were produced using an Olympus FV 1000 with a 40×/NA1.15 objective. Images in (A) and (G)–(H) were done using an Andor Spinning Disk Confocal with a 20×/NA0.7 objective.See also Figure S1 and Movies S3, S4, and S6.