Fig. 3

Optogenetic activity control influences THN migration.

(A) Overlay of fluorescent and transmitted-light images of a 30 hpf embryo co-expressing H2B-GCaMP6s and ChR2-YFP. At this developmental stage, only nuclei at the ventral end of the MHB express H2B-GCaMP6s at high enough levels to detect fluorescence signal increases over the background fluorescence from ChR2-YFP (nuclei 1 and 2, compare to nucleus 3 in the central region of the MHB). Nuclei at the URL, however, express only ChR2-YFP and serve as negative control (nucleus 4). Scale bar: 25 μm. (B) Nuclei co-expressing both markers show highly regular calcium transients (nuclei 1 and 2), while negative control THNs do not (nuclei 3 and 4). (C) Fourier transformation reveals that the frequency of calcium transients in nuclei 1 and 2 strongly coincides with the expected frequency of one signal per min, or 16.7 mHz (arrows). Nucleus 3 shows a weak peak at this frequency. (D) Tracking of THNs expressing either control (YFP-CAAX), ChR2-YFP, or SwiChR-YFP along the MHB (colored dots) demonstrates that hyperpolarization slows THNs down, while depolarization increases speed. Transmitted light images on the left provide positional information on THNs within the tissue; boxes indicate the region shown at greater magnification in the fluorescence images. Migration progress over time is schematically indicated on the right, in which arrows indicate the start and end points for the shown frames. Elapsed time is indicated at the top. Scale bar: 25 μm. See also S4 Video. (E) Individual THNs (orange) were tracked in SIMI°BioCell software for quantification. An example is marked in dark blue. The tracks were corrected for tissue shifts using reference points (cyan). See also S5 Video. (F) THNs reduce their migration speed as they progress along the MHB from approximately 25% to −35% of the distance to the ventral end. Zero percent is the dorsal and 100% is the ventral end of the MHB. Track distribution is given at the top. (G) Migration speeds are affected by de- and hyperpolarization irrespective of starting point. Means ± SEM are plotted per 10% MHB bin. (H) Tracks were grouped as either dorsal (≤ −27.5% MHB) or ventral (≥27.5% MHB) to test the significance of de- and hyperpolarization compared to control level. Only significant differences are indicated. Data depicted in the graphs can be accessed in S2 Data. CAAX, PM-targeting signal derived from K-Ras; ChR2, channelrhodopsin; F/F0, fluorescence over background; GCaMP6, circular permutated green florescent protein-Calmodulin-M13 peptide 6s; H2B, Histone 2B; hpf, hours post fertilization; MHB, midbrain-hindbrain boundary; n.s., not significant; PM, plasma membrane; SwiChR, mutated channelrhodopsin; THN, tegmental hindbrain nuclei neuron; URL, upper rhombic lip; YFP, yellow fluorescent protein.