Fig. 6.

Pioneer SAG neurons have a role in organizing the SAG. (A) Scheme of experimental design. At 16 hpf, pioneer SAG neurons are photoconverted and subsequently photoablated. The absence of magenta photoconverted cells at later stages indicates complete cell ablation. SAG development was imaged from 20-24 hpf to 34 hpf. OP, otic placode. (B) After partial (asterisk) or total ablation of Neurod⁺ cells anterior to the otic placode at 16 hpf, an altered shape of the SAG and an apparently reduced number of NBs can already be observed at 24 hpf. At 34 hpf, the formation of the posterior lobe is abrogated in the ablated conditions (blue dotted line; see also Fig. S10). The experiment was replicated three times. n control=4+3+6; n ablated condition=4+3+11 (n=number of embryos). (C) Migratory properties of otic NBs in control (green) or after ablation of pioneer SAG neurons (magenta) measured with DiPER (Breau et al., 2017; Gorelik and Gautreau, 2014). (C′) NB migration directionality and pathway are compromised in after pioneer SAG neuron ablation compared with controls. n=23 control cells, n=36 ablated condition cells, from 4 and 10 embryos, respectively for each condition. Each tick in axes represents a 15 µm increase. (C″) NB dispersion (95% confidence interval) according to the last timepoint position with tracks normalized to the origin in control (green dots) versus ablated (magenta dots) conditions. Non-overlapping regions illustrate the variance in dispersion, which is significant in the y-axis but non-significant in the x-axis (ns). Ticks in axes represent 10 µm. (C‴) NBs are able to migrate faster in the ablation condition. The mean migratory profile is not majorly affected, except that dispersion of velocity is increased in the ablated condition at initial stages of migration (Fig. S11 shows detailed individual migratory profiles). The number of cells populating the SAG is significantly reduced in the ablation condition, more than the number of cells ablated (Fig. S9A) and consequently SAG volume (Fig. S10). N number of embryos for this section (C‴) is 4 both for control and the ablated condition. *P<0.05, ***P<0.0005 (two-tailed Student's t-test). (D) Two examples of migration of NBs (photoconverted magenta cell, yellow tracks) between lateral and medial regions of the neurogenic domain. The otic vesicle is depicted within a white dashed line. Total control embryos n=6; total ablated embryos n=11 for this section (D). Scale bars: 20 µm.