Fig. S4

DoubleSwitch method to stochastically label and reconstruct Gsx1 R4 neurons.Related to Figure 5.

A. Top: tails of larvae (6 dpf) with either a Cre reporter (left, ?actin:Switch) or B3 reporter (right, HuC:Gal4, UAS:bloSwitch) after injection of 1 pg of recombinase RNA into the singlecell embryo. In both recombinase systems, the intact switch reporter expressed GFP (gray), and after recombinase-dependent excision, expressed RFP (red). Bottom: Western blot analyses of recombinase-dependent RFP fluorescence in larvae (6 dpf) with either a Cre reporter (Switch) or B3 reporter (bloSwitch) after injection of recombinase RNA into the single-cell embryo.

B. Lethality (% Death) of recombinase RNA injection into 1-cell stage embryos (Cre red, B3 blue). n=2 clutches for each recombinase. Error bars are s.e.m.

C. Reconstructions of Gsx1 neurons in the PPI-active region with a standard model of Mauthner cells in the coronal (left) and horizontal (right) views. Type A neurons projected bilaterally beyond R4 to rostral and caudal brain regions. Type B neurons, located in a lateral zone, confined their bilateral processes to rhombomeres 4-5, where they projected ventrally and ramified below the Mauthner lateral dendrite. Type C neurons projected ipsilaterally. D, dorsal. R, rostral. Scale bar 50 ?m.

D. Projections from type A Gsx1 neurons bilaterally appose the Mauthner cells at positions on the soma, with a strong correlation between the medio-lateral location of the apposition on the ipsi and contra-lateral Mauthner somas. n=5 neurons. Pearson correlation coefficient R2 = 0.9, P=0.04.