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Fig. S1. Correlation between physical landmarks and rhombomere positions. (A) A 5-dpf Islet1:GFP transgenic larva that labels cranial motor nuclei with GFP. This transgene is distinct from Isl1SS and uses a separate enhancer element of the islet1 gene. The positions of rhombomeres (r1-r8) are linked to the positions of various cranial motor nuclei. (B) Overlap of fluorescent image with bright-field image. Segments 1-15 are determined by bright-field physical landmarks. Va and Vp, anterior and posterior trigeminal motor nuclei; VII, facial motor nucleus; X, vagus motor nucleus. Scale bar: 100 µm.
Fig. S2. Trpa1b:GFP reporter line accurately marks trpa1b-expressing cells in trigeminal sensory neurons. trpa1b in situ hybridization was performed in 2 dpf Trpa1b:GFP transgenic fish. Images are single optical sections of trigeminal sensory ganglia, with GFP in green and trpa1b in red. All cells shown are positive for trpa1b. In total, seven ganglia were analyzed and 73 out of 74 fluorescent cells expressed trpa1b. Scale bar: 20 µm.
Fig. S3. Expression of trigeminal sensory neuron markers in Trpa1b and Isl1SS subtypes. (A,B) Optical sections of 2-dpf trigeminal ganglion (outlined in blue) stained with RNA probes for sensory neuron markers (red) and antibodies against transgenic reporters (green, Trpa1b:GFP in A and Isl1SS:Kaede in B). Left panels show the merged image. Right panels show only marker staining, with transgenic reporter-positive cells outlined. White outlines indicate double-positive cells and yellow outlines mark GFP-only or Kaede-only cells. Percentages of marker-positive cells for each subtype are shown in Fig. 1E. trkC1 and trkC2 staining appears as two dots in the nucleus. Asterisk marks the adjacent lateral line ganglion. Scale bar: 20 µm.
Fig. S4. Comparison of axonal varicosities and presynaptic puncta. Trigeminal neurons expressing both RFP and Syp-GFP were imaged and the number of varicosities and Syp-GFP puncta were measured independently. (A) The average number of total varicosities versus total puncta. There is no significant difference between these two measurements. Error bars indicate s.e.m. (B) Correlation of varicosities and Syp-GFP puncta. Each data point is measured from a single trigeminal axon segment, as defined in Fig. 4A. Linear regression shows that the two measures are highly correlated (P<0.0001) with a slope close to 1.
