kiaa0319 is specifically expressed in the notochord. The expression of kiaa0319 (a) and kiaa0319l (b) was examined at three embryonic stages (12, 24, and 48 hpf; WT zebrafish) using the RNAScope fluorescent multiplex assay with results shown for the head (a1, a2, a3, b1, b2, and b3) and the body (a4, a5, a6, b4, b5, and b6). kiaa0319 (red) is expressed throughout the three stages. High expression is detected in the developing brain and body midline. At 48 hpf, kiaa0319 is still highly expressed in the brain and strong signal is detected in the otic vesicles (a3; white arrows corresponding to the red arrow in the reference image) and in the notochord (a6). kiaa0319l shows a similar pattern of expression, including a strong signal in the otic vesicles (b3), but the expression in the notochord at 48hpf is very weak (b6). Black area in the brain at 48 hpf correspond to the pigmentation of the embryo (a3 and b3). (c) Longitudinal view of the body with 3D reconstructions from light‐sheet microscopy images at three developmental stages 72 (c1), 96 (c2), and 120(c3) hpf. The samples from a WT zebrafish are labeled with the kiaa0319 probe. kiaa0319 expression is localized to the notochord (white arrow). The signal diminishes as development progresses and the notochord regresses. See a 3D animation at 72hpf that allows assessing the signal from different orientations (Video SV1). See Figure S3 for the positive and negative controls acquired with light sheet microscopy (d) kiaa0319 expression in the notochord was confirmed with the Tg(gfap:GFP);Tg(Oligo2:dsRed) transgenic line, which express GFP (green) in the spinal cord (d1). No signal was detected for the negative control (d2) myoD1 (myogenic differentiation 1, a universal target for myogenic cells [Weinberg et al., 1996]) was used as positive control and demonstrates the specificity of the assay images were collected at 42 hpf by confocal microscopy. The scale bar indicates 50 μm in all panels
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