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Fig. S2 Zebrafish nf1a and nf1b are expressed during embryonic development. (A) Distribution of ESTs mapping to nf1a or nf1b in dbEST including tissue of origin. (B) RT-PCR analysis verifying maternal (one-cell) and zygotic (24, 72, and 84 hpf) expression of nf1a and nf1b. (C1–C6 and D1–D6) Whole-mount in situ hybridization of 24-hpf, 48-hpf, 3-dpf (all magnifications of insets in Fig. 2), and 4-dpf zebrafish embryos shows temporal and spatial expression patterns of nf1a and nf1b. (C5–C6 and D5–D6) Both nf1a and nf1b are expressed in cardiac tissue at 48 hpf and 3 dpf as seen in these anteroventral images. Notably, analysis of 14 μm (C7 and D7) cross-sections of whole-mount stained 4-dpf zebrafish embryos reveals strong nf1a and nf1b expression in the region of the dorsal vessel (arrows) directly below the notochord (NC). (E) Parameters used for sorting GFP-positive and GFP-negative cells from 3.5-dpf Tg(kdrl:GRCFP)zn1 zebrafish embryos. (F) Quantitative PCR analysis of the GFP-positive cell fraction reveals marked enrichment for endothelial-specific genes, including cdh5 and etsrp, relative to the GFP-negative fraction. (G) Reverse-transcription and PCR (RT-PCR) for nf1a and nf1b was performed using first-strand cDNA synthesized from endothelial cell RNA (GFP-positive cell fraction), confirming expression of both genes in vascular endothelium. (Scale bars: 25 μm.)