DEV02826 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Zebrafish have three Nkx6 proteins. (A) Nkx phylogeny demonstrating the three zebrafish Nkx6 proteins uniquely cluster within one node which is distinct from the Nkx2.2 and Nkx1 class of proteins. The PHYLIP software package was employed to create a neighbor-joining tree using the ClustalW algorithm. Sequences included in the tree were gathered from GenBank and bootstrap values are indicated. Expression of nkx6.3 mRNA begins at 20 hpf, persists until at least 72 hpf, and is limited to a small population of cells posterior to rhombomere 5 in the hindbrain. (B) Alignment of zebrafish Nkx6.1, Nkx6.2 and Nkx6.3 translated amino acid sequences. The dashed red line indicates the NK domain and the solid blue line indicates the homeodomain. (C) Whole-mount in situ RNA hybridization reveals that nkx6.3 is expressed in a subset of hindbrain cells at 25 hpf. (D) At 36 hpf, nkx6.3-expressing cells (purple) are Islet:GFP-negative and are located ventral to cells labeled with Islet:GFP (brown, GFP antibody staining). Lateral views, anterior to the left, dorsal to the top. ov, otic vesicle; sb, somite boundary. Scale bar: 40 μm.

• Supplemental Figure 2 -

  Fig. S2. Anti-Nkx6 detects both Nkx6.1 and Nkx6.2 proteins. 24-hpf embryos, anterior to the left. (A-D,H-J) lateral views; (E-G) dorsal views. The confocal images in E-G were all taken at the same settings to ensure comparability; likewise for the confocal images in H-J. The confocal images in H-J show only one side of the spinal cord; thus, there are fewer labeled cells than in the images in A and B, which show whole-mount embryos. nkx6.1 (A) and nkx6.2 (B) mRNA are expressed in the ventral spinal cord in similar patterns. Both nkx6.1 and nkx6.2 (C,D) mRNA are expressed in the ventral brain, although the expression patterns are different. Injection of nkx6.1 MO decreased Nkx6.1 antibody labeling in brain (F) and spinal cord (I) as compared to controls (E,H). However, some Nkx6.1-positive cells remained, suggesting that the antibody recognizes both Nkx6.1 and Nkx6.2 proteins. We tested this hypothesis by co-injecting nkx6.1 and nkx6.2 MOs, which extinguished nearly all Nkx6 antibody labeling in the spinal cord (J) and brain (G). Thus, we infer that the antibody recognizes both Nkx6.1 and Nkx6.2 proteins. Scale bars: 50 μm.