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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.