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Fig. 1. PMNs in tri;kny double mutants have hybrid identities. (A-D) islet1 RNA in situ hybridization at17-18 hpf. Dorsal view of tri;kny mutant (A) and lateral views of wild-type embryo (B), tri (C) and kny (D) mutants. The morphology of tri;kny mutants makes it difficult to obtain lateral views at these early stages. In lateral views, PMNs are ventral; dorsal cells are Rohon Beard sensory neurons (RBs) (see B). In dorsal views, all cells are PMNs; RBs are more lateral and outside the edges of these images. In wild types and single mutants, islet1-expressing PMNs (MiPs) are regularly spaced and their cell bodies are directly adjacent to the overlying somite boundaries (see schematic in E). In tri;kny mutants, islet1-expressing PMNs form almost continuous rows. In addition to the two major rows of PMNs, we also sometimes see some islet1-expressing and islet2-expressing cells more medial and slightly dorsal (arrowhead in A). Cross-sections (not shown) suggest that these PMNs form above a broader than normal floorplate. (E) Schematic of islet1 in situ hybridization showing MiPs adjacent to overlying somite boundaries. (F-I) islet2 in situ hybridization at 18-20 hpf. Dorsal view of tri;kny mutant (F) and lateral views of wild-type embryo (G), tri (H) and kny (I) mutants. In wild types and single mutants, islet2-expressing PMNs (CaPs) are adjacent to the middle of overlying somites (see schematic in J). In tri;kny mutants, islet2-expressing PMNs form almost continuous rows. (J) Schematic of islet2 in situ hybridization showing CaPs adjacent to overlying somite middles. (K-N) Islet antibody + islet2 in situ hybridization at 18-21 hpf. Dorsal view of tri;kny mutant (K) and lateral views of wild-type embryo (L) tri (M) and kny (N) mutants. Islet antibody staining is nuclear and brown; islet2 RNA is blue and cytoplasmic (see schematic in O). Brown-only cells (^*) express only islet1 and hence are MiPs; blue + brown cells express islet2 and possibly also islet1; these cells are either CaPs or hybrid PMNs. Comparison of double staining and single in situ hybridization shows that MiPs and CaPs are specified relatively normally in both single mutants. By contrast, the vast majority of PMNs in tri;kny mutants express islet2 (only one brown-only cell in K). (A) Shows that at least most of these PMNs also express islet1; this is confirmed by Islet antibody + islet1 in situ hybridization staining (U). (O) Schematic of Islet antibody + islet2 in situ hybridization. (P-S) znp1 antibody staining at 26-30 hpf. Lateral views of whole-mount wild-type embryo (Q), tri;kny (P), tri (R) and kny (S) mutants. Ventral CaP axons are clearly visible in all cases (examples indicated with circle). In wild-type embryos, and tri and kny mutants, MiP axons are visible in whole mounts (examples indicated with white arrow). However, MiP axons are very rare in tri;kny mutants and can be identified only in cross-section (X). (T) Schematic of a lateral view showing ventral CaP (blue) and dorsal MiP (red) axon trajectories. (U,V) Islet antibody + islet1 in situ hybridization at 18-21 hpf. Dorsal view of tri;kny mutant (U) and lateral view of wild-type embryo (V). In these embryos, brown-only cells express only islet2 and are therefore CaPs (#). Blue + brown cells express islet1, but possibly also islet2, and are therefore MiPs, or CaPs that have not yet completely downregulated islet1, or hybrid PMNs. We also see occasional cells that are blue only (+). These are probably RoPs or SMNs that have started to express islet1 RNA but not Islet protein. In tri;kny mutants (U), all of the PMNs express islet1 and have blue staining. The insert shows a higher magnification view of two of these PMNs. There are no brown-only cells (W,X) znp1 antibody staining at 26-30 hpf. Cross-sections of wild-type embryo (W) and tri;kny mutant (X). Ventral CaP axons (black circle) and dorsal MiP axons (white arrow) are visible in both cases. The MiP axon hugs the lateral surface of the spinal cord as shown in the schematic (Z). (Y) Schematic of Islet antibody + islet1 in situ hybridization staining. (Z) Schematic of a cross-section showing CaP (blue) and MiP (red) axon trajectories. The brown shading indicates znp1 immunoreactivity at the lateral surface of the spinal cord, caused by other znp1-immunoreactive spinal cord axons. Scale bar: 50 µm.

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