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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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