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Fig. 2 Nkx6 proteins regulate late islet1 expression. (A-D) Dorsal views at 11-12 hpf. (A) islet1 mRNA expression in PMNs of control embryos. islet1 mRNA expression is unaffected by nkx6.1 (B), nkx6.2 (C) or double nkx6 (D) MO-injection. (E-L) Lateral views of axial level 8-12 at 18 hpf. (E-H) islet2 mRNA expression is normal in control and nkx6 MO-injected embryos. (I-L) islet1 mRNA expression is confined to MiPs (arrows). In nkx6.1 MO-injected embryos, there are fewer islet1-positive cells (J; islet1-positive cells present in 74.4% of segments; n=133 segments in 14 embryos) as compared with controls (I; islet1-positive cells present in 82.4% of segments; n=97 segments in 12 embryos). The faint cell in J in the segment lacking an arrow is on the other side of the embryo; as far as we can tell the distribution of segments lacking islet1-positive MiPs is random and not correlated between the two sides of the embryo. (K) nkx6.2 MO-injected embryos also had a decrease in islet1 expression (islet1-positive cells present in 67.7% of segments; n=99 segments in 10 embryos). (L) The most dramatic loss of islet1 expression was in double nkx6 MO-injected embryos (islet1-positive cells present in 58.7% of segments; n=126 segments in 13 embryos). The number of islet1-positive cells might be an overestimate in some cases, because of islet1 RNA expression in RoP motoneurons (Appel et al., 1995). In some of these images, there appear to be abnormal numbers of Rohon-Beard (RB) spinal sensory neurons (islet1-positive cells in the dorsal spinal cord). This is an artifact of the way we mount the embryos to visualize PMNs in focus in many adjacent segments, which often requires tilting the embryos. The number of RBs appears within the normal range in nkx6 MO-injected embryos. Scale bar: 20 µm.