(A) Maximum intensity projection of an embryo injected with Sp-Eph MO1 (MeOH fixation). Pigmented immunocytes are dispersed, with some having inserted into the ectoderm. The immunocytes are more rounded and do not have their typical dendritic form. They are commonly within the blastocoel. The ventral ectoderm (VE) remains clear of immunocytes. Inset: DIC image of another specimen showing the overall healthy appearance of Sp-Eph morpholino injected embryos (MeOH fixation). (B,C) When Sp-EphMO1-injected embryos are prepared with an antibody that recognizes only the cleaved, or activated form of Caspase3, pigmented immunocyte precursors in the blastocoel are immunoreactive (MeOH fixation). (D) Control morpholino injected embryos have almost no pigmented immunocytes that are anti-Caspase3 immunoreactive (MeOH fixation). (E) Counts of the number of Sp1 immunoreactive cells indicate that there are no differences in the number of cells among treatments (NegControlMO, Sp-EphMO1, Sp-EfnMO1, DMSO, or NVP) in prism stages. However, in early plutei there are fewer pigmented immunocytes in Sp-EphMO-injected embryos, or embryos treated with NVP. (F) Interfering with expression of Sp-Eph blocks the transition to epithelial-inserted, dendritic morphology. In Sp-EphMO1 injected embryos, immunocytes have larger diameters, shorter filopodia, and fewer lamellipodia than NegControlMO injected embryos. (G) There are fewer pigmented immunocytes in the blastocoels of 72 hr embryos injected with control MO, or Sp-Efn MO1 than there are in 72 hr embryos injected with Sp-Eph MO1. (H) Preparations of Morpholino injected embryos with an antibody that recognizes the activated form of Caspase3 show that there are significantly more pigmented immunocytes in the blastocoel expressing activated Caspase3. (I–L) Expressing Sp-Efn throughout the embryo results in mislocalization of immunocytes. (I) Lateral view of an embryo injected with 200 ng/µl Sp-Efn RNA. The image is a projection of 8–1 µm optical sections centered on the mouth. Note that Sp1 labelled immunocytes have inserted, or are closely associated with the ventral ectoderm (VE) (MeOH fixation). (J) Maximum intensity projection of the ventral surface of an embryo injected with RNA encoding full length Sp-Efn. Pigmented immunocytes are inserted into the ventral ectoderm (MeOH fixation). See Figure 4—figure supplement 1 for a set of orthogonal projections at various levels through the image stack used to prepare this projection, which demonstrates that the Sp-1 labelled immunocytes are inserted in, or closely associated with the ventral ectoderm. The position of the oval outlining the ventral ectoderm was determined by the higher nuclear density of the cliary band. M; mouth K. Lateral view of an uninjected, control embryo prepared in the same manner as I (MeOH fixation). The image is a projection of 8–1 µm optical sections centered on the mouth. Note that Sp1 labelled immunocytes have not inserted in ventral ectoderm (VE). (L) A single mid-sagittal, optical section of an embryo injected with Sp-Efn RNA, showing immunoreactivity in the ventral ectoderm. Note that at this stage there is almost no expression of Sp-Efn in endoderm and the expression in ventral ectoderm appears graded (PEM fixation). (M) Maximum intensity projection of an embryo injected with Sp-Efn RNA showing ectopic expression of Sp-Efn in the ventral ectoderm (PEM fixation). * indicates significantly different outcomes Bars = 10 µm.