C Single optical section of dorsal ectodermal cells of 48 hr gastrula stage embryo. ( B) Z-projection of 5 optical sections of a 72 hr larva. ( B, C) Higher magnification showing cellular detail of basal surface of dorsal ectodermal cells. ( A) Fixation that preserves the cytoskeleton (PEM, Vielkind and Swierenga, 1989) reveals that Sp-Efn immunoreactivity localizes to long thin projections from the basal surfaces of dorsal ectoderm ( DE). The mean value for each region and S.E.M. The mean intensity per pixel within each rectangle was determined and the values were normalized to a percentage of the most posterior sample. The reticles were positioned so that they included only ectodermal cells and their associated cytonemes. Equal-sized reticles were positioned at 40 µm intervals along the dorsal surface. A median intensity projection of the stack of 8 optical sections from 6 specimens was prepared. Embryos in lateral orientations were selected and a set of 8 sections centered on an image plane that included the mouth was collected for each specimen. The distribution of Sp-Efn along the dorsal aspect of the ectoderm was determined from a set of mid-sagittal confocal optical sections from 72 hr embryos fixed with PEM and prepared for immunofluorescence with anti-Sp-Efn (4D2). The number of pigment cells within the volume bounded by the rectangular reticle was determined and plotted in Figure 1G. ( B) Sketch to demonstrate a projection of 8 optical sections centred mid-sagittally. A Sketch showing a mid-sagittal, lateral optical section of a pluteus. For each of these 6 projected images, the distance from the posterior-most tip of the larva along the dorsal surface was divided into 5 zones, each 40 µm in length the number of pigment cells in each zone was counted, and the mean and S.E.M. A maximum intensity projection of the 8 optical sections from 6 specimens was prepared. Embryos in lateral orientations were selected and a set of 8 optical sections centered on an image plane that included the mouth was collected for each specimen. The distribution of pigmented immunocytes on the dorsal aspect of the ectoderm was determined from a set of mid-sagittal confocal optical sections from 72 hr embryos fixed with methanol and prepared for immunofluorescence with Sp1. Mean intensity per pixel, normalized to the highest intensity per embryo was determined within each rectangle and Mean and S.E.M. Projections of 6-image stacks were prepared and equal-sized rectangles were positioned at 40 µm intervals along the ectoderm.
( H) The distribution of Sp-Efn in the same region of ectoderm was determined from a set of mid-sagittal images from 6 embryos prepared with anti-Sp-Efn (See Figure 1-figure supplement 1). The distance from the vertex of the larva along the surface to the animal pole domain was divided into five zones each 40 µm long and the number of pigmented immunocytes in each zone was counted.
Bar = 20 µm ( G) The distribution of pigmented immunocytes in the ectoderm between the animal pole domain and the vertex was determined from a set of mid-sagittal images from 6 embryos prepared with Sp1 (see Figure 1-figure supplement 1). M, mouth (PEM fixation) Bar = 20 µm ( F) Mid sagittal optical section of an early pluteus prepared with Sp1 to show the distribution of pigmented immunocytes (MeOh fixation). Arrows indicate high levels of immunoreactivity where the postoral larval arms are situated. Bar = 20 µm ( E) Maximum intensity projection of an early pluteus prepared with anti-Sp-Efn and oriented with the ventral surface foremost. Sp-Efn expression is not uniform throughout the dorsal ectoderm, there is an apparent gradation of expression that is highest at the vertex of the larva. BP, blastopore (PFA fixation) Bar = 20 µm ( C) Single sagittal optical section of a prism larva showing the expression of Sp-Efn only in the dorsal ectoderm and ciliary band and not in the ventral ectoderm (VE) (PEM fixation) Bar = 20 µm ( D) Single sagittal optical section of an early pluteus larva showing the expression of Sp-Efn and the ciliary band marker, Hnf6. The ligand is not expressed in the ventral ectoderm (VE). Bar = 15 µm ( B) Maximum intensity projection of confocal optical sections of a gastrula prepared with an antibody to Sp-Efn.
Immunocytes are most dense at the tips of larval arms, along the edge of the ciliary band, and the posterior tip of the larval body.
Immunocytes do not normally insert in the ventral ectoderm (VE), which is the larval surface surrounding the mouth (M) and encircled by the ciliary band. Pigmented immunocytes are scattered throughout the dorsal ectoderm (DE), which is the larval surface posterior to the ciliary band (CB). ( A) Maximum intensity projection of confocal optical sections of an early pluteus larva prepared with Sp1, a pigmented immunocyte cell surface marker, and pFAK, a marker for apical junctions that are prominent in ciliary band cells (MeOH fixation).
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