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Fig. 1. Explantation technique depicting how explants discussed in this paper are made. (A) This schematic shows the tissues relevant to our explants, including the notoplate, the neural plate (NP), the notochord (Ntc), the somitic mesoderm (S), endodermal epithelium (ee), and the midline (notoplate, notochord and underlying endodermal epithelium). (B) This schematic shows the expression of genes relevant to this paper, including Sonic Hedgehog in the deep notoplate, and N-β-tubulin in the deep neural plate. (C) Deep-neural-over-mesoderm explants: We first remove and discard the superficial neural tissue (dotted) at stage 13. We then explant the dorsal 180 degree sector of the embryo and culture it under coverslip. The dashed lines represent the edges of the notochord, slightly visible under the deep neural tissue. (D) Midlineless explants start as deep-neural-over-mesoderm explants, from which we subsequently remove the notochord, notoplate and underlying endoderm. We then push together the two lateral sections of deep neural tissue over somitic mesoderm and allow healing to occur. (E) Midlineless-explants-with-ectopic-midline require at least two embryos. The first embryo was previously injected with red dextran at stage 6 1/2 to produce a scatter of red labeled cells (red scattered cells). The second embryo was either injected with fluorescein dextran at the one-cell stage and was therefore entirely green fluorescent or was a transgenic Otx2:GFP embryo with a green fluorescent notochord (as depicted in this figure). NP, Neural plate; S, Somitic mesoderm; Ntc, Notochord; ee, Endodermal epithelium; sup, superficial; mdln tiss, midline tissues; h.k., hair knife. Davidson and Keller, 19991; Lance Davidson, unpublished observations.2
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Fig. 2. Cell paths and cell protrusive activity in deep-neural-over-mesoderm and midlineless explants. In (A, B), tips of arrows show the position of cells at the beginning of a time lapse, the arrowhead represents cell location at the end of the time lapse, and the stem depicts the path of the cell. (A) Lateral cells in deep-neural-over-mesoderm explants translocate medially toward the midline notochord/floor plate, resulting in convergence and extension. (B) Even though the midline tissues have been removed, lateral neural cells in midlineless explants also move medially toward the midline of the explants, albeit they do so less aggressively than cells in deep-neural-over-mesoderm explants. Arrows at the edge of the explants move outward. These are probably ectodermal cells, and can sometimes be seen in deep-neural-over-mesoderm explants, though more rarely than in midlineless explants because they are generally too wide to encompass ectodermal cells on the computer screen. (C, D) The protrusive activity of lateral neural cells in deep-neural-over-mesoderm and midlineless explants. (C) Lateral neural cells in deep-neural-over mesoderm explants have lamellopodia pointed toward the midline. Arrows point to protrusions. (D) A bullet diagram quantifies the protrusions of one cell traced from a control explant; it shows a clear bias in the direction of the midline tissues in the explant. Scale bar, 0.1 mm. (E, F) The protrusive activity of lateral neural cells in midlineless explants. (E) Frame from a time-lapse sequence, showing motile cells in midlineless explants. Arrows point to protrusions. (F) A bullet diagram shows the distribution of protrusions over time for one cell analyzed from a midlineless explant; the cell is aligned along the mediolateral axis of the explant. Scale bar, 0.1 mm.
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Fig. 3. Conservative intercalation occurs in midlineless explants. Colored cells show columns of cells that were traced to the end of the movie to see which cells intervened between them. Patterned cells are the cells that intervened between the colored cells. (A) Unbroken columns of cells at the beginning of a timelapse series. (B) By the end of the time lapse, patterned cells have intercalated between the colored cells; however, they were very close neighbors of the colored cells at t = 0.
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Fig. 4. Motility in midlineless-explants-with ectopic-midline. (A, B) A midlineless-explant-with-ectopic-midline made from an Otx2:GFP transgenic embryo converges and extends between t = 0 (A) and t = 4.5 h (B). The notochord lights up with GFP driven by the Otx2 promoter, demarcating the ectopic midline; the ghost midline is shown as a dotted white line. Labeled cells (white sprinkle) sandwiched between the ectopic midline and the ghost midline (section “bâ€) were followed for translocation (direction of their overall displacement) and protrusive activity. Scale bar in (A) and (B), 0.5 mm. (C) In midlineless-explants-with-ectopic-midline, cells in the neural plate of the donor midline (section “aâ€) converge to that midline. The two parallel dashed lines represent the edges of the notochord seen through the deep neural plate. Cells in the midlineless component of the explant (section “bâ€) still converge and extend, although their movement is erratic. Cells in section “c†get displaced toward their endogenous (ghost) midline through intercalation. The two parallel dashed lines represent the ectopic midline, and the dotted line is the ghost midline, at the beginning of the time-lapse sequence. The two panels, (1) and (2), come from two different embryos. (D) Frame from a movie shows protrusive cells in the section ‘b’ of an explant with an ectopic midline. Arrows point to protrusions. Scale bar; 0.1 mm.
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Fig. 5. Polarization and orientation of cells in midlineless-explants-with-ectopic-midline. The schematic at the top summarizes the state of polarization of cells in this style of explant. All cells become monopolar and oriented to whichever midline is closest to them, except for cells that are equidistant from the ghost and the ectopic midline, which instead point toward more random directions. Below the schematic of the explant, the two graphs categorize cells by distance from the ectopic or the ghost midline along the x-axis (0â3, 4â10, and over 10 cells away from the ectopic midline; 0â3 and over 3 cells away from the ghost midline). The top graph shows the percentage of cells in particular polarization states (monopolar and biased combined, bipolar, and random), and the bottom graph shows the percentage of monopolar cells that point in particular orientation.
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Fig. 6. Patterning of deep-neural-over-mesoderm explants, midlineless explants and midlineless-explants-with-ectopic-midline. Deep-neural-over-mesoderm explants maintain a very close to normal mediolateral pattern of gene expression as seen through Sonic Hedgehog, N-β-tubulin, and Xslug (A–C). Midlineless explants have no remaining midline, as shown by the absence of SHH expression (D). They express N-β-tubulin, but the pattern of expression of that gene is often disturbed (E), while Xslug is strongly expressed, though variable in breadth (F). Midlineless-explants-with-ectopic-midline have no SHH stripe in the midlineless portion, but it is present in the portion of the ectopic midline as expected (G). In addition, they express N-β-tubulin and Xslug in both the midline-free and the midline-containing parts of the explants (H, I). Pointers indicate the ectopic midline; all explants were fixed for in situ when whole embryos used as control reached stage 21 to 23.
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Fig. 7. The two-signal model for the mechanism through which the midline might polarize and orient neural cells. A persistent signal from the midline orients neural cells (red curves) and a distinct, short-lived signal monopolarizes them (black arrows). The monopolarizing signal passes unabated through the length of the explant, and neural cells respond by becoming monopolar protrusive. These monopolar protrusive cells direct their protrusions to the strongest source of orienting signal. This may be the ghost midline or the ectopic midline, and under normal circumstances, it is the endogenous midline. Cells that are equidistant between the ghost and the ectopic midline in midlineless-explants-with-ectopic-midline cannot clearly make out the direction of the orienting signal, and as a result, they protrude monopolarly but in random directions. The direction of monopolar cells in response to the orienting signal is shown with yellow arrows. Hypothetically, a high level of orienting factors is found near the ectopic midline, and the levels taper off with increasing distance from the midline, due to the effects of diffusion. Closest to the ghost midline, the leftover signal from the extracted midline still orients cells medially.
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