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First published online 15 June 2005
doi: 10.1242/dev.01862

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Lineage restriction maintains a stable organizer cell population at the zebrafish midbrain-hindbrain boundary

Department of Genetics, University of Technology, Dresden, c/o Max Planck Institute for Molecular Cell Biology and Genetics, Germany

View larger version (154K): [in a new window]   Fig. 1. Morphological changes and cell behavior during mhb formation. (A-C) Confocal optical sections of live embryos, stained with BODIPY-ceramide. From the 12-somite stage (A) to 24 hpf (C), a small indentation forms in the neural tube (nt) between the prospective midbrain (mb) and hindbrain (hb) (B, arrowheads). At 24 hpf (C), the now very deep invagination clearly separates the midbrain tectum (t) from the hindbrain cerebellum (cb). The AP axis folds up in the mhb area (dots in A and C). (D-F) Confocal optical sections of time-lapsed live embryos containing GFP-expressing cells in the mhb area. Cells form a pseudo-stratified epithelium, stretching from apical to basolateral (double arrow in E). Cell groups are pseudocolored according to their final position and traced backwards in the time-lapse: tectal cells are red, cerebellar cells are yellow. No mixing between the cell groups was observed (n=13 time-lapse movies analyzed). The asterisk indicates a gap containing unlabeled cells that forms between the cell groups. All images are dorsal views, anterior is towards the left. Arrowheads indicate the position of the early indentation (B,E) and late fold (C,F) in the mhb area. Scale bars: 50 µm.

View larger version (158K): [in a new window]   Fig. 2. Single nucleus tracking procedure. (A-C) Nuclei can be identified in both the anti-Otx staining and the live image. (A) One plane of an Otx antibody staining stack. (B) One plane of the last live stack of a time-lapse. (B,C) Three Otx-positive nuclei are marked by plus signs, three Otx-negative nuclei (histone-GFP positive) are marked by asterisks. Owing to the fixation and mounting, the embryo is slightly compressed in C, therefore more nuclei are visible in C than in B. (D,E) Individual nuclei are assigned a status (red, Otx-positive), numbered and tracked backwards to the start of the time-lapse. Imaged embryos displayed a normal morphology with only minor distortions because of the agarose embedding procedure (data not shown). (F) Cell position can be measured in rows distance from the Otx interface. (G-I) Nuclei can be tracked over divisions. Daughter cells of the dividing Otx-negative cell (asterisks) move a significant distance apart (H) but sort back into the Otx-negative domain (I). Elapsed time is indicated in minutes. (J-L) Morphological and lineage boundary do not match. Shown are stills from Movie 2 in the supplementary material. A strip of nuclei at the boundary and their progeny were marked throughout the time-lapse. Nuclei out of focus are marked by translucent dots. The morphological boundary (horizontal line) is always posterior to the lineage restriction boundary (tilted anterior lines in L, compare with Figs 1, 5). Gray dots indicate the borders of the neuroepithelium. All panels show dorsal views, anterior is towards the top. Scale bars: 20 µm.

View larger version (35K): [in a new window]   Fig. 3. Summary plots of nuclei positions in time-lapse 1. (A,B) Otx-positive and negative nuclei form coherent groups at the end (B, 24-somite stage) and at the beginning (A, 5-somite stage) of the time-lapse. The lines in B indicate the mhb on the basis of the posterior Otx2 boundary. (C,D) Upon shifting the Otx-positive/Otx-negative interface artificially by about one cell row anterior at the five-somite stage (C), the sharp interface of the two cell populations is lost at the 24-somite stage (D). The bracket indicates the zone of overlap. (E,F) Example of cell behavior at an arbitrary boundary in the middle of the tectum. Even though the cell populations are well separated at the five-somite stage (E), they show a marked zone of overlap at the 24-somite stage (F). (G,H) Plots showing the relative movement of cells with respect to their interface at the mhb from the beginning to the end of the time-lapse, expressed in rows (y-axis). The arrows indicate the position at the start of the time-lapse to the position at the end. Diamonds stand for cells that do not change their row position. Upper light-gray arrows represent Otx-positive cells, lower dark-gray arrows represent Otx-negative cells. Each daughter cell is treated as a separate cell (x-axis). (G) Cell movement over 10 hours. (H) Cell movement over 12 hours. Notice that cells move 10 and more cell rows away from their common interface. Most non-moving cells are found in the first cell rows (both Otx positive and Otx negative). Otx-positive nuclei are light grey, Otx-negative nuclei dark grey. Broken lines show the midline. (A-F) Dorsal views, anterior is towards the top, units are in µm in A-F. For ease of understanding, only a subset (leaving out the most dorsal and ventral planes) of the data of time-lapse 1 is shown in A-D. The plots are 2D projections along the z-axis and nuclei sizes are not drawn to scale.

View larger version (80K): [in a new window]   Fig. 4. Clonal analysis of single cell injections. (A) Schematic drawing of the iontophoretic single cell injection: current flows through an electrochemical Ag/AgCl half-cell (hc), a dye-filled glass capillary (c), by dye-flow (red) into the target cell, through the embryo, through the medium and back to the amplifier's headstage (hs). Current intensity is controlled via the amplifier. (B,C) Embryos at 24 hpf bearing labeled cells on one side of the morphological mhb, the midbrain or cerebellum, respectively. (D) An embryo with a clear two-sided label at 24 hpf. (E) Individual cases could not be resolved at the morphological level when cells were located directly in the boundary region. (F) Summary chart of single cell injections and transplantations. The number of two-sided clones decreases from shield stage (n=11/47), 80% epiboly (n=2/35) to tailbud stage (n=0/25). Shield stage statistics show a combination of single cell injections and transplantations. (B-E) Dorsal views, anterior is towards the left. Scale bar: 100 µm. t, tectum opticum; cb, cerebellum.

View larger version (113K): [in a new window]   Fig. 5. Gene expression domains at the mhb share a common border that coincides with the lineage restriction boundary. (A,B) The posterior gene expression boundary of otx2 shifts from perpendicular to the neural axis to oblique between the 16-somite stage and 24 hpf, reflecting morphological changes during mhb formation (compare with Fig. 1). (B,C) From dorsal to ventral, the opening (broken line) in the otx2 expression domain becomes broader. (D,E) Similar to otx2, the anterior gbx2 and fgf8 expression domain borders become oblique with respect to the neural axis at 24 hpf. Further ventrally, the expression domains of gbx2 and fgf8 extend more anteriorly, probably filling the opening in the otx2 expression domain (D,E, compare with C). (F) Summary scheme of gene expression boundaries in the mhb area at 24 hpf. Dots indicate co-expression. The lineage restriction boundary between the otx2 and gbx2 territories is marked with broken black lines. All panels show dorsal views, anterior is towards the left. In situ probes were detected with fluorescent FastRed substrate and embryos optically sectioned on a Zeiss confocal microscope. Scale bar: 50 µm.