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First published online May 17, 2004
doi: 10.1242/10.1242/dev.01140

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Single cell lineage and regionalization of cell populations during Medaka neurulation

¹ Kondoh Differentiation Signaling Project (ERATO/SORST), Japan Science and Technology Corporation, 14 Yoshida-Kawaracho, Sakyouku, Kyoto 606-8305, Japan
² Graudate School of Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
³ University Freiburg, Institute Biology 1 (Zoology), Department of Developmental Biology, Hauptstrasse 1, D-79104 Freiburg, Germany
⁴ Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

View larger version (73K): [in a new window]   Fig. 1. Cell positions in live Medaka embryos and their transfer to a computer graphics model. (A) Two labeled cells in an embryo at stage 15++ (20 hpf). (a,c) Fluorescence images of the same embryo viewed from different angles. Broken white lines indicate outline of embryo, blastoderm edge and cell thickening along the dorsal midline. Images in a,c were superimposed on stage- and size-matched computer graphics models in b,d, and positions of the two fluorescent cells were plotted on the model using red and black dots, respectively. The diameter of dots corresponds to 20 µm, approximating average cell size. Insets in a,b,d are enlargements of the squares indicated by broken lines. The cell coordinates l1 (along the dorsal midline shown by red broken line) and d1 (distance along the circumference from the midline) of the first measurement (b) agreed with those of the second (l2 and d2), and two plots overlapped well (d). (B) Three cells at stage 17 (25 hpf). (a,c,e) Fluorescence images of dorsal, lateral and frontal angles. (b,d,f) Cell positions in a,c,e are plotted on the computer graphics model. Insets in a,b are enlargements of the small squares. The cells indicated by the green dots later contributed to retina, while those indicted by orange dots contributed to mesencephalon. Error bars indicate standard deviations in embryo length along the dorsal midline and positions of the plotted cells (insets). Scale bars: 200 µm.

View larger version (118K): [in a new window]   Fig. 2. Distribution in stage 13 blastoderm of cell populations that later gave rise to neural or non-neural tissues. (A) A single labeled precursor cell (arrow) at stage 13 embryo in animal pole view. The arrowhead indicates embryonic shield. (B) Anterior region of the same embryo at stage 24 (44 hpf) in lateral view. The single cell shown in A divided, and its daughter cells (arrows) were later fated to mesencephalon (m) and rhombencephalon (rhombomere 1) (r). (C) One hundred and seven randomly chosen cells of stage 13 (13 hpf) embryos, one to two cell diameters beneath the envelope layer, were labeled and their location in stage 24 embryos (44 hpf) was recorded. Labeled neural progenitor cells (including CNS, placodes and neural crest) are indicated by red dots and shown in animal pole view. Light blue dots represent cells that contributed to epidermis and purple dots to somitic mesoderm. The arrowhead indicates dorsal midline (embryonic shield). Blastoderm margin (leading edge of epiboly) is indicated by a circle. The dot with an arrow indicates the labeled cell shown in A. Broken lines highlight the 140° neurogenic sector. Scale bars: 200 µm.

View larger version (255K): [in a new window]   Fig. 3. Time course of cells labeled at stage 13 (13 hpf) and their descendants until stage 24 (44 hpf). (A) Precursor cell populations at stage 13 and (C-E) their descendants at stage 24. Throughout, cells are indicated by dots and color-coded according to the location of cell fates at stage 24 as indicated in (C): telencephalon (Tel, red); retina (Re, green); diencephalon (Di, black); mesencephalon (Mes, orange); rhombencephalon (Rho, blue); spinal cord (SC, white); epidermis (Epi, light blue); peripheral nervous system including placodes and neural crest (yellow); trunk mesoderm (pink). Data of left and right halves are superimposed assuming bilateral symmetry of the embryo. (A) Plots of cells on the embryo model at stage 13 in animal pole view (dorsal midline towards the bottom). Double-colored dots (e.g. arrowhead) in A indicate cells that contributed to two different neural subdivisions or tissues as shown by cells in Fig. 2A,B. A pair of cells located adjacent to one another at stage 13 are indicated by black and white arrows. These cells are indicated in the panels of later embryos, and at stage 24 (D,E) are located distantly in the diencephalon and rhombencephalon. (B) Outline of territories occupied by the groups of cells later contributing to distinct neural subdivisions, which are indicated by the same color codes as the cells. (C) Neural subdivisions of Medaka at stage 24 illustrated with distinct colors. (D,E) Distribution of labeled cells in stage 24 embryo in side (D) and dorsal (E) views. (F-K) Distribution of color-coded cells on embryo model from stage 13 to stage 18. (F) Stage 13 (early gastrula, 13 hpf); (G) stage 15 (mid-gastrula, 17.5 hpf); (H) stage 16 (late gastrula, 21 hpf); (I) stage 16+ (late gastrula +, 22 hpf); (J) stage 17 (25 hpf); (K) stage 19 (two somites, 28 hpf). Cell groups forming individual neural subdivisions are clearly separated during the 1 hour period 21 hpf (stage 16) to 22 hpf (stage 16+). (A',G'-K') Enlargement of squares in A,G-K. Scale bars: 200 µm. Black and white arrows indicate two individual cells as in A-K

View larger version (86K): [in a new window]   Fig. 4. Trajectories of representative cells contributing to distinct neural subdivisions. (A) Distinct trajectories of cells that contributed to neural and non-neural tissues displayed on an embryo model in dorsal view from stage 13 (black dots) to stage 15 (blue dots), and then to stage 16+ (red dots). Trajectories of the cells are color-coded according to the later contributing neural subdivisions, as in Fig. 3: red, telencephalon; green, retina; orange, mesencephalon; blue, rhombencephalon; white, spinal cord; yellow, placode and neural crest derived tissue; light blue, epidermis. (B-H) Trajectories of ten randomly selected cells (with exception of telencephalon, five) that later contributed to different neural subdivisions: (B) telencephalon; (C) retina; (D) diencephalon; (E) mesencephalon; (F) rhombencephalon; (G) spinal cord; (H) tissue of placode and neural crest origin. Colored dots in black, blue, red and yellow indicate cell positions at stage 13 (25% epiboly), stage 15 (50% epiboly), stage 16+ (90% epiboly) and stage 24, respectively. Figures in the bottom right-hand corner indicate the average distance (µm) of migration among the cell groups from stage 13 to stage 15 (upper) and from stage 15 to stage 16+ (lower). Scale bar: 200 µm.

View larger version (60K): [in a new window]   Fig. 5. Establishment of the compartments for future neural subdivisions at stage 16+. (A) Location in embryo and size of the cell groups later contributing to distinct cephalic divisions, color-coded according to Fig. 3. Lateral view of tel-, di-, mes- and rhombencephalon, and spinal cord cell groups from stage 16+ (a) to stage 24 (f). Animal pole on the top and dorsal side on the right. White and red broken lines outline the boundaries for future neural subdivisions. The cell groups for tel-, di- and mesencephalon are displaced anteriorly without changing their AP length, while those for rhombencephalon and spinal cord elongate along the body axis. (B) Measurement of cell movement within cell groups. (a) Horizontal planes across the CNS-forming cell mass to measure cell movement along the AP axis. The planes were drawn on a computer graphics embryo model, so that planes 3 and 8 are placed at the position corresponding to the boundaries at stage 16+ between diencephalon- and mesencephalon-forming cell groups (plane 3) and between mesencephalon- and rhombencephalon-forming cell groups (plane 8). Other planes were drawn at intervals of 40 µm, corresponding to a two-cell width distance. (b) Number of cells crossing the transverse planes (1-10) in an hour was counted at stages indicated. (c) Number of cells crossing the midline at the stage indicated. (C) Occurrence of mitotic cell divisions giving rise to split fate daughter cells. Divisions that produced daughter cells that contributed to different neural subdivisions at stages 13-17 are indicated by two colored rectangles of the color code, as in Fig. 3. Numbers on the top indicate the split cell fate division in each stage. (D) Proliferation of labeled cell populations in cell groups that contributed to particular neural subdivisions at stage 24. Increase in cell number is robust between stage 17 and stage 19, but thereafter becomes moderate except in retina.

View larger version (94K): [in a new window]   Fig. 6. Spatial reorganization of the diencephalic and telencephalic compartments in relation to lateral splitting of retinal compartment. (A,D,G) Dorsal, (B,E,H) ventral and (C,F,I) lateral views of stage 16+, 18 and 19 embryos. (A-C) At stage 16+, retinal precursors form a single compartment overlying telencephalic and diencephalic compartment at their posterior and anterior halves. (D-F) Around stage 18, retinal precursors begin to split bilaterally starting from the posterior side, accompanied by intrusion of the anterior diencephalic compartment. (G-I) By stage 19, lateral splitting of retinal compartment is complete. (C,F,I) Lateral views demonstrating anterodorsal movement of the diencephalic compartment (white broken line) and dorsal movement of the telencephalic compartment (broken red line), relative to the retinal compartment. White lines indicate anterior and posterior limits of the retinal precursors. The white circle follows a single diencephalic precursor cell that moved in an anterior and dorsal direction (A,D,G). (C,F,I) The black arrows follow the anterior movement of a ventrally located diencephalic precursor cell. Scale bar: 100 µm.

View larger version (72K): [in a new window]   Fig. 7. Pax6 expression and establishment of neural compartments. (A,C,E) Pax6 expression and (B,D,F) corresponding regions of the computer graphics model. Broken lines surround cells of prospective retina. (A,B) At stage 16+ (22 hpf), Pax6 expression is first detected in a pair of lateral domains that include future lateral retinal cells, while prospective retinal cells in the middle do not express Pax6 yet. (C-F) After stage 16++ (23 hpf), Pax6 is now strongly expressed in the entire prospective retina, diencephalic cells and r1-r2 region of rhombencephalon, and moderately in the remaining part of the prospective rhombencephalon and spinal cord.