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First published online 28 February 2007
doi: 10.1242/dev.02822

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Specification of cell fate along the proximal-distal axis in the developing chick limb bud

¹ Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
² Division of Developmental Neuroscience, CTAAR, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
³ Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.

View larger version (57K): [in this window] [in a new window]   Fig. 1. Examples of DiI labeling of stage 19 limb buds. Four independent specimens, sample numbers 2 (A-C), 4 (D-F), 14 (G-I) and 19 (J-L), are shown. The top row (A,D,G,J) in each sample shows the limb bud immediately after labeling. The injected point is magnified in the right panel. Scale bar: 200 µm. The white dotted line shows the base of the AER. (B,E,H,K) The same limb bud as that shown in the top row after 2 days. (C,F,I,L) Cartilage pattern [visualized by proteoglycan-H (PGH) expression] of the same limb bud as that shown in the images in the middle row. Red lines indicate fluorescence-positive regions in B,E,H,K (indicated by brackets). Broken lines divide the zeugopod (Z) and autopod (A), estimated by PGH expression.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Two-step labeling of a stage 19 limb bud. (A-C) The distal limb bud (0-50 µm from the AER) was first labeled with DiI (A; an enlargement of the area is shown below, scale bar: 200 µm). After 2 days, the distal tip of the DiI-labeled limb bud was cut out and placed on a dish, and the proximal end of the DiI-labeled region (indicated by bracket in B) was then labeled with DiO (C). (D) Merged image of B and C, showing that the second labeling of DiO successfully marks the end of the first labeling of DiI. (E,F) The same sample as that in B-D was observed for DiO signal (E) and cartilage (Alcian Blue staining, F). The broken line in E divides the zeugopod (Z) and autopod (A) estimated from the Alcian Blue staining.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Diagrams of fate maps based on results of 27 independent labeling experiments at stage 19. Sample number is shown over each bar. (A) Representative stage 19 wing bud, showing positions at which DiI was injected. Graduations on the scale bar indicate the distance from the AER. Each colored bar under the scale shows the width of area labeled with DiI at 0 hours in each experiment. (B) Contribution of labeled cells 2 days later. Colors and numbers correspond in A and B. Note that the position of each bar on the anterior (top)-posterior (bottom) axis does not indicate the labeled position along the axis but that DiI was always injected in the prospective digit 3 region indicated by an arrow in A. S, stylopod; Z, zeugopod; A, autopod.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Double labeling with DiI and DiO in the same limb bud at stage 19. (A,B) Proximal two regions (A; 220-240 µm, B; 250-330 µm from the AER) were simultaneously labeled with DiI (red) and DiO (green). (C-F) Contribution of labeled cells 2 days later. The two signals are observed only within the zeugopod (Z) and stylopod (S), respectively (C,D), with a small overlap (E,F) (Alcian Blue staining, F). (G,H) Distal two regions (G; 0-70 µm, H; 120-170 µm from the AER) of the same limb bud were simultaneously labeled with DiI and DiO. (I-L) Contribution of labeled cells 2 days later. Note that overlapping of DiI (I) and DiO (J) signals (indicated in yellow in K) crosses the zeugopod-autopod boundary evident in the cartilage pattern (L). (M,N) Similar labeling experiment as that in G,H was performed. Labeled two regions are 10-50 µm (M) and 150-210 µm (N) from the AER of the same limb bud. (O-Q) Whole-mount observation of fluorescent signal in the same limb bud 2 days later than that shown in M,N. Fluorescence for DiI (O) and that for DiO (P) overlap. (R-T) Higher magnification of cross-sections of the overlapping area (detected in yellow and indicated by an arrow in Q) was sectioned. Both DiI (R) and DiO (S) are detected in the region of overlap (T). Scale bars: 100 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Fate mapping of a stage 23 limb bud. (A-O) Three independent specimens, sample numbers 2 and 9 (A-E), 3 and 15 (F-J), and 17 and 22 (K-O), are shown. (A,B,F,G,K,L) The limb buds immediately after labeling. In each sample, both DiI (A,F,K) and DiO (B,G,L) were injected into different levels (as indicated) at the same time. (C,D,H,I,M,N) The contribution of labeled cells after 2 days in the same limb bud as that shown in the top row. The fluorescence-positive region is indicated by brackets. (E,J,O) The cartilage pattern (visualized by Alcian Blue staining) of the same limb bud as that shown in the middle rows. Dashed lines divide the skeletal pattern into a series of proximal-distal parts. Scale bar: 300 µm. (P,Q) Diagrams showing a fate map based on results of 27 labeling experiments at stage 23. Graduations on the scale bar in P indicate the distance from the AER. Colored bars under the scale show the area labeled with DiI or DiO in each sample. Q shows the contribution of labeled cells in the middle finger region 2 days after labeling. S, stylopod; Z, zeugopod; A, autopod; c, carpal; m, metacarpal; d, digit.

View larger version (111K): [in this window] [in a new window]   Fig. 6. Non-mosaic expression of HOXA11 and HOXA13 in the distal mesenchymal cells. (A-E) Immunohistochemical double-staining for HOXA11 (green) and HOXA13 (red) at stage 20 (A), stage 21 (B), stage 22 (C), stage 23 (D) and stage 26 (E). All images are longitudinal sections of the distal limb bud at prospective third finger position, oriented with dorsal to the top and distal to the right. All images are at the same magnification. (Fa-Jc) Higher magnification of the distal limb buds. HOXA11 (Fa,Ga,Ha,Ia,Ja) and HOXA13 (Fb,Gb,Hb,Ib,Jb) do not show any mosaic expression at any of the stages we examined. This was confirmed by confocal microscopic observation at stage 24 (K-N). Both proteins are localized in all of the nuclei. Note that signals are detectable as particles, which appear yellow in the merged figure (N).

View larger version (27K): [in this window] [in a new window]   Fig. 7. Prospective fate of mesenchymal cells along the proximal-distal axis. (A,B) Diagram showing the prospective fate of different proximal-distal positions of the limb bud. (A) At stage 19, prospective zeugopod (yellow) and autopod (pink) regions have a large overlap (hatched) in the distal limb bud. In contrast to this, prospective stylopod (blue) and zeugopod regions are more regionalized in terms of developmental fate with a small overlap in the proximal limb bud. (B) At stage 23, the overlap between prospective zeugopod and autopod regions is reduced in the proximal limb bud. However, prospective metacarpus (green) and digit (light blue) regions still have a large overlap in the distal limb bud. (C,D) Diagram showing the contribution of mesenchymal cells to different proximal-distal positions of limb. At both stage 19 (C) and stage 23 (D), proximal mesenchymal cells show small dispersion and small degree of mixing along the PD axis. Meanwhile, distal mesenchymal cells are dispersed widely along the PD axis and show remarkable mixing. Red lines in C and D show the proximal end of the structure derived from the distal 150 µm region from the AER.

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