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First published online 22 August 2007
doi: 10.1242/dev.02873

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Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme

¹ Departamento de Biología del Desarrollo Cardiovascular, Centro Nacional de Investigaciones Cardiovasculares, Instituto de Salud Carlos III, E-28029 Madrid, Spain.
² Center for Genomic Regulation (CRG), Program on Systems Biology, Barcelona, Spain.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Clonal analysis identifies previously described compartments and lineage relationships. lacZ staining of embryos after 4OHTM injection at E8.5 during gestation. Clone distribution delineates lineage compartmentalization of mouse limb ectoderm into dorsal AER (A), ventral ectoderm (B) and dorsal ectoderm (C).

View larger version (62K): [in this window] [in a new window]   Fig. 2. D-V compartmentalization of limb mesenchyme. (A-H) lacZ staining of mouse embryos after 4OHTM injection at different gestational stages and recovery at various days of development, as indicated. (A,B) Dorsal and lateral views, respectively, of a large clone induced at E8.0. (C-H) Examples of D-V clone distribution at different embryonic stages, as indicated. The arrowhead in D indicates a local violation of the D-V restriction. (I) Bar chart indicating the position of the boundary respected by dorsal (left, red) and ventral (right, blue) clones, expressed as the percentage of the total D-V distance from dorsal. The x-axis indicates the percentage of specimens from each class.

View larger version (79K): [in this window] [in a new window]   Fig. 3. Optical projection tomography mapping of D-V clone expansion domains. Three-dimensional representations and virtual sections of ventral (A-G) and dorsal (H,I) clones in E14.5 mouse limbs. (A,F) Lateral and oblique views, respectively, of a ventral clone. (B) Ventral view of the same limb. Lines C'-E' indicate the planes of the virtual sections in C-E, respectively. (G) The three-dimensional clone domain in isolation. (H,I) Lateral and oblique views, respectively, of a dorsal clone.

View larger version (84K): [in this window] [in a new window]   Fig. 4. Vascular endothelial clones do not respect the D-V restriction. (A,B) Immunohistochemical detection of PECAM (brown) and ß-gal (blue) on a cross-section of an E11.5 mouse limb bud. B is an enlargement of the distal portion of the section shown in A.

View larger version (117K): [in this window] [in a new window]   Fig. 5. The D-V lineage restriction boundary correlates with the Lmx1b expression domain. (A-C) Immunodetection of Lmx1b protein. (A) An E13.5 autopod sectioned transversally. Arrowheads indicate the sites of chondrogenic condensation. (B) A transversal section of an E13.5 mouse limb at the level of the digital prechondrogenic condensations (circles). (C) A longitudinal section of the undifferentiated digit tip at E13.5. (D-I) Immunodetections on E11.5 limbs. (E,G,I) Lmx1b expression, together with the distribution of GFP-positive cells. (D,F,H) The same fields as in E, G and I, respectively, but excluding the GFP channel to reveal Lmx1b expression in clone cells (arowheads). D and E show in detail the distribution of cells belonging to a dorsal clone in relationship to the Lmx1b D-V boundary. Low-(F,G) and high-magnification (H,I) views showing the distribution of cells belonging to a ventral clone in relationship to the Lmx1b D-V boundary.

View larger version (144K): [in this window] [in a new window]   Fig. 6. The D-V restriction boundary runs across the chondrogenic condensations. (A-D) Paraffin sections of dorsal clone-containing E14.5 mouse limbs stained for lacZ expression. (B,C) Transverse sections of the specimen shown in A at the indicated positions. (E-J) Immunohistochemical detection of Lmx1b (brown) and ß-gal (blue) on cross-sections of E13.5 digits containing dorsal clones. Circles (B,F,I) indicate the sites of prechondrogenic condensations. Rectangles in E-G indicate the regions shown at higher magnification in H-J. Arrowheads (F) indicate the ventral boundary of Lmx1b expression, which is fading but still visible within the prechondrogenic condensation and coincides with the ventral-most position of lacZ-positive cells.

View larger version (77K): [in this window] [in a new window]   Fig. 7. Clone distribution along the A-P axis. (A-F) lacZ staining of mouse embryos after 4OHTM injection at different stages during gestation and recovery on different days of development, as indicated. The arrowhead (F) indicates lacZ-positive cells of an independent ectodermal clone.

View larger version (120K): [in this window] [in a new window]   Fig. 8. Clone distribution indicates no compartmentalization along the P-D axis. (A-F) lacZ staining of mouse embryos after 4OHTM injection at different stages during gestation and recovery on different days of development, as indicated. (A,C) Examples of violation of the stylopod-zeugopod frontier. (B,D) Examples of violation of the zeugopod-autopod frontier. (E,F) Examples of clones respecting the frontiers.

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