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Clonal separation and regionalisation during formation of the medial and lateral myotomes in the mouse embryo

Unité de Biologie moléculaire du Développement, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cédex 15, France

View larger version (82K): [in a new window]   Fig. 1. (A-E) Expression of the control R2AchnlsLacZ transgene (A,C) and of the R2AChnlsLaacZ transgene (B,D,E) in E11.5 embryos. (A,E) in toto X-Gal staining, (B) whole-mount in situ hybridization of a -2 embryo with a LaacZ probe, (C-D) transverse sections of X-Gal stained embryos. (F-K) Examples of monosegmented unilateral clones. (F-H) In toto; (I-K) transverse sections. Clones VG24 (F,I) and VG28 (G,J) are restricted to the medial part of the myotome and clone LM82 (H,K) to its lateral part. (L-N) Examples of bisegmented unilateral clones. Clone LM53 (L) is restricted to the medial part of the myotome and clone SC81 (N) to its lateral part. Clone VG27 (M) contributes to both the medial and to the lateral parts of the myotome and is contiguous across the morphological identation of the body wall. The dashed line indicates the morphological indentation of the body wall, which marks the limit between the epaxial and hypaxial domains of the myotome. (O) Contribution of the monosegmented and bisegmented clones to the thoracic segments of the embryo. For each of thoracic segments 12-24, we calculated the numbers of monosegmented and bisegmented, unilateral clones that contain ß-gal+ cells in this segment. m, medial; l, lateral; nt, neural tube.

View larger version (18K): [in a new window]   Fig. 2. (A-C) Mediolateral contribution of the thoracic unilateral monosegmented clones. Each white rectangle represents a clone, with its reference is indicated at the left. The medial border of the labelled segment is at the left, the lateral border at the right. Each segment is divided into 100 parts of equal length. Each part with labelled cells is represented by a bar of colour. The number of labelled cells is colour-coded: blue, one cell; green, 2 cells; yellow, 3 or 4 cells; red, 5 or more cells. (A) Monosegmented clones ordered by the middle position of their labelling, MidP=(MB+LB)/2, where MB is the medial border and LB the lateral border of the clone. (B-C) Classification of the monosegmented clones in relation to the clonal separation at 40-50% of the ML axis. The epaxial (B) and hypaxial (C) restricted clones are ordered by their lateral or medial border, respectively. m, medial; l lateral.

View larger version (22K): [in a new window]   Fig. 3. Mediolateral contribution of the thoracic unilateral bisegmented clones. (A) Definition of the maximal mediolateral extension (MLE) in pooled segments of a bisegmented clone. Each white rectangle represents a segment and the closed circles are labelled cells. MB, medial border; LB, lateral border of the clone. (B-D) Mediolateral position of the labelled cells in the thoracic unilateral bisegmented clones. Each white rectangle represents a clone with its two labelled segments. The code of colours is the same as in Fig. 2. The clones are classified by their property of not crossing (B-C) or of crossing (D) the clonal separation at the middle of the segment. m, medial; l, lateral.

View larger version (21K): [in a new window]   Fig. 4. Distribution of the crossing index (C) along the mediolateral axis. (A) Definition of the crossing index, C. The large rectangle represents a segment with a hypothetical clone, and the filled circles represent the ß-gal+ cells of the clone. The mediolateral axis of the segment is divided into parts of equal length. For each clone, the probability of crossing the limit between two parts, C(i), is equal to 1 if the clone has ß-gal+ cells on both sides of the limit, and is equal to 0 if all the labelled cells are on the same side of the limit. C is the sum of C(i) for all the clones. (B,C) Probability of crossing for the unilateral monosegmented (B) and bisegmented (C) clones. The mediolateral axis is divided into 20 parts of equal length. m, medial; l, lateral.

View larger version (29K): [in a new window]   Fig. 5. Test of the hypothesis that two permanent stem cell systems produce epaxial and hypaxial clones. (A,B) Hypothetical clones that would be obtained with two permanent stem cell systems. The dermomyotome (green), and the myotome (blue) are represented at three stages of their mediolateral growth. Stem cells are represented by closed circles and the myocytes by open circles. The clones that would result from the labelling of a stem cell are represented by thin horizontal lines below. (A) Model of two stem cell systems located in the edges of the dermomyotome. (B) Model of two stem cell systems located in the center of the dermomyotome. (C) Mediolateral extension of epaxial (closed diamonds) and hypaxial (open rectangles) monosegmented clones in function of their medial (epaxial clones) or lateral border (hypaxial clones). (D) Clonal complexity of the myotome along the mediolateral axis. The mediolateral axis is divided into 20 parts of equal length, and the number of contributing monosegmented clones is shown. Insets: (a,b) expected graphs for the two opposing stem cell systems hypothesized, respectively, in A and in B. m, medial; l, lateral.

View larger version (21K): [in a new window]   Fig. 6. Models for the spatial relationship between myocytes and their precursors in the pool for two adjacent segments. The pool of precursors before the formation of the clonal boundary is shown above two consecutive myotomal segments. The colour gradient symbolises the regionalisation of the precursors. In the inverted model (A), cells in the extreme parts of the pool of precursors (closed circles) give rise to myocytes in the middle of the myotome, and cells in the middle part of the pool of precursors (open circles) to myocytes in the extreme parts of the myotome, resulting in an inversion of the gradient. Consequently, labelling of a precursor cell in the middle of the pool (open circle) before the establishment of the clonal separation will give rise to cells on both sides of the boundary, and will result in a clone contributing only to the two medial and lateral extremities of the myotome. In the direct model (B), precursors in the pool for two segments give rise to myocytes, keeping the orientation of the gradient. Labelling of a cell in the middle of the pool of precursors (open circle) will result in a clone that is contiguous across the boundary.

View larger version (40K): [in a new window]   Fig. 7. Models for the production of the myotome from the dermomyotome. (A-F) Represented above are the two pools of precursors in the dermomyotome (d), in the middle the myotome (m), and below (horizontal thin lines), the clones resulting from labelling of a precursor in the dermomyotome. The clonal boundary in the dermomyotome is represented as a black vertical line. Arrows indicate the genealogical relations between precursors and the myocytes. (A) Non-regionalised model based on extensive cell mixing of the precursors and of the myocytes. (B) Non-regionalised model based on two stem cell systems located in the edges of the dermomyotome. (C) Non-regionalised model based on two stem cell systems located in the central part of the dermomyotome. (D) Regionalised model with a direct relationship between the precursors and the myocytes. (E) Regionalised model with an inverted relationship between the precursors and the myocytes. (F) Regionalised and temporal model based on two stem cell systems with rapid recruitement and loss of new cells. The pool is first composed of black cells that contribute to the myotome (vertical arrows) and self-renew in the dermomyotome (horizontal arrows). These black cells are progressively replaced by the gray cells and then by the white cells.