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First published online 17 August 2005
doi: 10.1242/dev.01975


Development 132, 4143-4154 (2005)
Published by The Company of Biologists 2005


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Hair follicle renewal: organization of stem cells in the matrix and the role of stereotyped lineages and behaviors

Emilie Legué and Jean-François Nicolas*

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



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Fig. 1. Experimental design. (A) The method of temporal induction of clones: OHT binds to the mutated estrogen receptor (ERT) inducing nuclear translocation of CreERT and excision of the LoxP-flanked-stop-sequence, allowing lacZ transcription and leading to inheritable ß-galactosidase expression. (B) Experiments 1-3: D0, day of depilation; induction, OHT injection on D8 (day 8) in experiment 1, D11 in experiment 2 and D3 in experiment 3. Skin biopsies and observations are as indicated. (C) HF growth is linear. The length of 40 HFs proximodistal axis (y-axis) was recorded after depilation (x-axis). (D-K) Cell morphologies. (D) Transverse section of ROSA26 HF. (E-K) In toto views of CMV Cre ERTxR26R F1 HF displaying labeling in medulla (E), cortex (F), hair cuticle (G), IRS (H), ORS (I,J). (K) GATA-3 nls lacZ HF; ß-galactosidase stains IRS (Huxley and IRS cuticle layers) cell nuclei (red arrowheads). The adjacent hair cuticle and Henlé layers are negative (white arrowheads). Scale bars: 25 µm in D,E,I,J; 50 µm in F-H.

 


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Fig. 2. Inner HF structure clones of permanent precursors. Clones induced on D8 and observed on D14. (A,B) IRS clones; (C,D) cuticle clones; (E) a medulla clone. ß-Galactosidase+ cells are present in the matrix. Scale bars: 100 µm.

 


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Fig. 3. Inner HF structure clones of transient progenitors. Clones induced on D11 and observed on D14. (A-F) Clones of transient progenitors (absence of labeling in the matrix). The number of labeled cells (in red rectangles) reveals the proliferative potential of the progenitors. (A,B) IRS progenitor divided once. (C,D) Cuticle progenitor divided once. (E,F) Medulla progenitor divided two or three times. (G-L) Clones of permanent precursors (labeled cells in the matrix, arrowheads) in the same experiment. (G,H) IRS; (I,J) cuticle; (K,L) medulla. Scale bars: 100 µm in A,C,E; 25 µm in B,D,F.

 


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Fig. 4. The permanent precursors of the inner structures are organized into distinct sectors and are arranged with different orientations after division. (A) Position of the most distal cell (in µm) in the clones induced at D11. This position is more distal for clones produced by transient progenitors than for clones produced by permanent precursors. (B,C,E-G) In toto views of the matrix. Same clones as in Fig. 2. Arrowheads indicate the cell juxtaposed to the DP. Scale bars: 25 µm. (B,C) IRS clones showing two related pairs of cells (1, 2 and 3, 4) and progressive intercalation of ß-galactosidase+ cells between the pairs in layer 3 (see Fig. 6). 1, permanent precursor; 2, transient progenitor; 3,4, cells originating from division of the progenitor (see text); (D) Diagram of IRS mode of growth. (E,F) Cuticle clones; (G) A medulla clone. (H) Measurement of the position (a) of the permanent precursor and of the angle of the alignment of the permanent precursor and the transient progenitor ({alpha}) with respect to the proximodistal axis of the DP. (I) position (y-axis, in % of the length of the proximodistal axis of the DP, `a' value in H) of the permanent precursors of the IRS (blue lozenge), cuticle (pink square), medulla (green circle). y-axis is the value (in degrees) of {alpha} in G of the IRS (blue cross) and the cuticle (pink cross). x-axis gives the clone reference (from experiments 1 and 2).

 


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Fig. 5. The ORS follows a coherent regional mode of growth and shows apoptosis. (A-D) In toto views of ORS clones; ORS precursors are dispersed (brackets) along the proximodistal axis of the HF. Scale bar: 100 µm. (E-M) Anti-cleaved caspase 3 immunostaining on 20 µm anagen HF sections. Scale bar: 25 µm. (E,H,K) Bright field. (F,I,L) DAPI staining in blue. (G,J,M) Cleaved caspase 3 staining in green. Apoptotic cells are marked by red arrowheads. (N) Clonal complexity (y-axis) of the 100 subdivisions of equal length of the ORS proximodistal axis (x-axis). Clonal complexity is the number of times a subdivision is labeled (calculated from the 60 clones in O, Experiment 1). (O) Classification of the 60 ORS clones according to their proximodistal extension. Each horizontal line corresponds to a clone (the colored rectangles). The numbers indicate the position of the clone according to the subdivision of the proximodistal axis in 100 equal parts. Each color refers to the number of cells in the clone. See color code in P. (P) Number of clones in relation to the number of ß-galactosidase+ cells. The x-axis represents the number of ß-galactosidase+ cells for each class of clones. The y-axis represents the number of clones. ORS clones differ considerably in size. (Q) Percentage of ORS clones of the total number of HFs observed (labeled and unlabeled) (y-axis) calculated for D7, D9, D11 and D14 (experiment 3). Percentage decrease indicates the loss of ORS clones during anagen owing to apoptosis.

 


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Fig. 6. Cell behavior and cell fate organization in the HF matrix. The hypothesis of behavior and fate uncoupling. (A) Cell outlines were drawn from an optical confocal section of a HF from a farnesylated-GFP transgenic mouse in which GFP labels the cell membranes. The right side is the mirror image of the picture of the optical confocal slice. Cell behaviors are organized along the radial dimension of the matrix: the stem cells form a germinative layer juxtaposed to the DP (darkest colors). They undergo self-renewal and generate the transient progenitors in the second layer (in lighter colors). The next layers contain the postmitotic cells (lightest colors) that intercalate to produce columns. Cell fates are assigned according to the position of the stem cells along the proximodistal axis. Each structure is produced by several cells forming a sector. The most proximal cells are not involved in the formation of any inner structure; they are clonally related to the ORS (pink) (data not shown). Within a sector, the stem cells and the transient progenitors form a characteristic angle (63° for the IRS and 20° for the cuticle). This organization of matrix cells prefigures the organization of the HF in concentric layers (red arrows). Cell fates and cell behaviors therefore seem to be uncoupled and organized in two orthogonal systems. (B) Clonal hierarchy in the matrix (see text).

 





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