application note, Kopan, genetic mosaic analysis, VayTek, medical imaging, scientific software, VoxBlast, stem cells, epithelium

Three frames extracted from movies generated as part of Dr. Raphael Kopan's research are shown above. Genetic mosaic analysis reveals that the bulb region of anagen hair follicles contains several active multipotent stem cells. VayTek's VoxBlast software was used to render 3-D volumes from registered stacks of 2-D images. The complete movies may be viewed at http://molecool.wustl.edu/kopanlab/movies.html

Please note that the information presented here is excerpted from a longer paper by Raphael Kopan, John Kesterson, Meei-Hua Lin, Andrew J. Syder, Neil Crutchfield, Jason Books, and Jeffrey I. Gordon: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and VayTek, Inc. of Fairfield, Iowa. For a complete and detailed presentation, see the article published in Developmental Biology, available online at http://www2.elsevier.co.uk/links (search for <Kopan>). Higher resolution images than those presented above may also be viewed in the Developmental Biology article.

Digital Photography and 3-Dimensional Reconstruction of Chimeric Follicles: Black and white images were collected, from comparable regions of 150 adjacent serial sections/skin sample, using a Quantix Digital Camera (Photometrics). Each region in each section was photographed three times using color separation filters. 8-bit images were then merged (Image-Pro Plus software; Media Cybernetics, Silver Spring, Maryland) to generate 24-bit RGB color images. RGB images from 24-50 serial sections were used for each 3-D reconstruction. These images were first aligned to a fiduciary reference image using an alignment software package written for this study by John Kesterson and now available from VayTek. Aligned images (x- and y-axes) were stacked, together with blank images, to reconstruct the z-axis using utilities incorporated into this software package. The stacks were imported into the volume rendering program, VoxBlast, which generates 3-D projections from the registered 2-D images. The 3-D projections were viewed using an alpha blending or surface rendering algorithms. Particularly revealing views were saved as a series of TIFF images. Movies were generated from these image series with QuickTime Pro (downloadable at http://www.apple.com/quicktime/).

Abstract: The hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal-distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia. ©2002 Elsevier Science.

Introduction: The hair follicle is a distinctive stratified epithelium that renews itself in anatomically distinct compartments, has well defined patterns of cellular organization, and periodically undergoes cycles of destruction and reconstruction. Each follicle, irrespective of its location in the body, displays radial symmetry along its anterior-posterior (A/P) axis and has an asymmetric proximal-to-distal axis. At its proximal end, the follicle contains an epithelium that surrounds the mesenchymal teardrop-shaped dermal papilla (DP). Cells in the DP are the source of inductive signals required for growth and differentiation of the epithelium (Hardy, 1992; Kishimoto et al., 2000; Sengel, 1976)....

Wholemount preparations of fixed and X-Gal-stained skin were used to identify and photograph regions containing chimeric follicles. These regions were then isolated under a dissecting microscope, embedded in paraffin, and serial 5 micron sections prepared, either parallel or perpendicular to the anterior-posterior axis of the hairs. Sections were then counter stained with nuclear fast red (NFR).

The digital 3-dimensional reconstruction of these follicles allowed us to address largely unanswered questions in the field of mammalian stem biology that have been articulated and pursued by Mintz and coworkers (Bradl et al., 1991; Gearhart and Mintz, 1972; Mintz, 1967; Mintz and Palm, 1969; Mintz and Silvers, 1970; Moore and Mintz, 1972; Porter et al., 1991). How many active stem cells participate in the generation of a particular structure? In structures that are supplied by more than one oligo- or multipotent stem cell, is the fraction contribution of each stem cell to the structure fixed, or does the activity of a given stem cell fluctuate over time?....

Rafi Kopan Picture

Raphael Kopan, Associate Professor
Department of Molecular Biology and Pharmacology
and Department of Medicine (Division of Dermatology)
Washington University,
http://molecool.wustl.edu/Kopan.html

Acknowledgements - We are grateful to David Bebee, Rhiner Strorb, Arthur Eisen, Melissa Wong, Emily Garabedian, Darlene Stewart and Teresa Tolley for their advice and assistance during the course of these studies. This work was supported in part by grants from the National Institutes of Health (PO1-AR45254 and DK30292). MHL is the recipient of a post-doctoral fellowship from the Dermatology Foundation and Pharmacia-Upjohn.

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Active Stem Cell Participation in the Generation and Maintenance of an Epithelium

Active Stem Cell Participation in the
Generation and Maintenance of an Epithelium