Science 12 March 2010:
Vol. 327. no. 5971, pp. 1385 – 1389
DOI: 10.1126/science.1184733 Lgr6 Marks Stem Cells in the Hair Follicle That Generate All Cell Lineages of the Skin Hugo J. Snippert,1,* Andrea Haegebarth,1,* Maria Kasper,2 Viljar Jaks,2 Johan H. van Es,1 Nick Barker,1 Marc van de Wetering,1 Maaike van den Born,1 Harry Begthel,1 Robert G. Vries,1 Daniel E. Stange,1 Rune Toftgård,2 Hans Clevers,1 Mammalian epidermis consists of three self-renewing compartments: the hair follicle, the sebaceous gland, and the interfollicular epidermis. We generated knock-in alleles of murine Lgr6, a close relative of the Lgr5 stem cell gene. Lgr6 was expressed in the earliest embryonic hair placodes. In adult hair follicles, Lgr6+ cells resided in a previously uncharacterized region directly above the follicle bulge. They expressed none of the known bulge stem cell markers. Prenatal Lgr6+ cells established the hair follicle, sebaceous gland, and interfollicular epidermis. Postnatally, Lgr6+ cells generated sebaceous gland and interfollicular epidermis, whereas contribution to hair lineages gradually diminished with age. Adult Lgr6+ cells executed long-term wound repair, including the formation of new hair follicles. We conclude that Lgr6 marks the most primitive epidermal stem cell.
1 Hubrecht Institute–KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, Netherlands.
2 Karolinska Institutet, Center for Biosciences and Department of Biosciences and Nutrition, Novum, SE-141 57 Huddinge, Sweden. * These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: h.clevers@hubrecht.eu

Dev Cell. 2006 Oct;11(4):459-70. Multicellular rosette formation links planar cell polarity to tissue morphogenesis. Blankenship JT, Backovic ST, Sanny JS, Weitz O, Zallen JA. Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA. Elongation of the body axis is accompanied by the assembly of a polarized cytoarchitecture that provides the basis for directional cell behavior. We find that planar polarity in the Drosophila embryo is established through a sequential enrichment of actin-myosin cables and adherens junction proteins in complementary surface domains. F-actin accumulation at AP interfaces represents the first break in planar symmetry and occurs independently of proper junctional protein distribution at DV interfaces. Polarized cells engage in a novel program of locally coordinated behavior to generate multicellular rosette structures that form and resolve in a directional fashion. Actin-myosin structures align across multiple cells during rosette formation, and adherens junction proteins assemble in a stepwise fashion during rosette resolution. Patterning genes essential for axis elongation selectively affect the frequency and directionality of rosette formation. We propose that the generation of higher-order rosette structures links local cell interactions to global tissue reorganization during morphogenesis. PMID: 17011486 F1000 reviews

Nat Cell Biol. 2010 Jan;12(1):60-5; sup pp 1-9. Epub 2009 Dec 6. An actomyosin-based barrier inhibits cell mixing at compartmental boundaries in Drosophila embryos. Monier B, Pélissier-Monier A, Brand AH, Sanson B. Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. bm343@cam.ac.uk Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting

V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets Hanchuan Peng, Zongcai Ruan, Fuhui Long, Julie H Simpson & Eugene W Myers Nature Biotechnology (2010) doi:10.1038/nbt.1612 Received 30 November 2009
Accepted 08 February 2010
Published online 14 March 2010 The V3D system provides three-dimensional (3D) visualization of gigabyte-sized microscopy image stacks in real time on current laptops and desktops. V3D streamlines the online analysis, measurement and proofreading of complicated image patterns by combining ergonomic functions for selecting a location in an image directly in 3D space and for displaying biological measurements, such as from fluorescent probes, using the overlaid surface objects. V3D runs on all major computer platforms and can be enhanced by software plug-ins to address specific biological problems. To demonstrate this extensibility, we built a V3D-based application, V3D-Neuron, to reconstruct complex 3D neuronal structures from high-resolution brain images. V3D-Neuron can precisely digitize the morphology of a single neuron in a fruitfly brain in minutes, with about a 17-fold improvement in reliability and tenfold savings in time compared with other neuron reconstruction tools. Using V3D-Neuron, we demonstrate the feasibility of building a 3D digital atlas of neurite tracts in the fruitfly brain.

Preparation of Fab-QDs and tagging single EGFRs with the conjugate AntiEGFR–Fab-biotin was produced by pepsin digestion of an anti-EGFR IgG (AB-11; clone 199.12, LabVision) followed by reduction of the resulting F(ab′)2 to yield a Fab′ fragment. The Fab′ was then conjugated with biotin through the unique -SH at its carboxy-terminal end. To prepare antiEGFR–Fab-QD conjugates, a solution of 0.7 nM antiEGFR–Fab-biotin in 1 ml of DMEM/F12 was added dropwise to 1 ml of 2 nM QD605-streptavidin conjugate (Invitrogen). Cell monolayers (50% confluent) previously serum starved for >1 h were incubated with 500 μl of the final antiEGFR–Fab-QD solution at 25 °C for ~2 min. The cells were then washed twice with PBS at 25 °C, placed in serum-free medium, and loaded onto the microscope stage at 37 °C in 5% CO2 for imaging. Imaging system and analysis Total internal reflection fluorescence microscope (TIRFM) imaging was performed using a Nikon Eclipse TE2000 inverted microscope with 100×/1.49NA Plan Apo objectives (Nikon). The sample was illuminated by the 488 nm line of a solid-state laser (Andor Technology) and images were acquired by the iXon back-illuminated EMCCD camera (Andor Technology). Individual trajectories of antiEGFR–Fab-QD were constructed by manual tracking plug-in in Image-J in parallel with the tracking module in Imaris (Bitplane). 10Hz, 100 ms per frame, not that fast.