A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells.

BACKGROUND: Pax7 is a transcription factor involved in the specification and maintenance of muscle stem cells (MuSCs). Upon injury, MuSCs leave their quiescent state, downregulate Pax7 and differentiate, contributing to skeletal muscle regeneration. In the majority of regeneration studies, MuSCs are isolated by fluorescence-activated sorting (FACS), based on cell surface markers. It is known that MuSCs are a heterogeneous population and only a small percentage of isolated cells are true stem cells that are able to self-renew. A strong Pax7 reporter line would be valuable to study the in vivo behavior of Pax7-expressing stem cells. METHODS: We generated and characterized the muscle properties of a new transgenic Pax7EGFP mouse. Utilizing traditional immunofluorescence assays, we analyzed whole embryos and muscle sections by fluorescence microscopy, in addition to whole skeletal muscles by 2-photon microscopy, to detect the specificity of EGFP expression. Skeletal muscles from Pax7EGFP mice were also evaluated in steady state and under injury conditions. Finally, MuSCs-derived from Pax7EGFP and control mice were sorted and analyzed by FACS and their myogenic activity was comparatively examined. RESULTS: Our studies provide a new Pax7 reporter line with robust EGFP expression, detectable by both flow cytometry and fluorescence microscopy. Pax7EGFP-derived MuSCs have identical properties to that of wild-type MuSCs, both in vitro and in vivo, excluding any positional effect due to the transgene insertion. Furthermore, we demonstrated high specificity of EGFP to label MuSCs in a temporal manner that recapitulates the reported Pax7 expression pattern. Interestingly, immunofluorescence analysis showed that the robust expression of EGFP marks cells in the satellite cell position of adult muscles in fixed and live tissues. CONCLUSIONS: This mouse could be an invaluable tool for the study of a variety of questions related to MuSC biology, including but not limited to population heterogeneity, polarity, aging, regeneration, and motility, either by itself or in combination with mice harboring additional genetic alterations

Innervation: The Missing Link for Biofabricated Tissues and Organs

Innervation plays a pivotal role as a driver of tissue and organ development as well as a means for their functional control and modulation. Therefore, innervation should be carefully considered throughout the process of biofabrication of engineered tissues and organs. Unfortunately, innervation has generally been overlooked in most non-neural tissue engineering applications, in part due to the intrinsic complexity of building organs containing heterogeneous native cell types and structures. To achieve proper innervation of engineered tissues and organs, specific host axon populations typically need to be precisely driven to appropriate location(s) within the construct, often over long distances. As such, neural tissue engineering and/or axon guidance strategies should be a necessary adjunct to most organogenesis endeavors across multiple tissue and organ systems. To address this challenge, our team is actively building axon-based living scaffolds that may physically wire in during organ development in bioreactors and/or serve as a substrate to effectively drive targeted long-distance growth and integration of host axons after implantation. This article reviews the neuroanatomy and the role of innervation in the functional regulation of cardiac, skeletal, and smooth muscle tissue and highlights potential strategies to promote innervation of biofabricated engineered muscles, as well as the use of living scaffolds in this endeavor for both in vitro and in vivo applications. We assert that innervation should be included as a necessary component for tissue and organ biofabrication, and that strategies to orchestrate host axonal integration are advantageous to ensure proper function, tolerance, assimilation, and bio-regulation with the recipient post-implant

Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration

Tissue development and regeneration depend on cell-cell interactions and signals that target stem cells and their immediate progeny. However, the cellular behaviours that lead to a properly regenerated tissue are not well understood. Using a new, non-invasive, intravital two-photon imaging approach we study physiological hair-follicle regeneration over time in live mice. By these means we have monitored the behaviour of epithelial stem cells and their progeny during physiological hair regeneration and addressed how the mesenchyme influences their behaviour. Consistent with earlier studies, stem cells are quiescent during the initial stages of hair regeneration, whereas the progeny are more actively dividing. Moreover, stem cell progeny divisions are spatially organized within follicles. In addition to cell divisions, coordinated cell movements of the progeny allow the rapid expansion of the hair follicle. Finally, we show the requirement of the mesenchyme for hair regeneration through targeted cell ablation and long-term tracking of live hair follicles. Thus, we have established an in vivo approach that has led to the direct observation of cellular mechanisms of growth regulation within the hair follicle and that has enabled us to precisely investigate functional requirements of hair-follicle components during the process of physiological regeneration. © 2012 Macmillan Publishers Limited. All rights reserved

An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

Here we use the motile ventral cilia of the planarian S. mediterranea to examine the role of outer arm dynein in the generation and maintenance of metachronal synchrony. We demonstrate that a single dynein light chain plays a mechanosensory role necessary to entrain and maintain the metachronal synchrony of motile cilia

Assembly, structure and regulation of dyneins in cilia

Cilia are conserved cellular organelles with important roles in human physiology. Dyneins are a family of minus-end directed, microtubule motors that are crucial for cilia function. For example, an isoform of cytoplasmic dynein drives the retrograde intraflagellar transport (IFT); a process required for cilia assembly and maintenance. In addition, a diverse class of dynein motors, namely the outer and inner dynein arms, associate with the axoneme making the entire structure motile. In this study we investigated the role of the lissencephaly protein (Lis1) on the regulation of the outer arm dynein complex. We showed that Lis1 has a conserved function in motile cilia and that a Chlamydomonas ortholog (CrLis1) interacts with the a heavy chain (or the associated LC5 subunit) of the outer arm complex. We also provided evidence to suggest that the association of CrLis1 with the outer arm is regulated by other axonemal subsystems, including the central pair apparatus, radial spokes and inner arm system, as part of singling pathway that controls outer arm motor activity. Light chain subunits that belong to the family of LC8/DYNLL, are also important for outer arm motor function. Here, we have characterized a conserved third member of this family of light chains and showed that each of these three subunits has a distinct role in the assembly and regulation of the outer arm motor. In this study we also investigated the architecture of the dynein motor that powers retrograde IFT; a motor complex whose organization, structural composition and regulation is poorly understood. We showed that this motor consists of a heavy chain dimer, and other conserved subunits including a novel intermediate chain (FAP133) that we identified and characterized. FAP133 is an integral component of the IFT dynein and directly binds LC8, forming a complex that is essential for the loading of the dynein complex onto the anterograde transport system or onto other IFT cargoes. Finally, we explored the use of Schmidtea mediterranea as a biological system to study cilia and cilia-based motility, and demontrated that ciliary motility is required for planarian gliding locomotion.

Assembly, structure and regulation of dyneins in cilia

Cilia are conserved cellular organelles with important roles in human physiology. Dyneins are a family of minus-end directed, microtubule motors that are crucial for cilia function. For example, an isoform of cytoplasmic dynein drives the retrograde intraflagellar transport (IFT); a process required for cilia assembly and maintenance. In addition, a diverse class of dynein motors, namely the outer and inner dynein arms, associate with the axoneme making the entire structure motile. In this study we investigated the role of the lissencephaly protein (Lis1) on the regulation of the outer arm dynein complex. We showed that Lis1 has a conserved function in motile cilia and that a Chlamydomonas ortholog (CrLis1) interacts with the a heavy chain (or the associated LC5 subunit) of the outer arm complex. We also provided evidence to suggest that the association of CrLis1 with the outer arm is regulated by other axonemal subsystems, including the central pair apparatus, radial spokes and inner arm system, as part of singling pathway that controls outer arm motor activity. Light chain subunits that belong to the family of LC8/DYNLL, are also important for outer arm motor function. Here, we have characterized a conserved third member of this family of light chains and showed that each of these three subunits has a distinct role in the assembly and regulation of the outer arm motor. In this study we also investigated the architecture of the dynein motor that powers retrograde IFT; a motor complex whose organization, structural composition and regulation is poorly understood. We showed that this motor consists of a heavy chain dimer, and other conserved subunits including a novel intermediate chain (FAP133) that we identified and characterized. FAP133 is an integral component of the IFT dynein and directly binds LC8, forming a complex that is essential for the loading of the dynein complex onto the anterograde transport system or onto other IFT cargoes. Finally, we explored the use of Schmidtea mediterranea as a biological system to study cilia and cilia-based motility, and demontrated that ciliary motility is required for planarian gliding locomotion.

The Dynamic Duo: Niche/Stem Cell Interdependency

Most tissues in our bodies undergo constant cellular turnover. This process requires a dynamic balance between cell production and elimination. Stem cells have been shown in many of these tissues to be the major source of new cells. However, despite the tremendous advances made, it still remains unclear how stem cell behavior and activity are regulated in vivo. Furthermore, we lack basic understanding for the mechanisms that coordinate niche/stem cell interactions to maintain normal tissue homeostasis. Our lab has established a novel imaging approach in live mice using the skin as a model system to investigate these fundamental processes in both physiological and pathological settings such as cancer, with the goal of understanding how tissues successfully orchestrate tissue regeneration throughout the lifetime of an organism

A Multiple Hypothesis Based Method for Particle Tracking and Its Extension for Cell Segmentation

In biological studies, it is often required to track thousands of small particles in microscopic images to analyze underlying mechanisms of cellular and subcellular processes which may lead to better understanding of some disease processes. In this paper, we present an automatic particle tracking method and apply it for analyzing an essential subcellular process, namely clathrin mediated endocytosis using total internal reflection microscopy. Particles are detected by using image filters and subsequently Gaussian mixture models are fitted to achieve sub-pixel resolution. A multiple hypothesis based framework is designed to solve data association problems and handle splitting/merging events. The tracking method is demonstrated on synthetic data under different scenarios and applied to real data. We also show that, by equipping with a cell detection module, the method can be extended straightforwardly for segmenting cell images taken by two-photon excitation microscopy