The Basics About Research in the Fuchs Laboratory
Why Use Skin as a Model System for Study?
As basic scientists with an interest in applying our knowledge to human medicine, we chose skin as a model system, because skin epithelium is one of the few tissues of the body whose human and mouse stem cells (keratinocytes) can be maintained and propagated long-term in culture, and where terminal differentiation can be induced. This feature has been exploited for nearly 4 decades to treat burn patients with epidermal sheets generated from cultured stem cells.
At the body surface, skin is readily accessible and particularly well-suited for mouse genetics and screens. Our prior collaborative studies with the Mombaerts lab have shown that hair follicle stem cell nuclei can be reprogrammed epigenetically by nuclear transfer, enabling us to clone mice. When coupled with the ability to generate induced pluripotent stem cells (iPS cells) by genetic manipulation of skin cells, the potential for the use of skin stem cells in regenerative medicine is broad.
On the basic science side, we study the molecular biology of how skin stem cells behave in vitro, and then exploit transgenic, gene knockout and lentiviral knockdown (shRNA)/knockout (Crispr/CAS) technologies to test skin protein function in vivo. To explore how skin biology changes during wound-healing, we monitor keratinocyte migration in skin explant cultures as well as carry out wound-repair studies with mice. To explore how skin biology changes in mouse or human genetic skin disorders, we culture the stem cells from skin biopsies of affected individuals. Because no immortalization is necessary, normal skin stem cells can be studied, and the link to human genetic diseases, including cancers, is greatly facilitated.
Cloned mice from epidermal skin stem cells
What kinds of questions does the laboratory address?
We continue to expand upon our knowledge of skin stem cells. In the past decade, we devised novel methods to mark, lineage-track and purify the epithelial stem cells from mouse skin, and we employed RNAseq technology to determine their global patterns of gene expression in vivo. Many of these changes involve transcription factors and we've used conditional knockout technology to dissect their functions. We're also employing in vivo Chip-seq and ATAC-seq to understand how skin stem cells coordinate transcription factors and epigenetic modifiers to change their chromatin landscape as stem cells receive signals from their environment and transition from a non-tissue generating (quiescent) state to one where they are either making or repairing a tissue. The hair follicle is ideal for these studies, since in the mouse, every HF has a stem cell niche, and all the HF stem cell niches act synchronously, either remaining in quiescence for weeks, or actively regrowing the HF and making hair.
Quiescent HF stem cells are in a WNT-inhibited and BMP-rich environment and become activated to make upon directional WNT signaling and inhibition of BMP signaling. We've begun to dissect the downstream targets of these pathways, and have shown that while WNT inhibition represses stem cell fate specification, BMP signaling maintains quiescence by a mechanism that involves NFATc1, FOXc1 and ID transcription factors, downstream of canonical pSMAD1/SMAD4. Elevated BMP signaling also occurs in aging skin, providing a mechanism for why our hair becomes sparser as we age.
We'd like to know more about the molecular coordination of these and other signaling pathways that operate on skin stem cells, and how they elicit changes in chromatin and transcriptome dynamics. We want to elucidate the molecular cross-talk between epidermal/HF stem cells and other permanent or transient residents of the skin, including dermal fibroblasts, immune cells, blood vessels, sensory neurons and melanocytes. As we dissect the cross-talk that elicits dramatic changes in chromatin, transcriptional and translational landscapes in the skin stem cell niches, we also want to elucidate the significance of the myriad of downstream changes in proliferation, cell adhesion, cytoskeletal dynamics and cell polarity.
The global question we are addressing is how stem cells become mobilized from their niches to form tissues, how stem cells know when to stop making tissue after wound-repair and how these processes are deregulated in aging, hyperproliferative disorders and in cancer. To do so requires a multifaceted approach to skin biology. Elaine's formal training is as a chemist, biochemist and cell biologist. However, her lab’s research encompasses a broader repertoire of expertise including mouse and human genetics, molecular biology, developmental biology, live imaging, biophysics, biomedical engineering, immunology, neurobiology and cancer biology.
Nearly all lab members use cell culture, transgenic/knockout mice and in vivo lentiviral-mediated knockdown (shRNA) and knockout (sgRNA/CRISPR/Cas) approaches as tools to study skin. However, it's not surprising to find that there is a broad range of expertise and research approaches taken by individual lab members. Some in the lab employ approaches such as genome-wide in vivo ChIP/ATAC-seq, RNA-seq, and ribosomal profiling to study how stem cells interpret environmental signals to alter their chromatin, transcriptional and translational landscapes during embryonic development, homeostasis, wound-repair, aging and cancer progression. Others employ live imaging, proteomics, single cell analyses, biophysical and bioengineering approaches to study tissue dynamics during these processes. Their main focus is on cell-cell and cell-substratum interactions, cell migration, and cytoskeletal dynamics, including spindle orientation and its contribution to morphogenesis.
Irrespective of the questions asked and the approaches taken, the lab routinely employs mouse genetics, screens and other functional tests to explore the physiological relevance of our findings. We also routinely try to take what we are learning from the basic biology of mouse skin stem cells and translate this knowledge to human conditions. For instance, with our newfound lentiviral approaches, we’ve been able to carry out genome-wide screens for oncogenes, tumor-suppressors and oncomiRs of skin and head and neck cancers. This strategy also allows us to exploit that cancer genome atlas (TCGA) of databases on human cancers. While these databases miss epigenetic changes which play a major role in cancers, they do provide insights into gene mutations, amplifications and deletions that occur in human cancers. Our strategies allow us to sift through functionally to determine which of these changes are drivers in cancers and which are mere bystanders. As we delve further into the relation between normal and cancerous stem cell behavior, we hope to be able to contribute to the overarching goal of identifying cancer therapeutics that target the cancer’s tumorigenic cells without harming the normal stem cells of the tissue.
What kinds of approaches are taken in the Fuchs' lab?
Elaine's formal training is as a chemist, biochemist and cell biologist. Her lab's research utilizes molecular, cell and developmental biology as well as mouse and human genetics. Hence, while nearly all lab members use cell culture, transgenic/knockout mice and now lentiviral in vivo knockdown approaches as tools to study skin, it's not surprising to find that there is a broad range of expertise and research approaches taken by individual lab members. Those in the lab who study skin stem cells and signaling employ approaches such as gene expression and promoter (ChIP-seq) profiling to study how stem cells alter their transcriptional program in response to changes in their microenvironment during development, differentiation and wound-healing. We combine RNA and ChIP-sequencing with molecular approaches to study transcriptional regulation. We use lentiviral knockdowns and mouse genetics to explore the functional relevance of our findings. In addition, we cloned out the microRNAs that are differentially expressed during skin development. After demonstrating that miRs are globally important, we've now begun to dissect the functions of individual miRs.Others in the lab focus on cytoskeletal and adhesion dynamics in morphogenesis, and howthese processes change in wound- healing and cancer. Postdocs and students in this area probe the regulation of actin, microtubule and intermediate filament dynamics during skin development and epithelial sheet formation, and cytoskeletal interactions with integrins and cadherins in adhesion. We've engineered transgenic mice expressing fluorescently labeled cytoskeletal and adhesive proteins and use videomicroscopy, whole embryo imaging, mouse genetics and biochemistry, molecular and cell biology to study changes in cytoskeletal dynamics and adhesion in living cells and tissues. Such approaches led us to our discovery that mammalian epidermal cells stratify and differentiate by using a mechanism found in worms and flies to orient their mitotic spindle and divide asymmetrically.
Finally, we’ve launched a major effort to elucidate how malignant progression hijacks the basic mechanisms that normal stem cells use to make and repair tissues. With our newfound lentiviral approaches, we’ve been able to carry out genome-wide screens for oncogenes and tumor-suppressors of skin and head and neck cancers. This strategy also allows us to exploit that cancer genome atlas (TCGA) of databases on mutations found in different types of human cancers. While these databases miss epigenetic changes which play a major role in cancers, they do provide insights into gene mutations, duplications and deletions that occur in human cancers. Our strategies allow us to sift through functionally to determine which of these changes are drivers in cancers and which are mere bystanders.
Understanding the molecular mechanisms involved in asymmetric divisions in stem cells and in changes in cytoskeletal-adhesive dynamics in epidermal migration, wound repair and cancer are major efforts of this side of the lab's spectrum. As we delve further into the relation between normal and cancerous stem cell behavior, we hope to be able to contribute to the overarching goal of identifying cancer therapeutics that target the cancer’s tumorigenetic cells without harming the normal stem cells of the tissue.