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The Basics About Research in the Fuchs Laboratory

Why Use Skin as a Model System for Study?

Culturing human skin cellsCulturing human skin cells

lab skinAs 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, without losing the ability to regenerate skin. This feature has been exploited for nearly 4 decades to treat burn patients with epidermal sheets generated from cultured stem cells. With gene therapy and Crispr/Cas technologies, devastating skin disorders can now be treated. The research opened similar doors for treating corneal blindness.

At the body surface, skin is readily accessible and particularly well-suited for mouse genetics and screens. After 25 years of performing classical knockouts and conditional knockouts to explore gene function, we developed methodology to perform gene manipulations specifically in the skin epithelium, but in a fraction of the time of conventional genetics. This revolutionary technology involving ultrasound-guided delivery to expose the early embryo to lentivirus in utero, now enables us to perform genome-wide screens to unearth the physiological relevance of genes in the skin of mice. LV injection techWith this technology, we’ve performed screens to identify novel oncogenes, tumor-suppressors, translational regulators, cancer-causing microRNAs and more. We’ve also exploited the technology in our studies on squamous cell carcinomas, the second most common and 6th most life-threatening of cancers world-wide. This work led us to new mechanisms elucidating how cancers evade chemo and immunotherapies.

 

 

What kinds of questions does the laboratory address and what kinds of approaches do we take to address them?

We continue to expand upon our knowledge of skin stem cells. The global questions we are addressing center on 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, inflammation and cancer. To do so requires a multifaceted approach to skin biology and the realization that there are many different cell types in the skin, many of which interact with stem cells and impact their behavior.

A long-standing focus of the lab has been on cytoskeletal and adhesion dynamics in morphogenesis. 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.  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.

 

In the past decade, we also devised novel methods to mark, lineage-track and purify the epithelial stem cells from mouse skin, and we employed RNAseq and single cell 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.

Elaine's formal training is as a chemist, biochemist and cell biologist, but she focuses on questions that involve molecular, cell and developmental biology as well as mouse and human genetics. It's not surprising to find that there is also a broad range of expertise and research approaches taken by individual lab members. The lab routinely uses high throughput technology and bioinformatics in their research.   

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.

 

We’ve also 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. By taking what we are learning from the basic biology of mouse skin stem cells and translating this knowledge to human conditions, we’ve begun to illuminate how cancer cells resist chemo and immunotherapy. Our studies in mice are beginning to inform how human cancer patients should be treated. As we delve further into the relation between normal and cancerous stem cell behavior, we hope to be able to continue 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.

 

 

 

 

Why Use Skin as a Model System for Study?Culturing human skin cellsAs basic scientists with an interest in applying our knowledge to human medicine, we chose skin as a model system because sk