Although primary keratinocyte cultures have been instrumental for elucidating many features of epidermal biology, they undergo significant morphological and biochemical changes that limit their value for studying normal tissue physiology. This is true even for organotypic cultures and in vivo
engraftment approaches, which generate wound-like perturbations in tissue integrity. Although in vivo
xenotransplantation makes it possible to test relationships between human and mouse biology and hence represents an important approach to skin cancer research30
, engraftment procedures require immunocompromised mice, and thus can not recapitulate the full complement of cellular behaviors likely to play a role in cancers.
Our strategy for conducting comprehensive functional analyses couples the accessibility of epidermis with the utility and expediency of RNAi and commercially available shRNA libraries, and thus greatly expands the molecular toolbox for dissecting complex genetic pathways in mammalian tissue biology. In its simplest form as a single-gene functional analysis, our method necessitates only a few weeks between target selection and phenotypic analysis. As such, it offers a distinct advantage over classical mouse genetics, the conventional methods currently used to study embryonic development and tissue homeostasis in an unperturbed physiological setting.
The ability to selectively target and label epidermal progenitors allowed us to develop the CGI assay as a quantitative tool for dissecting pathways that regulate cell growth. Given that the epidermis has long served as a major model in cancer studies, this new strategy becomes particularly powerful. In addition, the combination of RNAi-mediated knockdown and lentiviral Cre-mediated knockout allows for a rapid assessment of genetic epistasis. Moreover, at least four different viruses can be used for simultaneous tissue infection, expanding the utility of this system, for example, for eliminating functional redundancies or conducting knockdown and/or replacement studies.
It is noteworthy that epidermal transduction with LV-Cre provides temporal and spatial benefits over the existing epidermis-specific Cre lines. The earlier and more uniform generation of epidermal- specific gene knockouts with LV-Cre permits future exploration of early developmental functions such as stratification, planar cell polarity and epithelial-mesenchymal interactions. The ability to control infection levels by varying lentiviral titers offers (i) an ideal source of animal-matched internal control cells, (ii) a means of analyzing gene function in adult skin, which is often precluded by newborn lethality, and (iii) the ability to distinguish between cell-autonomous and non-cell-autonomous protein roles in vivo.
Finally, using Cnnta1
as the archetype, we have shown how our technology can be used to uncover new insights into the genetic interplay between intercellular adhesion and growth control. Our analyses demonstrated a measurable effect of Ras-MAPK–dependent cell proliferation and Trp53-dependent cell death on Ctnna1
loss-of-function phenotypes during skin morphogenesis. It is tempting to speculate that the genetic interactions we uncovered between these two opposing pathways allow the epidermis to suppress neoplastic growth and sustain homeostasis following loss of α1-catenin. A similar phenomenon has been observed following loss of TGF-β signaling in the skin31
. Given our new findings, we posit that this genetic interplay between opposing pathways may be a common feature of tumor suppressors in skin epithelium. In this scenario, tipping the balance toward cell survival through pro-survival signals or alterations in Trp53 pro-apoptotic function could subsequently lead to development of epidermal tumors, reinforcing why the frequent occurrence of Trp53
-null mutations following chronic UVB exposure contributes so greatly to skin cancers.