Abstract
The epidermis is the outermost tissue of our body and has to be constantly renewed. To guarantee the constant renewal, the stem cells of the epidermis are kept in a balance between cells that stay stem cells and others that give rise to differentiating epidermal cells. Different mechanisms have been described how this regulation is achieved, however it remains unclear to what extend the integrity of the epidermis itself is actively contributing to the maintenance of epidermal cell fate. Therefore, in this study we assessed the role of keratin intermediate filaments, which are the main structural proteins of the epidermal cells, in epidermal stem cell regulation.
Using the zebrafish epidermis as a model system, we analyzed the spatiotemporal expression and function of 15 different keratins. For this, the mRNA expression of individual keratins in the whole epidermis was studied using RNA-FISH and confocal light microscopy. To address its functions, single and combination of keratins were knocked-out using CRISPR/Cas9. The analysis revealed specific functions and expression patterns for specific keratins. Further, some keratins, such as the basal epidermal cell keratins krtt1c19e and krt91 appeared to act redundantly in maintaining the integrity of the epidermis.
Strikingly, krtt1c19e and krt91 appeared not only to be involved in the structural integrity of the epidermis, but also in the regulation of epidermal stem cell differentiation. The simultaneous loss of both keratins leads to differentiation of epidermal stem cells, as suggested by analysis of various epidermis markers. As epidermal stem cells in a wild type epidermis remain undifferentiated at this developmental stage, this indicates a precocious differentiation. To understand the role of the two keratins krtt1c19e/krt91 in cell fate regulation and differentiation timing, we addressed different described mechanisms how epidermal stem cells are regulated. Using in vivo live imaging and various mRNA and protein staining, we analyzed epidermal stem cells at the onset of the precocious differentiation. Further, we used CRISPR/Cas9 genetic knockouts and genetically encoded tools to perturb cell adhesion and morphology. Additionally, electron microscopy was performed of krtt1c19e/krt91 deficient epidermis. However, the findings of those experiments suggest that not a classical mechanism drives precocious differentiation of epidermal stem cells upon krtt1c19e/krt91 loss.
To understand the basis of the precocious differentiation, we examined the role of the epidermal master transcription factor p63. CRISPR/Cas9 genetic knockout of p63 and p63 expression in krtt1c19e/krt91 deficient epidermis revealed that upon p63 loss, epidermal stem cells start to differentiate precociously. Interestingly, results of this study together with previous findings indicate that krt1c19e itself is regulated by p63 and supports the existence of a feedback loop, where p63 expression leads to krtt1c19e/krt91 expression, which in turn helps to maintain p63 expression.
Together, the results of this study demonstrate that keratins of epidermal cells appear to contribute not only to the structural integrity, but in addition can regulate the cell fate and stemness. This reveals a potential new mechanism for how the differentiation of epidermis is timed and maintained.
Bill, Robert Theofanis