A balancing act between stratification and EMT in cultured human thymic epithelial cells

The thymus is the primary organ for T cell differentiation and maturation. Its stroma forms a characteristic sponge-like 3D structure mainly composed of thymic epithelial cells. Despite of this unconventional epithelial architecture, TECs express markers associated with epidermal specification and differentiation. We have uncovered that the human thymus contains a population of clonogenic TECs that can be extensively expanded in a culture system originally developed for skin keratinocyte stem cells. In vitro, human TECs (hTECs) can give rise to four morphologically distinct colony-types and express markers of stratified epitheliaâs basal layers, such as P63, K5/K14 and CD49f. We were able to demonstrate that cultured hTECs can be split in two distinct subpopulations based on their EpCAM expression level. EpCAM+ hTECs only give rise to stratified colonies that contain squame-like cells and express markers of epidermal differentiation, whereas EpCAM- hTECs mostly give rise to non-stratifying colonies but have the capacity to generate EpCAM+ hTECs. EpCAM- hTECs maintain a basal epithelial identity but display hallmarks of EMT, such as the upregulation of ZEB1, the loss of CDH1 and a reduced expression of the miR-200 family members. We were also able to show that miR-200c overexpression is sufficient to convert EPCAM- hTECs into EpCAM+ ones, implying a crucial role for the ZEB/miR-200 double-negative feedback loop in the control of stratification and EMT in cultured hTECs. Our work suggests that hTECs possess an intrinsic stratification program of functional importance, not likely to result from promiscuous gene expression. We speculate that the maintenance of the thymic tridimensional epithelial network requires a fine balance between stratification and EMT, which could be regulated by the ZEB/miR-200 double-negative feedback loop. In this context, cultured hTECs represent an insightful system to better understand the mechanisms governing epithelial stratification and plasticity

RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

April Bezdek Pomey

The evolutionarily conserved Notch signaling cascade plays a pivotal role in the regulation of many fundamental processes such as stem cell maintenance, proliferation, and differentiation. Recently, we aimed to identify downstream target genes regulated by Notch1 during epidermal differentiation. For this purpose an Affymetrix mouse gene chip array of 14.000 genes was performed in both Notch1 gain and loss of function experiments using murine primary keratinocytes. Thirteen genes positively or negatively regulated, based on expression levels and relevance of the involved signaling pathways, were further validated by real-time PCR. Among these, we decided to focus on the Rho GTPase RhoV because it showed robust up- and down-regulation under both gain- and loss-of-function conditions. Therefore, we generated different transgenic mice expressing either a dominant active or a dominant negative form of RhoV specifically in the skin. Morphological and histological analyses did not reveal any overt skin phenotype in transgenic mice. In addition, challenging the system by performing wound healing assays demonstrated that the healing process in transgenic mice occurred normally and that the migration and/or differentiation of keratinocytes was unimpaired. Taken together, our data suggest that gain or loss of RhoV function does not perturb skin development and homeostasis, and does not affect the wound healing process. In addition to the investigation of the potential Notch target gene RhoV, we sought to generate both a floxed Jag2 gene targeted mouse line and a Notch2-specifc reporter mouse. The former was intended to enable conditional deletion of the Notch ligand Jagged2 and therefore allow its role to be studied in a loss-of-function context. The Notch2 reporter mouse is a tool enabling the in vivo study of cells that receive a Notch2-mediated signal. The strategy employs elements of a Tet-Off system and allows cells receiving a Notch2 specific signal to be identified in any tissue at any given time point.

EPFL2010

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells

Yann Barrandon

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.

2008

Asymmetric Cell Division in the Skin

Wan-Hung Fan

An asymmetric cell division (ACD) is defined as a division that gives rise to two daughter cells with different fates. It is the most common type of division during development. Analogous to Drosophila neuroblasts, proteins involved in ACD such as Insc and LGN are found to be asymmetrically localized during perpendicular cell divisions in mammalian skin, suggesting that the cutaneous tissue homeostasis may be controlled by using similar mechanisms. Most cell divisions in the basal layer of the skin are perpendicular to the basal membrane (BM), but when a skin tumor is formed, most cell divisions would become parallel to the BM. Previous studies in our lab have shown that regressing skin tumor caused by β-catenin depletion have increased perpendicular cell divisions accompanied by reduced stem cell numbers and enhanced mTrim2 expression. Furthermore, induced expression of mTRIM2 in skin tumors led to tumor regression. Taken together, this suggests that progenitor cells in the basal layer of the epidermis may use vertical ACDs to maintain progenitor cell number while using horizontal SCDs to expand progenitor cell number and mTrim2 may function as a cell fate determinant in skin. To validate the hypothesis that vertical divisions of basal keratinocytes to the BM are ACDs, I utilized light sheet based fluorescence microscopy (LSFM) to perform the time lapse recording of murine embryonic skin development between E14.5 and E17.5 and to monitor individual dividing cells and to track their fates after division. For visualizing the dividing cells, protein localization and differentiating cells, I have generated several transgenic mouse lines and combined them together. My study showed that ACD of basal keratinocytes can be either vertical or horizontal to the BM and divisions in the suprabasal layer can be both ACDs and SCDs. To study the potential function of mTRIMs in skin, I generated the knock-out (KO) mice. Neither mTrim2 KO, mTrim3 KO or mTrim32 KO mice showed any phenotype in skin, therefore I combined these mice to generate mTrim2/3 double KO and mTrim2/3/32 triple KO mice. Still, no skin phenotype was observed in these mice, indicating that mTrim2/3/32 might not function as a cell fate determinant in skin.