In JoVE (1)
Articles by Aya Amitai-Lange in JoVE
A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice Aya Amitai-Lange*1, Eran Berkowitz*2, Anna Altshuler1, Noora Dbayat2, Waseem Nasser1, Edith Suss-Toby3, Beatrice Tiosano2, Ruby Shalom-Feuerstein1 1Department of Genetics and Developmental Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, 2Department of Ophthalmology, Hillel Yaffe Medical Center, 3Bioimging Center, Biomedical Core Facility, The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.
Other articles by Aya Amitai-Lange on PubMed
Lineage Tracing of Stem and Progenitor Cells of the Murine Corneal Epithelium Stem Cells (Dayton, Ohio). Jan, 2015 | Pubmed ID: 25187087 Accumulating evidence supports the dogma that the corneal epithelium is regenerated by stem cells located exclusively in the limbal niche, at the corneal periphery. Accordingly, limbal stem cells (LSCs) give rise to progenitors that proliferate and migrate centripetally to repopulate the corneal epithelium, which has a short turnover. Moreover, LSC loss leads to corneal opacity and blindness, while limbal grafting restores patients' vision. However, contradicting data suggested that the limbus does not participate in corneal homeostasis and that the cornea contains stem cells. As of today, only indirect evidence for limbal cell migration under homeostasis or injury has been demonstrated. Here, we performed lineage tracing experiments using R26R-Confetti mice to follow K14+ limbal/corneal epithelial cells stochastically induced to express one out of four fluorescent genes. In homeostasis, radial limbal stripes of slow migrating cells proceeded toward the corneal center while, infrequently, slow cycling limbal clones resembling quiescent stem cells were observed. Additionally, rare corneal clones that did not migrate centripetally, but survived for over 4 months, were inspected. In contrast to limbal stripes, corneal clusters had minor contribution to tissue replenishment in homeostasis. Corneal cells, however, significantly contributed to mild wound repair while large limbal streaks appeared within a week following severe wounding that coincided with partial loss of corneal transparency. This data suggest that the mouse limbus largely contributes to corneal renewal while corneal progenitor cells have a long turnover and, therefore, may be able to maintain the corneal epithelium for several months.
Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis Via Notch Signaling Inhibition of MITF Molecular Cell. Aug, 2015 | Pubmed ID: 26236014 The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.