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In JoVE (1)
Other Publications (3)
Articles by Debra Ellisor in JoVE
JoVE General
A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo
1Department of Neuroscience, Division of Biology and Medicine, Brown University, 2Department of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University
Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.
Other articles by Debra Ellisor on PubMed
Comparative Analysis of Conditional Reporter Alleles in the Developing Embryo and Embryonic Nervous System
A long-standing problem in development is understanding how progenitor cells transiently expressing genes contribute to complex anatomical and functional structures. In the developing nervous system an additional level of complexity arises when considering how cells of distinct lineages relate to newly established neural circuits. To address these problems, we used both cumulative marking with Cre/loxP and Genetic Inducible Fate Mapping (GIFM), which permanently and heritably marks small populations of progenitors and their descendants with fine temporal control using CreER/loxP. A key component used in both approaches is a conditional phenotyping allele that has the potential to be expressed in all cell types, but is quiescent because of a loxP flanked Stop sequence, which precedes a reporter allele. Upon recombination, the resulting phenotyping allele is 'turned on' and then constitutively expressed. Thus, the reporter functions as a high fidelity genetic lineage tracer in vivo. Currently there is an array of reporter alleles that can be used in marking strategies, but their recombination efficiency and applicability to a wide array of tissues has not been thoroughly described. To assess the recombination/marking potential of the reporters, we utilized CreER(T) under the control of a Wnt1 transgene (Wnt1-CreER(T)) as well as a cumulative, non-inducible En1(Cre) knock-in line in combination with three different reporters: R26R (LacZ reporter), Z/EG (EGFP reporter), and Tau-Lox-STOP-Lox-mGFP-IRES-NLS-LacZ (membrane-targeted GFP/nuclear LacZ reporter). We marked the Wnt1 lineage using each of the three reporters at embryonic day (E) 8.5 followed by analysis at E10.0, E12.5, and in the adult. We also compared cumulative marking of cells with a history of En1 expression at the same stages. We evaluated the reporters by whole-mount and section analysis and ascertained the strengths and weaknesses of each of the reporters. Comparative analysis with the reporters elucidated complexities of how the Wnt1 and En1 lineages contribute to developing embryos and to axonal projection patterns of neurons derived from these lineages.
Tamoxifen Dose Response and Conditional Cell Marking: is There Control?
Conditional marking and gene inactivation experiments are valuable approaches used to understand developmental and molecular mechanisms. CreER(T) is a fundamental component in recombinase-based conditional strategies and is used to gain temporal control subsequent to tamoxifen administration. We tested the hypothesis that tamoxifen dose linearly correlates with recombination efficiency in vivo. Wnt1-CreER(T) and tamoxifen administration were used to mark progenitors that contributed to the trigeminal ganglia. We executed a dose response study to determine the number of neurons that had undergone recombination in response to tamoxifen administered at doses ranging from 50 to 500 mg/kg. Our findings show a substantial variability in the amount of recombination within and between dose groups with no clear correlation between tamoxifen dose and the number of marked cells. This is the first study that we are aware of in which cell counts, robust quantitative data, and statistical analyses were performed on sections obtained from embryos marked in response to a wide range of tamoxifen dose in vivo. We provide an important quantitative and statistical framework for designing CreER(T)-based experiments and choosing tamoxifen dosing paradigms.
The Lineage Contribution and Role of Gbx2 in Spinal Cord Development
Forging a relationship between progenitors with dynamically changing gene expression and their terminal fate is instructive for understanding the logic of how cell-type diversity is established. The mouse spinal cord is an ideal system to study these mechanisms in the context of developmental genetics and nervous system development. Here we focus on the Gastrulation homeobox 2 (Gbx2) transcription factor, which has not been explored in spinal cord development.
