Chapter Eleven - Mapping Cell Fate and Function Using Recombinase-Based Intersectional Strategies
Introduction
For any map—whether it be a road map, a genetic cell-fate map, or a functional neuronal circuitry map—improving resolution improves accuracy of the mapped relationships and thus the usefulness of the information conveyed. For murine cell-fate or cell-function maps, one means to improve resolution is by directing expression of the cell-lineage tracer or cell effector molecule, respectively, to smaller more uniquely defined subsets of cells. This allows a tighter correspondence to be drawn, for example, between a specific progenitor cell subtype and the kinds of progeny cells it produces, or between a specific neuron subtype in the adult brain and its contribution to a specific animal behavior. In this chapter, we discuss a general approach for driving conditional expression of a reporter or effector molecule with high cell-subtype selectivity in the mouse—called intersectional transgene activation (Awatramani et al., 2003, Branda & Dymecki, 2004, Farago et al., 2006, Jensen et al., 2008, Kim et al., 2009)—and we detail how this strategy can be applied to enhance cell-subtype resolution in murine genetic fate maps and in functional neuronal circuitry maps. In brief, order-of-magnitude improvements in map resolution can be achieved simply through increasing, by one, the conditions on which cell subtypes are selected for genetic manipulation: instead of selecting cells based on their capacity to drive the expression of one particular gene, as is the case for most conventional transgenics, they are now selected based on a capacity to drive expression of pairwise gene combinations. In other words, transgene-encoded tracer or effector molecule expression is directed exclusively to cells residing within the intersection of the patterns driven by two independent promoter/enhancer fragments (envision the partial overlap of two circles in a Venn diagram). Here, we discuss concepts, reagents, and methods underlying this “intersectional” approach, beginning first by introducing the basics of genetic fate mapping (previously reviewed in Branda & Dymecki, 2004, Dymecki & Kim, 2007, Dymecki et al., 2002, Joyner & Zervas, 2006) and the recombinase enzymes involved and follow with implementation of currently available intersectional fate mapping and effector alleles. Relevant methods and materials are detailed in Section 8 and are applicable across many kinds of recombinase-based genetic experiments.
Section snippets
Accessing Mouse Embryonic Cells In Utero for Tracer Molecule “Delivery” Using Transgenesis and Site-Specific DNA Recombination
Genetic fate mapping, being noninvasive and transgene-based, has opened wide the door to fate mapping studies in mice. Hindering murine studies in the past has been the access barrier of in utero development, making it difficult, if not impossible, to inject mouse embryos with lineage tracers (Cepko et al., 1990, Galileo et al., 1990, Keller, 1975, Walsh & Cepko, 1988, Wetts & Fraser, 1988) or cell grafts (Le Douarin, 1982) without also disturbing development—the very process under
Improving Cell-Subtype Selectivity in Genetic Fate Maps Using a Dual-Recombinase Intersectional Method
For intersectional genetic fate mapping, two site-specific recombinases rather than one are required to activate a reporter transgene (Fig. 11.1B) (Awatramani et al., 2003, Farago et al., 2006, Dymecki & Kim, 2007, Jensen et al., 2008, Miyoshi et al., 2010, Yamamoto et al., 2009). Two STOP cassettes, disrupting the reporter transgene, must be removed: one STOP cassette is loxP-flanked and removable by Cre, the other is FRT-flanked and removable by Flpe (Fig. 11.1B). Thus only cells contained in
Transgenes Enabling Subtractive as well as Intersectional Genetic Fate Mapping
A typical intersectional indicator allele can be configured as cartooned in Figs. 11.1B and 11.2A, where the intersectional reporter (e.g., GFP)-encoding sequence is separated from R26/CAG transcriptional regulatory and initiation elements by the presence of two STOP cassettes, the first loxP-flanked and the second FRT-flanked. Advantageously, a second cell population lying outside the intersectional subset can be mapped simultaneously simply by incorporating a second reporter gene, such as one
Exploiting Different Reporter Molecules to Reveal Different Features of Mapped Cell Populations
Depending on the type of reporter molecule employed for genetic fate mapping, whether it is intersectional or single-recombinase based, different features of the mapped cell population may be uncovered in addition to their genetic history. Nuclear localized versions of reporters (e.g., nGFP or nβ-gal) can allow visualization of individual cells in a highly sensitive way, often not achievable by cytoplasmic reporters when cells are tightly clustered. On the other hand, cytoplasmic or
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In addition to providing improved cell-subtype selectivity, intersectional alleles offer a further practical advantage. Three different mouse lines can be generated from one initial transgene construction and strain generation: the intersectional indicator mouse line, but also two derivative single recombinase responsive lines (Table 11.1). The latter two are readily generated through germ line deletion of either the loxP- or FRT-flanked cassette from the original intersectional allele (Farago
Intersectional Transgene Activation Reaches from Mapping Cell Fate to Mapping Cell Function
Genetic fate maps are indeed advancing our understanding of mammalian development and disease, ushering in new molecular views of tissue architecture and anatomy and their underlying construction. Importantly, genetic fate maps are also templating an entirely new class of tools where various cell functions can be probed in addition to cell fate. This is achieved by modifying the intersectional indicator allele to now include a genetically encoded effector molecule, for example, a molecule
Comparing recombinase versus endogenous driver gene expression by in situ hybridization
It is critical to establish the extent to which recombinase expression mirrors spatiotemporally the expression profile of the chosen driver gene or driver enhancer elements. This is because unexpected recombinase expression could lead to erroneous inclusion of cells into the fate map that are unrelated genetically. We therefore recommend performing in situ hybridizations on adjacent tissue sections prepared from a developmental series of staged transgenic embryos (e.g., embryos collected at
Concluding Remarks
Our goal for this chapter has been to introduce how the site-specific recombinases, Cre and Flpe (or Flpo) can be used together along with a responsive target transgene to effectively “deliver” virtually any genetically encoded molecule to highly selective cell subsets in the mouse—and to achieve this noninvasively, reproducibly, and with versatility. Emphasis has been placed on exploiting intersectional genetics to improve the resolution by which cells are selected for tracking or
Acknowledgments
This work was supported by grants from the National Institutes of Health and the Harvard Neurodiscovery Center. J. C. K. has been a fellow of the Foundation for Fighting Blindness of Canada. We thank members of the Dymecki lab for critical reading of the manuscript and valuable suggestions.
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