Fig. 2.
Intersectional and subtractive genetic fate mapping strategy. (a) Multiple uniquely coded molecular subdomains may comprise a single gene-expression domain. Shown are schematics of the neural tube (gray cylinder), with different gene-expression domains depicted in different colors. The expression domain for hypothetical gene A (yellow) restricts along the dorsoventral (DV) axis but extends along the anteroposterior (AP) axis; by contrast, the expression domains for genes B (pink) and C (red) restrict along the AP axis but extend along the DV axis. Thus, the gene A expression domain (yellow) is subdivided into three molecularly distinct subdomains: one in which genes A and B are coexpressed (tan domain), another in which genes A and C are coexpressed (orange domain), and finally, that territory (yellow) marked by gene A expression, but not B or C. Similarly, both the gene B and C expression domains are each subdivided. (b) Structure of a prototypical dual recombinase (Cre and Flpe)-responsive indicator allele. In contrast to a single recombinase-responsive indicator allele (Fig. 1b), a dual recombinase-responsive indicator allele has two stop cassettes, one flanked by directly oriented loxP sites (triangles) and the other by FRT sites (vertically oriented rectangles). Cre-mediated stop cassette removal results in expression of nβ-gal, while the remaining FRT-flanked stop cassette prevents GFP expression. Following removal of both stop cassettes, requiring Cre- and Flpe-mediated excisions, GFP expression is turned on and nβ-gal expression off. (c) Illustration of intersectional and subtractive populations and the latter dependency on stop-cassette order. In the “PF” configured allele, the loxP-flanked stop cassette precedes the FRT-flanked cassette (left panel), while the reciprocal order characterizes the “FP” configuration (right panel). Shown are schematics of the neural tube (gray cylinder), with the expression domain for hypothetical gene A and Flpe recombinase (yellow) restricting along the DV axis but extending along the AP axis (top row); in contrast, the expression domain for gene B (pink) restricts along the AP axis but extends along the DV axis (middle row). When gene A::Flpe and gene B::cre are coupled with a PF dual recombinase-responsive indicator allele (bottom row, left), cells expressing cre and Flpe activate production of GFP (green domain, intersectional population), while cells expressing only cre activate production of nβ-gal (blue domain, subtractive population). When gene A::Flpe and gene B::cre are coupled with an FP-configured allele (bottom row, right), cells expressing cre and Flpe still activate production of GFP in the same intersectional population (green domain), but now cells expressing only Flpe (rather than cre) activate production of nβ-gal (blue domain, new subtractive population). (d) Illustration of the selective fate mapping achievable using an intersectional and subtractive approach. Cylinders represent the neural tube at early (left) to late (right) developmental stages. Top row illustrates transient Flpe expression, driven by gene A, in progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row illustrates transient Cre expression, driven by gene B, in progenitor cells located at a particular anteroposterior level of the neural tube at an early developmental stage (pink domain). Bottom row illustrates coupling gene A::Flpe and gene B::cre are coupled with a dual recombinase-responsive indicator allele (FP configuration), cells expressing Flpe and Cre activate production of GFP, while cells expressing only Flpe activate production of nβ-gal. Activation of reporter molecule expression is permanent, and all cells descended from Flpe-expressing or Flpe- and cre-expressing progenitors will continue expressing the blue or green marker, respectively. Descendant cells from the intersectional domain are denoted by green circles, those from the subtractive population by blue circles. (Reproduced from ref. 4 with permission from Elsevier Science) (Color figure online)