Molecular neuroanatomy's "Three Gs": a primer

Abstract

New genetic technologies are transforming nervous system studies in mice, impacting fields from neural development to the neurobiology of disease. Of necessity, alongside these methodological advances, new concepts are taking shape with respect to both vocabulary and form. Here we review aspects of both burgeoning areas. Presented are technologies which, by co-opting site-specific recombinase systems, enable select genes to be turned on or off in specific brain cells of otherwise undisturbed mouse embryos or adults. Manipulated genes can be endogenous loci or inserted transgenes encoding reporter, sensor, or effector molecules, making it now possible to assess not only gene function, but also cell function, origin, fate, connectivity, and behavioral output. From these methodological advances, a new form of molecular neuroscience is emerging that may be said to lean on the concepts of genetic access, genetic lineage, and genetic anatomy – the three ‘Gs’ – much like a general education rests on the basics of reading, ‘riting and ‘rithmetic.

Figure 1

Site-Specific DNA Excisions Serve as “On-Off” Switches for Gene Activity and as the basis for Genetic Fate Mapping. (A) Structure of a generic SSR-responsive transgene inserted as a single copy into the mouse genome. SSR-mediated recombination between directly repeated SSR recognition sites (triangles) results in deletion of intervening transcriptional stop sequences (red octagonal stop sign) and consequent expression of a reporter molecule. Depending on the type of promoter incorporated, either constitutive or tissue-specific reporter expression can be achieved. Spatial control of transgene activation is conferred by the regulatory elements used to drive SSR expression. (B) Two prototypical SSR-based manipulations of an endogenous locus: conditional gene removal versus repair. Depending on recognition site (triangle) placement, SSR-mediated excision can be exploited to remove (B, upper panel) or repair (B, lower panel) endogenous gene sequences. Light gray boxes represent untranslated exon regions (UTRs); dark gray boxes, coding exons; ATG, translation initiation codon; TAA, translation stop codon. (C) Illustration of how site-specific recombination can be used to study the deployment of progenitor cells and their descendants during development. This method is referred to as genetic fate mapping. The generic SSR-responsive transgene of panel A is modified here (C, upper panel) by incorporation of a broadly active promoter (BAP) ideally capable of driving transgene expression in any cell type at any stage in development, such that after a recombination event in a given cell, that cell and all its progeny cells should be marked by reporter expression regardless of subsequent cell differentiation. We refer to conditional target transgenes for genetic fate mapping as ‘indicator’ transgenes because they indicate or provide a permanent record of all earlier occurring recombination events. Lower panel, strategy for SSR-based genetic fate mapping with development of the neural tube rendered as a simple cylinder and progressing left to right in each row. Top row: hypothetical gene A is expressed transiently by progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row: SSR-expressing transgene utilizes enhancer elements from gene A. Bottom row: When geneA::SSR is coupled with an indicator transgene, cells expressing the SSR will activate production of the reporter molecule (for example, ßgal). Activation of reporter molecule expression is permanent, and all cells descended from the SSR-expressing (gene A-expressing) progenitors will continue expressing the reporter, thereby marking a genetic lineage as it contributes to different brain regions during development. Descendant cells are depicted here as blue circles.

Figure 1

Site-Specific DNA Excisions Serve as “On-Off” Switches for Gene Activity and as the basis for Genetic Fate Mapping. (A) Structure of a generic SSR-responsive transgene inserted as a single copy into the mouse genome. SSR-mediated recombination between directly repeated SSR recognition sites (triangles) results in deletion of intervening transcriptional stop sequences (red octagonal stop sign) and consequent expression of a reporter molecule. Depending on the type of promoter incorporated, either constitutive or tissue-specific reporter expression can be achieved. Spatial control of transgene activation is conferred by the regulatory elements used to drive SSR expression. (B) Two prototypical SSR-based manipulations of an endogenous locus: conditional gene removal versus repair. Depending on recognition site (triangle) placement, SSR-mediated excision can be exploited to remove (B, upper panel) or repair (B, lower panel) endogenous gene sequences. Light gray boxes represent untranslated exon regions (UTRs); dark gray boxes, coding exons; ATG, translation initiation codon; TAA, translation stop codon. (C) Illustration of how site-specific recombination can be used to study the deployment of progenitor cells and their descendants during development. This method is referred to as genetic fate mapping. The generic SSR-responsive transgene of panel A is modified here (C, upper panel) by incorporation of a broadly active promoter (BAP) ideally capable of driving transgene expression in any cell type at any stage in development, such that after a recombination event in a given cell, that cell and all its progeny cells should be marked by reporter expression regardless of subsequent cell differentiation. We refer to conditional target transgenes for genetic fate mapping as ‘indicator’ transgenes because they indicate or provide a permanent record of all earlier occurring recombination events. Lower panel, strategy for SSR-based genetic fate mapping with development of the neural tube rendered as a simple cylinder and progressing left to right in each row. Top row: hypothetical gene A is expressed transiently by progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row: SSR-expressing transgene utilizes enhancer elements from gene A. Bottom row: When geneA::SSR is coupled with an indicator transgene, cells expressing the SSR will activate production of the reporter molecule (for example, ßgal). Activation of reporter molecule expression is permanent, and all cells descended from the SSR-expressing (gene A-expressing) progenitors will continue expressing the reporter, thereby marking a genetic lineage as it contributes to different brain regions during development. Descendant cells are depicted here as blue circles.

Figure2

Intersectional and Subtractive Genetic Fate Mapping Strategy and an Enabling Prototypical Dual Recombinase-Responsive Indicator Allele. (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 co-expressed (tan domain); another in which genes A and C are co-expressed (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. By contrast to a single recombinase-responsive indicator allele (Figure 1C), 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 nßgal, 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 nßgal 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 dorsoventral (DV) axis but extending along the anteroposterior (AP) axis (top row); by contrast, the expression domain for gene B (pink) restricts along the AP axis but extends along the DV axis (middle row). When geneA::Flpe and geneB::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 nßgal (blue domain, subtractive population). When geneA::Flpe and geneB::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 nßgal (blue domain, new subtractive population). (D) Illustration of the selective fate mapping achievable using an intersectional and subtractive approach. Development of the neural tube is again rendered as a simple cylinder progressing left to right in each row. Top row: gene A drives transient Flpe expression in progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row: gene B drives transient cre expression in progenitor cells located at a particular AP level of the neural tube at an early developmental stage (pink domain). Bottom row: when geneA::Flpe and geneB::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 nßgal. 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.

Figure2

Intersectional and Subtractive Genetic Fate Mapping Strategy and an Enabling Prototypical Dual Recombinase-Responsive Indicator Allele. (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 co-expressed (tan domain); another in which genes A and C are co-expressed (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. By contrast to a single recombinase-responsive indicator allele (Figure 1C), 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 nßgal, 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 nßgal 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 dorsoventral (DV) axis but extending along the anteroposterior (AP) axis (top row); by contrast, the expression domain for gene B (pink) restricts along the AP axis but extends along the DV axis (middle row). When geneA::Flpe and geneB::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 nßgal (blue domain, subtractive population). When geneA::Flpe and geneB::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 nßgal (blue domain, new subtractive population). (D) Illustration of the selective fate mapping achievable using an intersectional and subtractive approach. Development of the neural tube is again rendered as a simple cylinder progressing left to right in each row. Top row: gene A drives transient Flpe expression in progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row: gene B drives transient cre expression in progenitor cells located at a particular AP level of the neural tube at an early developmental stage (pink domain). Bottom row: when geneA::Flpe and geneB::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 nßgal. 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.

Figure2

Intersectional and Subtractive Genetic Fate Mapping Strategy and an Enabling Prototypical Dual Recombinase-Responsive Indicator Allele. (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 co-expressed (tan domain); another in which genes A and C are co-expressed (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. By contrast to a single recombinase-responsive indicator allele (Figure 1C), 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 nßgal, 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 nßgal 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 dorsoventral (DV) axis but extending along the anteroposterior (AP) axis (top row); by contrast, the expression domain for gene B (pink) restricts along the AP axis but extends along the DV axis (middle row). When geneA::Flpe and geneB::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 nßgal (blue domain, subtractive population). When geneA::Flpe and geneB::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 nßgal (blue domain, new subtractive population). (D) Illustration of the selective fate mapping achievable using an intersectional and subtractive approach. Development of the neural tube is again rendered as a simple cylinder progressing left to right in each row. Top row: gene A drives transient Flpe expression in progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row: gene B drives transient cre expression in progenitor cells located at a particular AP level of the neural tube at an early developmental stage (pink domain). Bottom row: when geneA::Flpe and geneB::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 nßgal. 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.

Figure 3

Nonconcurrent driver genes add temporal resolution to intersectional fate maps. Development of the neural tube is again rendered as a simple cylinder progressing left to right in each row. Top row: gene A drives transient Flpe expression in progenitor cells located in the dorsal neural tube (yellow domain) at an early developmental stage. Middle row: gene B drives transient cre expression in a population of later-stage progenitor cells located at a particular AP level of the neural tube (pink domain). Bottom row: when geneA::Flpe and geneB::cre are coupled with a dual recombinase-responsive indicator allele (FP configuration), expression of nßgal, as a lineage tracer of geneA-expressing progenitor cells, is activated first. Cells with a history of gene A expression that go on to express gene B, and therefore cre, will, following Cre-mediated recombination, turn-off nßgal expression and turn on GFP expression (the intersectional population).

Figure 4

Addressing temporal aspects of lineage allocation by controlling SSR activity. (A) Schematic of a transgene encoding the recombinase-steroid fusion protein, SSR-ER^T2, whose activity is regulated posttranslationally by the ligand 4-hydroxy tamoxifen (4-OHT, red circle). (B) Inducible recombination and cell marking using SSR-ER^T2. In the absence of 4-OHT, SSR-ER^T2 is inactive due to sequestration of the fusion protein into an Hsp90 complex. Binding of 4-OHT to SSR-ER^T2 results in a conformational change that disrupts the Hsp90 interaction, freeing the recombinase to enter the cell nucleus and mediate recombination at its target sites (triangles) positioned within an indicator transgene. Excisional recombination renders cells positive for reporter expression (for example, cytoplasmic ßgal as indicated in dark blue). (C) Cumulative versus inducible genetic fate mapping. Development of the neural tube is again rendered as simple cylinders progressing left to right in each row. Cumulative genetic fate mapping is schematized in the top two rows, much as done previously in Figure 1C. Top row: transient, midgestation expression of cre recombinase in progenitor cells of the dorsal neural tube defined by their expression of gene A. Second row: activation of nßgal, for example, as a lineage tracer in all cells that ever in their history expressed gene A::cre. Inducible genetic fate mapping is schematized in the bottom two rows. Third row: transient, midgestation expression of SSR-ER^T2 in progenitor cells of the dorsal neural tube defined by expression of gene A. Bottom row: induction of recombinase activity and consequent indicator transgene expression following administration of 4-OHT permits selective tracking of late-emerging cohorts in virtual isolation.

Figure 4

Addressing temporal aspects of lineage allocation by controlling SSR activity. (A) Schematic of a transgene encoding the recombinase-steroid fusion protein, SSR-ER^T2, whose activity is regulated posttranslationally by the ligand 4-hydroxy tamoxifen (4-OHT, red circle). (B) Inducible recombination and cell marking using SSR-ER^T2. In the absence of 4-OHT, SSR-ER^T2 is inactive due to sequestration of the fusion protein into an Hsp90 complex. Binding of 4-OHT to SSR-ER^T2 results in a conformational change that disrupts the Hsp90 interaction, freeing the recombinase to enter the cell nucleus and mediate recombination at its target sites (triangles) positioned within an indicator transgene. Excisional recombination renders cells positive for reporter expression (for example, cytoplasmic ßgal as indicated in dark blue). (C) Cumulative versus inducible genetic fate mapping. Development of the neural tube is again rendered as simple cylinders progressing left to right in each row. Cumulative genetic fate mapping is schematized in the top two rows, much as done previously in Figure 1C. Top row: transient, midgestation expression of cre recombinase in progenitor cells of the dorsal neural tube defined by their expression of gene A. Second row: activation of nßgal, for example, as a lineage tracer in all cells that ever in their history expressed gene A::cre. Inducible genetic fate mapping is schematized in the bottom two rows. Third row: transient, midgestation expression of SSR-ER^T2 in progenitor cells of the dorsal neural tube defined by expression of gene A. Bottom row: induction of recombinase activity and consequent indicator transgene expression following administration of 4-OHT permits selective tracking of late-emerging cohorts in virtual isolation.

Figure 5

Turning genetic fate maps into functional connectivity maps using SSR technology as template. (A) Schematic of a modular, ‘plug and play’ vector designed for assembly of dual recombinase responsive transgenes that offer highly selective, conditional expression of effector molecules of choice (J. Kim and S. Dymecki, unpublished reagent). BAP, broadly active promoter (for example, CAG/R26); MCS, multiple cloning sites; STOP, transcriptional stop cassette; loxP site, triangle; FRT site vertical rectangle; sequence encoding an effector molecule and pA, red rectangle. (B) A sample of ‘plug-in’ genetically encoded effector molecules that have shown some degree of efficacy in mice.