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WIREs Dev Biol
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Versatile genetic paintbrushes: Brainbow technologies

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Abstract Advances in labeling technologies are instrumental to study the developmental mechanisms that control organ formation and function at the cellular level. Until recently, genetic tools relied on the expression of single markers to visualize individual cells or lineages in developing and adult animals. Exploiting the expanding color palette of fluorescent proteins and the power of site‐specific recombinases in rearranging DNA fragments, the development of Brainbow strategies in mice made it possible to stochastically label many cells in different colors within the same sample. Over the past years, these pioneering approaches have been adapted for other experimental model organisms, including Drosophila melanogaster, zebrafish, and chicken. Balancing the distinct requirements of single cell and clonal analyses, adjustments were made that both enhance and expand the functionality of these tools. Multicolor cell labeling techniques have been successfully applied in studies analyzing the cellular components of neural circuits and other tissues, and the compositions and interactions of lineages. While being continuously refined, Brainbow technologies have thus found a firm place in the genetic toolboxes of developmental and neurobiologists. WIREs Dev Biol 2015, 4:161–180. doi: 10.1002/wdev.166 This article is categorized under: Technologies > Generating Chimeras and Lineage Analysis
Brainbow techniques take advantage of DNA rearrangements mediated by site‐specific recombinases. (a, b) Two aims of multicolor labeling approaches are illustrated in the developing Drosophila nervous system, but also apply to other cell types and tissues. Neural stem cell equivalents, the neuroblasts (NBs), self‐renew and produce ganglion mother cells (GMCs). These divide to produce postmitotic neuronal progeny, which can be visualized with specific enhancers (ON) driving the expression of reporters. (a) Anatomical studies of overlapping neuron branches require sparse labeling of individual cells. In mosaic approaches, single (i) or lineage‐related cells (ii) can be visualized, if recombination events by site‐specific recombinases (SSR) are triggered in GMCs or NBs, respectively. Unlike monochrome reporters (ii), sparse multicolor labeling enables the tracing of several neurons in the same sample (iii), even if they are lineage‐related or targeted by the same enhancer. SSR*, multicolor labeling can be achieved by recombinase activity in precursors or progeny. (b) Lineage analyses require comprehensive labeling of entire sets of progeny. Activity of SSRs in one precursor allows the labeling of a single clone (i). If a single reporter is used, progeny of multiple lineages can no longer be discerned (ii). Multicolor labeling makes it possible to study several lineages in the same sample (iii). Prior to enhancer activation, FPs are indicated as colored outlines of cells; full expression is indicated by filled cells. (c–h) Cre or FLP SSRs catalyze specific recombination events between pairs of target recombination sites (RS). Each RS consists of two inverted repeats (IR) and a spacer, determining RS directionality. (c) Two RS sites with the same orientation positioned in trans trigger the exchange of sequences between homologous chromosome arms. This configuration is used for mosaic approaches, such as MARCM. (d) SSRs mediate excision of DNA fragments between RS pairs with the same orientation and positioned in cis. (e) SSRs catalyze reversible inversions of DNA fragments located between RS pairs with opposite orientations. (f) SSRs mediate recombination between identical pairs of heterospecific site variants that differ in the spacer sequence (blue and cyan). (g) SSR variants are specific for target site pairs with distinct IR sequences (light blue, green). (h) ϕC31 mediates irreversible recombination events between attB and attP sites, characterized by distinct imperfect IR sequences, to generate new attL and attR sites.
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Four examples of Brainbow technologies at work. (a) Purkinje cells in the mouse cerebellum are visualized in seven colors (i–vii) using Brainbow‐3.1 and L7‐Cre transgenes, as well as antibody amplification. (Reprinted with permission from Ref . Copyright 2013 Nature Publishing Group) Scale bar, 20 µm. (b) Pyramidal neurons in the P28 cortex of a CAG‐CreERTM mouse are labeled by combinations of co‐electroporated MAGIC Cytbow and Nucbow markers at E15. The image was acquired by two‐photon microscopy. (Reprinted with permission from Ref . Copyright 2014 Elsevier Ltd.) Scale bar, 100 µm. (c) Neurites of lamina and medulla neuron subtypes (ln, mn) in the adult Drosophila optic lobe are visualized by endogenous fluorescent protein signals using a Flybow‐2.0B transgene, activated by hs‐mFLP5 and NP4151‐Gal4—an enhancer trap insertion into the Netrin B locus. The image represents a single optical section. Several neurons (arrowheads) are suitable for tracing in stacks. Photoreceptor axons are visualized by immunolabeling with mAb24B10 (blue). Scale bar, 20 µm. (d) Nuclei of epithelial cell clones in a 3rd instar larval wing disc of Drosophila are labeled by four fluorescent proteins using a Raeppli‐NLS transgene, activated by tubulin‐Gal4 and UAS‐FLP. This approach facilitates the comprehensive analysis of clones in the entire tissue. (Reprinted with permission from Ref . Copyright 2014 The Company of Biologists Ltd.) Scale bar, 50 µm.
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Multicolor labeling tools for use in zebrafish and mouse, as well as for electroporation in mouse and chicken. Constructs following the Brainbow‐1 strategy are indicated in blue and constructs based on the Brainbow‐2 strategy in purple. (a) In zebrafish, the mouse Brainbow‐1.0L cassette has been placed downstream of four regulatory elements: the cytomegalovirus enhancer (CMV), the βactin2 (βact2) enhancer, upstream activation sequences (UAS), or the ubiquitin (ubi) enhancer. UAS‐Zebrabow‐B uses non‐repetitive (nr) tandem UAS sites. V, variegated; B, broad; S, single; M, multiple. (b) In Confetti, a stop cassette precedes the two invertible cassettes of the original mouse Brainbow‐2.1 (R) transgene. Expression is controlled by CAG, the chicken β‐actin promoter with cytomegalovirus (CAG) enhancer. Grey lines only indicate a subset of possible recombination events. Thy1‐controlled Brainbow‐3.0, 3.1, 3.2 trangenes use farnesylated (f) FPs – mOrange2 (mO2), EGFP, and mKate2. In Brainbow‐3.1 and 3.2, a stop cassette prevents default FP expression. In Brainbow‐3.2, a woodchuck hepatitis virus posttranscriptional regulatory element (W) has been placed downstream of each FP. In Autobow, Thy1 controls expression of lox‐flanked Cre to trigger recombination events and self‐excision. In Flpbow‐3.0 and 3.1 transgenes, FLP mediates recombination of spacer variant pairs FRT3, FRT5T2, and FRT545. Flpbow‐3.1 uses a stop cassette and FP fused with a SUMOstar tag (S) for immunodetection. mCer, mCerulean; PhiYFP, Phialidium YFP; tdTom, tandem dimer Tomato. (c) MAGIC and CLoNe plasmids are transposon‐based vectors suitable for electroporation experiments in mouse and chicken. Tol2 or PiggyBac (PB) transposases promote the genomic integration of vectors. Ubiquitous or tissue/cell‐type specific Cre is provided by co‐injected vectors (chick and mouse) or by expression from genomic insertions (mouse). FP expression is controlled by the CAG regulatory element. In MAGIC markers, four different FPs are expressed from single vectors. FP are localized in the cytoplasm (Cytbow), in nuclei using Histone‐2B (H2B) fusions (Nucbow), in the membrane using a palmitoylation (p) signal (Palmbow) or in mitochondria (mit) using a targeting signal from human COX8 (Mitbow). H2B‐EBFP2 is expressed in unrecombined cells. In the CLoNe approach, four FPs [EGFP, mT‐Sapphire (mT‐Sap), mEYFP, and mCherry] are expressed from twelve separate labeling vectors. FPs are either cytoplasmic (cy), or nuclear (nc), and membrane‐bound (mb) using H2B or palmitoylation tags, respectively. A stop cassette prevents default expression of markers in the absence of Cre. Stable multicolor labeling is achieved by different random combinations of vector insertions and expression in individual cells. Asterisk indicates that mT‐Sapphire was assigned the color blue, although the maximum emission is in the green/yellow range. References for transgenes are provided in Table .
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Multicolor labeling tools in Drosophila. Transgenes following the excision‐based Brainbow‐1 strategy are highlighted in blue. Transgenes modeled on the inversion/excision‐based Brainbow‐2 strategy are shown in purple. Constructs are downstream of upstream activation sequences (UAS). (a) In dBrainbow, a stop cassette prevents marker expression prior to Cre activation. FPs are detected with three epitope‐tags. Native fluorescence signals can be collected for EGFP and mKusabira Orange2 (mKO2); EBFP2 requires detection by immunolabeling (asterisk). (b) In Flybow, FPs are membrane‐tethered using cd8 or myristoylation‐palmitoylation (mp) sequences. Flybow B transgenes use mTurquoise (mTq) instead of V5‐tagged mCerulean (mCer), which requires immunodetection (asterisk). Flybow‐1.0, 1.1, 1.0B, and 1.1B transgenes show default expression of mCherry (mCher) or EGFP. Flybow‐2.0 and 2.0B require FLP‐mediated excision of a FRT‐site flanked stop cassette. Recombination events between mFRT71 sites are triggered by mFLP5. (c‐e) UAS‐Brainbow, LOLLIbow, and UAS‐Brainbow2.1R‐2, are derived from the mouse Brainbow transgenes M and R. Recombination events are mediated by Cre. LOLLIbow relies on photo‐activated split‐Cre. p, palmitoylation signal. (f) In TIE‐DYE, FLP mediates the excision of stop cassettes in three separate transgenes controlled by ubiquitin (ubi) or actin (act) enhancers. Gal4 leads to expression of mRFP1. lacZ requires detection with an antibody against βGal (asterisk). Seven color outcomes are possible for the combination of these markers, targeted by a nuclear localization signal (nls) or Histone‐2A (H2A). (g) Raeppli transgenes are downstream of lexAop or UAS. Cre‐mediated excision converts transgenes into exclusively Gal4 or LexA controlled constructs. FLP‐mediated excision of a FRT‐flanked stop cassette, enables ϕC31 transcription. Integrase expression is controlled by the full heat shock protein 70 (hsp70) promoter or UAS. This leads to recombination between the attB site and one of the four attP sites preceding each FP and to integrase self‐excision. E2‐Or, E2‐Orange. FPs label cell nuclei in Raeppli‐NLS, and cell membranes using a farnesylation (f) signal in Raeppli‐CAAX. References for transgenes are provided in Table .
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Principles of mouse Brainbow blueprints. (a) Brainbow‐1 strategy transgenes (blue) build on the ability of Cre to mediate excisions between heterospecific lox pairs orientated in the same direction. In Brainbow‐1.0 (L), dTomato (dTom) is expressed by default. Cre catalyzes recombination events between lox2272 or loxP pairs, resulting in the stochastic expression of mCerulean (mCer) or mEYFP, respectively. In Brainbow‐1.1 (M), the default marker is mKusabira Orange (mKO). Cre mediates recombination between loxN, lox2272, and loxP pairs, allowing the expression of mCherry (mCher), mEYFP or mCerulean. A palmitoylation signal (p) targets these FPs to the membrane. pA, polyadenylation signals. (b) Brainbow‐2 strategy transgenes (purple) use the ability of Cre to mediate inversions and excisions between loxP sites oriented in the opposite and the same direction, respectively. Brainbow‐2.0 consists of one invertible cassette. tdimer2 is expressed by default. Cre triggers reversible inversions between loxP sites, inducing expression of palmitoylated ECFP. Brainbow‐2.1 (R) consists of two invertible cassettes. Nuclear (nls) GFP is expressed by default. Cre‐mediated inversions and excisions between loxP pairs allow expression of mEYFP, tdimer2 or palmitoylated mCerulean. All transgenes are under the control of the nervous system specific Thy1 enhancer. (c) Combinatorial expression of blue, green, and red FPs from three transgene copies increases the color palette from 3 to 10 hues. References for transgenes are provided in Table .
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