Multicolor strategies for investigating clonal expansion and tissue plasticity

Abstract

Understanding the generation of complexity in living organisms requires the use of lineage tracing tools at a multicellular scale. In this review, we describe the different multicolor strategies focusing on mouse models expressing several fluorescent reporter proteins, generated by classical (MADM, Brainbow and its multiple derivatives) or acute (StarTrack, CLoNe, MAGIC Markers, iOn, viral vectors) transgenesis. After detailing the multi-reporter genetic strategies that serve as a basis for the establishment of these multicolor mouse models, we briefly mention other animal and cellular models (zebrafish, chicken, drosophila, iPSC) that also rely on these constructs. Then, we highlight practical applications of multicolor mouse models to better understand organogenesis at single progenitor scale (clonal analyses) in the brain and briefly in several other tissues (intestine, skin, vascular, hematopoietic and immune systems). In addition, we detail the critical contribution of multicolor fate mapping strategies in apprehending the fine cellular choreography underlying tissue morphogenesis in several models with a particular focus on brain cytoarchitecture in health and diseases. Finally, we present the latest technological advances in multichannel and in-depth imaging, and automated analyses that enable to better exploit the large amount of data generated from multicolored tissues.

Keywords: Brain; Fluorescent reporters; Lineage tracing; Morphogenesis; Multichannel imaging; Transgenic mouse.

Fig. 1

Scheme of different constructs used to generate multicolor mice: MADM (A), Brainbow (B) and Brainbow derivatives (C). The recombinase excises or inverts the DNA fragment between compatible target sequence with identical or opposite orientation, respectively. Compatible target sequence of Cre and Flp recombinase is pictured by pairs of triangles with the same color (loxP: black, lox2272: dark gray, loxN: light grey, 5T2: dark blue, 545: cyan, F3: green). The cell can express no FP (_CEGFP, _CtdTom, _CDsred2, _NEGFP, _NtdTom, _NDsred2, PhiNFP) or blue (EBFP2), cyan (ECFP, mCer, mTFP1, mTurq) yellow (mEYFP, EYFP, PhiYFP), green (_NEGFP + _CEGFP, hrGFPII, EGFP), orange (mKO, mOr) or red (mCher, _NDsred2 + _CDsred2, _NtdTom + _CtdTom, tdTom, tdimer2, mKate2) FP. To enhance the endogenous FP expression, WPRE element (W) has been added to Brainbow-3.2 and ifgMosaic constructs in addition to a Sumo start epitope (*) in Flpbow-3.1. The endogenous signal can be specifically amplified by immunostaining targeting antigenically distinct FP (Brainbow-3.0, 3.1, 3.2, Flpbow-3.1) or tags (T, ifgMosaic) as symbolized by an antibody scheme. Abbreviations used: 2A bicistronic element, GOI gene of interest, mCer mCerulean, mCher mCherry, mOr mOrange2, mTurq mTurquoise2, NeoR neomycin resistance cassette, pA polyadenylation stop signal, PhiNFP non fluorescent mutant protein detectable after immunostaining, dTom dTomato, tdTom tdTomato, T tag (V5, His, HA), W WPRE element, * Sumo start epitope

Fig. 2

Scheme of constructs used to create acute transgenesis models such as StarTrack (A), iOn (B), CLoNe (C), MAGIC Markers (D), LeGO (E), Brainbow, VAST and Tetbow AAVs (F). Antiparallel terminal repeats frame the StarTrack, CLoNe, MAGIC Markers constructs while parallel terminal repeats are used in iOn plasmids. The endogenous signal is increased by inclusion of a WPRE (W) element in the LeGO, Brainbow, VAST and Tetbow viral vectors. Ubiquitous (CAG, CMV, SFFV, UbC), neuronal (Syn1) or glial (GFAP, NG2) promoters drive FP (co-)expression in the host cell and progeny after (co-)electroporation or (co-)transduction. Abbreviations used: ATG start codon, CAG composite promoter composed of the fusion of CMV enhancer, chicken ß actin promoter and rabbit ß globin splice acceptor site, CMV cytomegalovirus promoter, cPPT central polypurine tract, GFAP glial fibrillary acidic protein, ITR inverted terminal repeats, TR long terminal repeat used in viral constructs, mCer mCerulean, mCher mCherry, mRub mRuby2, mt-S mt-Sapphire, mTurq mTurquoise2, NeoG mNeonGreen, NG2 neural glial antigen 2, pA polyadenylation stop signal, PBase piggyBac transposase, RRE rev response element, SFFV spleen focus-forming virus promoter, Syn1 human Synapsin1, tdTom tdTomato, TR terminal repeats recognized by piggyBac (pink) or Tol2 (blue) transposases, TRE tetracycline response element, tTA tetracycline transactivator, UbC ubiquitin C promoter, W WPRE element, Ψ psi packaging signal

Fig. 3

Typical labeling scheme of the main multicolor approaches used to study clonality in the mouse cerebral cortex. (A) Mosaic analysis with MADM labels mutant cells in green, heterozygous cells in yellow and wild type cells in red in order to both study clonal behavior and cell-autonomous gene function. (B) With a limited number of colors after signal amplification in the nervous system, the Confetti reporter mouse is suitable to follow slowly proliferating and poorly migrating cells, such as adult astrocytes. (C) StarTrack offers versatile tools to perform multiclonal analysis of brain development after electroporation of 12 integrative reporter vectors and transposase into neural progenitors driven by a ubiquitous UbC promoter (left side) or GFAP promoter (right side). (D) MAGIC Markers is useful for generating rare color codes that unambiguously identify clones such as sibling protoplasmic and fibrous astrocytes exhibiting a yellow nuclei and red cytoplasm after in utero co-electroporation with Cre recombinase and transposases. Abbreviations used: KO mutant green cell, Het heterozygous yellow cell, WT wild type red cell, αFP (Fluorescent Protein) signal after immunostaining against FP, MADM mosaic analysis with double markers, MAGIC markers Multiaddressable Genomic Integrating Color markers, mCer mCerulean, ^mmCer membrane mCerulean, mCher mCherry, mOr mOrange2, mt-S mt-Sapphire, mTurq mTurquoise2, NG2 neural glial antigen 2, ⁿGFP nuclear GFP, tdim tdimer2, tdTom tdTomato

Fig. 4

MAGIC Markers labeling in the adult mouse cortex enables multicolor clonal analysis. (A) Maximal projection of 80 μm thick sagittal section displaying the cerebral cortex of a 3 month-old mouse. Cortical progenitors were targeted at E14 by in utero co-electroporation of MM Nucbow and Cytbow, their respective transposases Tol2 and piggyBac, and a self-excisable Cre to trigger multicolor labeling. Neurons born at the time of electroporation express a wide palette of colors resulting from the expression of FP from both integrated and episomal vectors and have migrated to the upper cortical layers, while the oligodendrocyte and astrocyte progenies, which have colonized the entire cortex, have diluted episomes and only express integrated, stable, and clonally reliable markers. Mosaic image stacks were obtained by sequential confocal acquisition of mTurquoise2/mCerulean, mEFYP, mCherry/tdTomato (rendered in blue, green and red, respectively), with a 20 × 0.8NA oil objective on an Olympus FV1000 microscope, and stiched with Fiji. (B) Close-ups of neurons (first panel), oligodendrocytes and astrocytes clusters. (C) Examples of cytoplasm and nucleus-cytoplasm color combinations of cortical glial cells found in this one 80 μm section. IUE in utero electroporation. Scale bar: 200 μm