FIGURE 2.

Experimental paradigms to genetically dissect non-cell-autonomous mechanisms in radial cortical neuron migration. (A) Chimeras. A chimera is an animal that has two or more populations of genetically distinct cells. Depending on the degree of chimerism (i.e., ratio of wild-type versus mutant cells), such assay offers one way to distinguish between cell-autonomous gene function and non-cell-autonomous mechanisms in vivo. Any phenotypic difference seen between the neurons of the same genotype, but present in distinct genotypic environments indicate non-cell-autonomous effects. (B) Retroviral infection. Retroviral infection allows to sparsely target developing neurons by either expression of the reporter only (e.g., in a wild-type or mutant environment) or using a viral vector that encodesa wild-type or mutant version of the gene of interest in combination with a reporter. This facilitates the inactivation or rescue of the gene of interest in either wild-type or mutant environments, allowing for the distinction of cell-autonomous gene function and non-cell-autonomous effects. Appropriately diluted retrovirus encoding the reporter and gene of interest allows for the discrimination of individual neurons and one can adjust the viral titer to generate more or less sparsely targeted neuronal populations. (C) In utero electroporation. Timed in utero electroporation for inactivation of a gene allows the sparse targeting of nascent migrating neurons in an otherwise wild-type environment. The inactivation of a specific gene can either be achieved by gene knockdown in combination with a reporter in a wild-type mouse or by electroporation of an expression vector which drives expression of CRE and a reporter into a mouse carrying a conditional floxed allele. In this paradigm one can mainly dissect the cell-autonomous gene function in the targeted neurons, although the presence of non-cell-autonomous effects provided by the wild-type environment will be present (mutant cells in wild-type environment). To investigate non-cell-autonomous effects, it is necessary to electroporate of a separate set of tissue only with the fluorescent reporter in an otherwise mutant environment (mutant cells in mutant environment). Thus, neurons mutant for the same gene in two different environments allows for the distinction of non-cell-autonomous effects, provided that a different phenotype is observed between the mutant cells in each specific environment. Wild-type neurons in an otherwise mutant background by (over)expression of a rescue construct would further allow determination of non-cell-autonomous effects originating from the mutant environment (wild-type cells in mutant environment). The comparison of these three distinct paradigms will facilitate detailed description of cell-autonomous gene function and non-cell-autonomous effects. (D) Consecutive electroporation. Consecutive electroporation enables labeling, genetic manipulation and the monitoring of two or more distinct neuronal populations in the developing embryonic brain. The first neuronal population is electroporated for gene knockdown and the consecutive population with control fluorescent markers or vice versa (first mutant, then wild-type). In such assay, the phenotype of the first cohort of electroporated cells can reflect cell-autonomous gene function whereas the phenotype of the second cohort of cells could reflect a combination of directed non-cell-autonomous effects originating from the first cohort and more global community effects. (E) MADM. Mosaic analysis with double markers (MADM) allows for the analysis of sparse genetic mosaic (sparse mosaic) versus global/whole tissue (full-KO) ablation of a candidate gene with single cell resolution. This allows to quantitatively analyze non-cell-autonomous effects by subtracting the phenotype present in the sparse mosaic from the full-KO (cell-autonomous + non-cell-autonomous) versus cell-autonomous (sparse mosaic). It is important to note that the background cells in a MADM sparse mosaic are heterozygous and may need adjustment of the paradigm in the case of investigation of a dosage-sensitive gene (haploinsufficiency). In that case, the MADM experiment can also provide a solution by comparing all genotypes/colors, e.g., green –/–, red +/+ and yellow ±. For details of such application the reader is referred to Hippenmeyer et al., 2010. (F) Optogenetics. Optogenetics facilitates the use of genetically encoded tools to temporally control gene expression or protein function with light. Viral infection approaches and transgenic mice expressing optogenetic effector proteins in a Cre-dependent manner can be utilized to generate photoactivatable tissue. These approaches can create experimental paradigms which enable investigation of mutant neurons in an otherwise wild-type environment vs. wild-type neurons in a mutant environment in a spatiotemporal manner (G) In toto imaging. In toto live-imaging can visualize the movement of individual cells and their interactions with the surrounding cells within the whole developing tissue. This would allow for a direct assessment of non-cell-autonomous effects exerted by the neighboring cells on an individual cell or vice versa. In toto imaging mostly involves labeling of all cell membranes so each cell in the organism/microenvironment can be tracked and segmented. Here, a two-color combination of a membrane-localized fluorescent protein and a histone-fused fluorescent protein labeling chromatin which allows for tracking the cell membrane morphologies and nuclei movement has been displayed. Tracking the exact cell boundaries of the neurons spatiotemporally would enable the mapping of the physical interactions and forces which are exerted by the individual cell and that of the surrounding cells.