Reconstruction of Rat Retinal Progenitor Cell Lineages in Vitro Reveals a Surprising Degree of Stochasticity in Cell Fate Decisions Francisco L
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DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 227 Development 138, 227-235 (2011) doi:10.1242/dev.059683 © 2011. Published by The Company of Biologists Ltd Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions Francisco L. A. F. Gomes1, Gen Zhang2, Felix Carbonell3, José A. Correa4, William A. Harris5, Benjamin D. Simons2,6 and Michel Cayouette1,7,8,* SUMMARY In vivo cell lineage-tracing studies in the vertebrate retina have revealed that the sizes and cellular compositions of retinal clones are highly variable. It has been challenging to ascertain whether this variability reflects distinct but reproducible lineages among many different retinal progenitor cells (RPCs) or is the product of stochastic fate decisions operating within a population of more equivalent RPCs. To begin to distinguish these possibilities, we developed a method for long-term videomicroscopy to follow the lineages of rat perinatal RPCs cultured at clonal density. In such cultures, cell-cell interactions between two different clones are eliminated and the extracellular environment is kept constant, allowing us to study the cell-intrinsic potential of a given RPC. Quantitative analysis of the reconstructed lineages showed that the mode of division of RPCs is strikingly consistent with a simple stochastic pattern of behavior in which the decision to multiply or differentiate is set by fixed probabilities. The variability seen in the composition and order of cell type genesis within clones is well described by assuming that each of the four different retinal cell types generated at this stage is chosen stochastically by differentiating neurons, with relative probabilities of each type set by their abundance in the mature retina. Although a few of the many possible combinations of cell types within clones occur at frequencies that are incompatible with a fully stochastic model, our results support the notion that stochasticity has a major role during retinal development and therefore possibly in other parts of the central nervous system. KEY WORDS: Cell fate choice, Lineage, Live imaging, Retina, Rat INTRODUCTION molecular mechanisms at work in invertebrate lineages are also Classic experiments in the nematode C. elegans have shown that apparent in the developing vertebrate CNS, but to what extent progenitor cells undergo stereotyped patterns of cell division to stereotypic lineages play a part remains an open question. generate specific cell types at particular stages of development Retroviral lineage tracing and single-cell dye injections in the (Sulston and Horvitz, 1977; Sulston et al., 1983). Achieving developing cortex and retina have shown that single progenitors are different fates in such lineages depends on asymmetrically multipotent and that clones vary widely in size and composition inherited intrinsic determinants or predictable interactions between (Holt et al., 1988; Price et al., 1991; Reid et al., 1997; Turner and sister cells and, consequently, mutations that affect any regulators Cepko, 1987; Turner et al., 1990; Walsh and Cepko, 1990; Wetts of such components alter the lineage trees and have a major effect and Fraser, 1988). These studies, however, did not provide on cell fate decisions (Rose and Kemphues, 1998). These results information about how the clones developed over time. In an effort indicate that lineage-dependent ‘developmental programs’ operate to tackle this problem, Temple and colleagues pioneered in vitro over multiple rounds of cell division and are crucial regulators of clonal-density cultures of cortical progenitor cells that allowed development in C. elegans. Similarly, in Drosophila neuroblasts, long-term time-lapse recordings that charted every cell division, highly complex reproducible lineages play an essential part in death and differentiation event in the entire lineage trees (Qian et neurogenesis, and their molecular mechanisms are beginning to be al., 1998; Qian et al., 2000; Shen et al., 2006). The order of elucidated (Kao and Lee, 2010). Many of the cellular and neuronal cell production and the final cell compositions in these isolated lineages were strikingly consistent with what is known about cortical neurogenesis in vivo, suggesting that these cortical progenitors have intrinsically programmed lineages. However, 1 Cellular Neurobiology Research Unit, Institut de recherches cliniques de Montréal, detailed analysis of the lineage trees of these progenitors still Montréal, QC H2W 1R7, Canada. 2Department of Physics, Cavendish Laboratory, J J Thomson Avenue, Cambridge CB3 0HE, UK. 3The McConnell Brain Imaging Centre, showed large variability in size and composition, suggesting that Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada. stochastic decisions played a part. Indeed, a recent mathematical 4 Department of Mathematics and Statistics, McGill University, Montreal, QC H3A analysis of the cortical lineage data indicated that the distribution 2K6, Canada. 5Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. 6Wellcome of lineage tree sizes is consistent with a stochastic model in which Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court the probabilities of undergoing a division are weighted according 7 Road, Cambridge CB2 1QN, UK. Département de Médecine, Université de to cell generation (Slater et al., 2009). Montréal, Montreal, QC H3T 3J7, Canada. 8Department of Biology, Department Anatomy and Cell Biology, and Division of Experimental Medicine, McGill University, Although stochastic models might help to explain the variability Montreal, QC H3A 1B1, Canada. in the number of cells within clones, understanding how specific neuronal cell types are generated at the right time within lineages *Author for correspondence ([email protected]) of various cellular compositions is more challenging. It seems Accepted 15 November 2010 clear, however, that cortical and retinal progenitors intrinsically DEVELOPMENT 228 RESEARCH ARTICLE Development 138 (2) change their potential to give rise to specific cell types over time. connected to a computer equipped with Volocity software (Perkin Elmer) For example, mouse cortical progenitors follow the correct programmed to capture a frame every 5 minutes for a period of 9-14 days. sequence of neuronal cell type production, even when cultured at The cells were kept in a 37°C, 8% CO2, 12% O2 environment. clonal density (Shen et al., 2006). Strikingly, mouse embryonic Immunostaining stem (ES) cells that are directed toward cortical fates also generate After the recording, the cells were fixed with 4% paraformaldehyde for 10 the different types of cortical neurons in the appropriate minutes and the following antibodies were used for immunostaining: chronological order in culture (Gaspard et al., 2008). Much like in monoclonal mouse anti-Islet1 (1:2000; produced by T. Jessell and obtained the cortex, the different cell types of the vertebrate retina are from the Developmental Studies Hybridoma Bank) and rabbit anti-Pax6 generated from progenitors in a conserved, but overlapping, (1:10,000; Santa Cruz Biotech). Primary antibodies were detected using chronological order (Rapaport et al., 2004; Young, 1985). The goat anti-mouse IgG2b Alexa Fluor 488 and goat anti-rabbit IgG Alexa variability of the lineages generated from individual retinal Fluor 594. In all cases, nuclei were stained with Hoechst 33342. progenitor cells (RPCs), combined with the temporal program of Reconstruction of RPC lineage trees histogenesis, originally led to the suggestion that RPCs make For lineage tree reconstructions, the time-lapse movies were replayed and ‘lineage-independent’ fate decisions based on changing the movement and divisions of each individual RPC were followed environmental influences that operate at different stages of manually over time. To generate the lineage tree, the progenies of a single development to instruct the production of the various retinal cell cell were tracked and their fates were identified based on their specific types (Holt et al., 1988; Turner and Cepko, 1987; Turner et al., morphologies and validated by the corresponding immunostaining data. 1990; Wetts and Fraser, 1988). Such instructional cues, however, Numerical simulation of the RPC lineage trees have not been found so far. Moreover, retinal clones generated in The clone size distribution and lineage composition predicted by the clonal-density cultures are, as a population, indistinguishable in stochastic models discussed in the text were determined by numerical size and composition from clones generated in explants of retina of simulation using a Monte Carlo analysis based on the Gillespie algorithm the same age (Cayouette et al., 2003). Thus, RPCs seem (Gillespie, 1977). The balance between proliferation and differentiation, intrinsically programmed to generate a distribution of clones of and the specification of the different differentiated cell types, were set by different sizes and composition, which, amazingly, when added fixed probabilities as defined in the main text. Where necessary, the distribution of cell cycle times was taken to be log-normal with an average together, generate the correct number and proportions of cell types and variance fixed by the fit to the experimental data as defined in the main in the retina. text. There are at least two possible explanations for how RPCs generate clones