Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions

Abstract

SUMMARYIn 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.

Mapping differentiation kinetics in the mouse retina reveals an extensive period of cell cycle protein expression in post‐mitotic newborn neurons

“…6A). In line with live imaging studies indicating that M-phase is $30 min (Cayouette et al, 2003;Gomes et al, 2011), most PH3 þ cells became BrdU þ in a single 20-min chase window (Fig. 6A, Table 7).…”

In vivo evidence for unbiased ikaros retinal lineages using an ikaros‐cre mouse line driving clonal recombination

“…Moreover, when RPCs are plated at clonal density, they generate clones that are of similar size and composition as clones that develop in situ, and the general order of cell type production in the population is reproduced (Cayouette et al, 2003). Although feedback inhibition signals from the environment can clearly influence the proportion of specific cell types produced (Reh and Tully, 1986;Reh, 1992;Waid and McLoon, 1998;Wang et al, 2005), even in clonal cultures, these results are consistent with a model in which RPCs do not require specific instructive environmental signals for proper cell fate specification, but rather depend on cell intrinsic cues acting to bias their developmental potential over time (Cayouette et al, 2006;Gomes et al, 2011).…”

“…Considering the significance of this data for the field, the analysis of a larger number of clones from an earlier time point would be desirable, and in particular mandatory to test various models of retinogenesis. For example, a large number of clones will be required to test in vivo a stochastic model of retinal cell fate specification that we recently proposed from the analysis of lineages in culture (Gomes et al, 2011). This project further asks for lineage data at several intermediate stages of retinogenesis, and these results will be best compared to adult clone composition in the context of a unique and consistent mouse line.…”

Direction-selective retinal ganglion cells arise from molecularly specified multipotential progenitors

“…For example, recent studies have shown that amacrine subtypes arise at distinct times, as predicted by the competence model (38,43), and that further distinctions among them may result from postmitotic fate choices (44). Similarly, analysis of clones containing late-born retinal cell types (rods, amacrines, bipolars, and Müller glia) indicates that stochastic mechanisms acting on a homogeneous population of progenitors can account for the much of the diversity observed (1,45). The observation that cdh6-positive progenitors produce multiple subtypes of bipolar and amacrine cells could also be explained by stochastic mechanisms acting to produce the diversity of later-born cell types.…”