RESUMEN
A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, because temporal changes in intracellular protein abundances cannot be measured directly in live cells, fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systematically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog, we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight the problem of measurement in live cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteína Homeótica Nanog/metabolismo , Animales , Biología Celular , Células Madre Embrionarias/citología , Citometría de Flujo , Expresión Génica/fisiología , Regulación de la Expresión Génica/fisiología , Técnicas de Sustitución del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Cinética , Masculino , Ratones , Microscopía Fluorescente , Modelos Moleculares , Proteína Homeótica Nanog/genética , ARN Mensajero/metabolismoRESUMEN
Populations of mammalian stem cells commonly exhibit considerable cell-cell variability. However, the functional role of this diversity is unclear. Here, we analyze expression fluctuations of the stem cell surface marker Sca1 in mouse hematopoietic progenitor cells using a simple stochastic model and find that the observed dynamics naturally lie close to a critical state, thereby producing a diverse population that is able to respond rapidly to environmental changes. We propose an information-theoretic interpretation of these results that views cellular multipotency as an instance of maximum entropy statistical inference.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Modelos Biológicos , Células Madre Multipotentes/fisiología , Animales , Ataxina-1/biosíntesis , Entropía , Células Madre Hematopoyéticas/metabolismo , Ratones , Células Madre Multipotentes/metabolismoRESUMEN
A number of key regulators of mouse embryonic stem (ES) cell identity, including the transcription factor Nanog, show strong expression fluctuations at the single-cell level. The molecular basis for these fluctuations is unknown. Here we used a genetic complementation strategy to investigate expression changes during transient periods of Nanog downregulation. Employing an integrated approach that includes high-throughput single-cell transcriptional profiling and mathematical modelling, we found that early molecular changes subsequent to Nanog loss are stochastic and reversible. However, analysis also revealed that Nanog loss severely compromises the self-sustaining feedback structure of the ES cell regulatory network. Consequently, these nascent changes soon become consolidated to committed fate decisions in the prolonged absence of Nanog. Consistent with this, we found that exogenous regulation of Nanog-dependent feedback control mechanisms produced a more homogeneous ES cell population. Taken together our results indicate that Nanog-dependent feedback loops have a role in controlling both ES cell fate decisions and population variability.
