Futile attempts to differentiate provide molecular evidence for individual differences within a population of cells during cellular reprogramming

Abstract

The heterogeneity of cell populations and the influence of stochastic noise might be important issues for the molecular analysis of cellular reprogramming at the system level. Here, we show that in Physarum polycephalum, the expression patterns of marker genes correlate with the fate decision of individual multinucleate plasmodial cells that had been exposed to a differentiation-inducing photostimulus. For several hours after stimulation, the expression kinetics of PI-3-kinase, piwi, and pumilio orthologs and other marker genes were qualitatively similar in all stimulated cells but quantitatively different in those cells that subsequently maintained their proliferative potential and failed to differentiate accordingly. The results suggest that the population of nuclei in an individual plasmodium behaves synchronously in terms of gene regulation to an extent that the plasmodium provides a source for macroscopic amounts of homogeneous single-cell material for analysing the dynamic processes of cellular reprogramming. Based on the experimental findings, we predict that circuits with switch-like behaviour that control the cell fate decision of a multinucleate plasmodium operate through continuous changes in the concentration of cellular regulators because the nuclear population suspended in a large cytoplasmic volume damps stochastic noise.

A Next-Generation Sequencing Approach to Study the Transcriptomic Changes during the Differentiation of Physarum at the Single-Cell Level

“…All these belong to the same hierarchy of ontologies, so all these can be summarized with the lower and more specific category, i.e., the ‘calcium ion binding’ GO term. Both analyses of GO annotations correlate well with previous studies that point to the upregulation of genes associated with the ion transport in the light-induced plasmodium 13,20…”

“…In these works, both the assembly and mapping procedures were achieved using the mouse genome as a reference. Previously, we succeeded in measuring the expression on a set of 35 differentially regulated genes in populations of single-cells, using the GeXP RT-PCR platform 20. Here, using the power of RNA-seq to obtain whole transcriptomes without relying on previous genomic information, we were able to characterize a large set of expressed genes in different samples during the sporulation of Physarum , an organism without a known genomic sequence.…”

Physarum polycephalum mutants in the photocontrol of sporulation display altered patterns in the correlated expression of developmentally regulated genes

“…) as well as quantitative multiplex RT–PCR (GeXP) analyses with RNA samples prepared from individual plasmodial cells (Hoffmann et al . ) have shown that at 6 h after induction by a far‐red light pulse, that is, shortly downstream of the point of no return, the expression level of many genes has already changed drastically. Some genes are differentially regulated even at 2 h (Hoffmann et al .…”

“…Afterwards the RNA was precipitated with ethanol, and digested with rDNase to remove DNA contamination as described (Hoffmann et al . ).…”

Motif for controllable toggle switch in gene regulatory networks

“…Systems-level biological modeling is a necessary and powerful tool for understanding how the regulatory network achieves a switch between the two stable states of a bistable behavior. Bistability is discussed with respect to all-or-none cellular processes such as differentiation, proliferation, survival and apoptosis [1][2][3][4][5][6]. However, much evidence about reversible switches has been observed in the regulatory network in biological experiments [7][8][9][10][11].…”