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Multistable switches and their role in cellular differentiation networks.

ABSTRACT: Cellular differentiation during development is controlled by gene regulatory networks (GRNs). This complex process is always subject to gene expression noise. There is evidence suggesting that commonly seen patterns in GRNs, referred to as biological multistable switches, play an important role in creating the structure of lineage trees by providing stability to cell types.To explore this question a new methodology is developed and applied to study (a) the multistable switch-containing GRN for hematopoiesis and (b) a large set of random boolean networks (RBNs) in which multistable switches were embedded systematically. In this work, each network attractor is taken to represent a distinct cell type. The GRNs were seeded with one or two identical copies of each multistable switch and the effect of these additions on two key aspects of network dynamics was assessed. These properties are the barrier to movement between pairs of attractors (separation) and the degree to which one direction of movement between attractor pairs is favored over another (directionality). Both of these properties are instrumental in shaping the structure of lineage trees. We found that adding one multistable switch of any type had a modest effect on increasing the proportion of well-separated attractor pairs. Adding two identical switches of any type had a much stronger effect in increasing the proportion of well-separated attractors. Similarly, there was an increase in the frequency of directional transitions between attractor pairs when two identical multistable switches were added to GRNs. This effect on directionality was not observed when only one multistable switch was added.This work provides evidence that the occurrence of multistable switches in networks that control cellular differentiation contributes to the structure of lineage trees and to the stabilization of cell types.

SUBMITTER: Ghaffarizadeh A

PROVIDER: S-EPMC4110729 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Project description:Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGF?)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGF?-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation.Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGF?1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGF?-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (?-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis.Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGF?1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in ?-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGF?1 showed the expected low expression of ?-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGF?-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGF?-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein.Conclusions: Id3 regulates TGF?-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.

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Multistable switches and their role in cellular differentiation networks.

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