An Ovol2-Zeb1 EMT-Regulatory Circuit Governs Mammary Basal-Luminal Binary Differentiation

The capacity of epithelial cells to acquire enhanced lineage plasticity could depend on their ability to undergo EMT. Investigations performed on cultured epithelial cells support a link between EMT and bestowment of stem cell (SC)-like properties, raising the possibility that regulators of EMT may be responsible for producing an intermediate cellular identity between epithelial and mesenchymal states and is compatible with SC potential. The goal of my thesis project is to identify and characterize key transcriptional regulators of the dynamic EMT process that facilitate the production and maintenance of epithelial SCs, using the MG as a model system. The Dai laboratory identified Ovol2 as a TF that is required for mammary and epidermal development. My work contributed to the discovery of Ovol2 as a master negative regulator of EMT that directly represses the expression of various EMT-related genes, the most important being Zeb1, a critical mediator of Ovol2 loss-of-function effects. Zeb1 is a potent EMT-TF implicated in conferring SC-like traits to differentiated cells in mammary epithelial tumors. However, its in vivo role within normal mammary epithelia has not been studied. I found that Zeb1 also directly represses Ovol2, leading to the identification of an Ovol2-Zeb1 cross-repression circuit, which is shown by mathematic modeling to support intermediate cellular states between terminal epithelial and mesenchymal identities. Additionally, my data shows that Zeb1 expression is activated during early pregnancy in the basal cells of the mammary epithelium, which are known to gain multipotency upon pregnancy or transplantation. Using in vivo and ex vivo approaches to determine how perturbations to the Ovol2-Zeb1 circuit regulate stemness, I found this circuit to be important in modulating mammary SC basal-luminal differentiation. In addition to protecting basal cells from precocious differentiation toward a luminal fate, Zeb1 functions in regulating SC self-renewal/proliferative activity. Both mechanisms may contribute to the observed, Zeb1 loss-induced defect in ductal branching during mammary regeneration. My findings uncover a previously unknown role of Zeb1 and its associated molecular circuit in regulating mammary SC activity and basal/luminal differentiation, offering new insights into how epithelial plasticity contributes to stemness and identify novel transcriptional regulators of epithelial SCs

An Ovol2-Zeb1 EMT-Regulatory Circuit Governs Mammary Basal-Luminal Binary Differentiation

The capacity of epithelial cells to acquire enhanced lineage plasticity could depend on their ability to undergo EMT. Investigations performed on cultured epithelial cells support a link between EMT and bestowment of stem cell (SC)-like properties, raising the possibility that regulators of EMT may be responsible for producing an intermediate cellular identity between epithelial and mesenchymal states and is compatible with SC potential. The goal of my thesis project is to identify and characterize key transcriptional regulators of the dynamic EMT process that facilitate the production and maintenance of epithelial SCs, using the MG as a model system. The Dai laboratory identified Ovol2 as a TF that is required for mammary and epidermal development. My work contributed to the discovery of Ovol2 as a master negative regulator of EMT that directly represses the expression of various EMT-related genes, the most important being Zeb1, a critical mediator of Ovol2 loss-of-function effects. Zeb1 is a potent EMT-TF implicated in conferring SC-like traits to differentiated cells in mammary epithelial tumors. However, its in vivo role within normal mammary epithelia has not been studied. I found that Zeb1 also directly represses Ovol2, leading to the identification of an Ovol2-Zeb1 cross-repression circuit, which is shown by mathematic modeling to support intermediate cellular states between terminal epithelial and mesenchymal identities. Additionally, my data shows that Zeb1 expression is activated during early pregnancy in the basal cells of the mammary epithelium, which are known to gain multipotency upon pregnancy or transplantation. Using in vivo and ex vivo approaches to determine how perturbations to the Ovol2-Zeb1 circuit regulate stemness, I found this circuit to be important in modulating mammary SC basal-luminal differentiation. In addition to protecting basal cells from precocious differentiation toward a luminal fate, Zeb1 functions in regulating SC self-renewal/proliferative activity. Both mechanisms may contribute to the observed, Zeb1 loss-induced defect in ductal branching during mammary regeneration. My findings uncover a previously unknown role of Zeb1 and its associated molecular circuit in regulating mammary SC activity and basal/luminal differentiation, offering new insights into how epithelial plasticity contributes to stemness and identify novel transcriptional regulators of epithelial SCs

Mammary Morphogenesis and Regeneration Require the Inhibition of EMT at Terminal End Buds by Ovol2 Transcriptional Repressor

Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition [EMT]). However, how epithelial plasticity is kept in check in epithelial cells during tissue development and regeneration remains to be fully understood. Here we show that restricting the EMT of mammary epithelial cells by transcription factor Ovol2 is required for proper morphogenesis and regeneration. Deletion of Ovol2 blocks mammary ductal morphogenesis, depletes stem and progenitor cell reservoirs, and leads epithelial cells to undergo EMT in vivo to become nonepithelial cell types. Ovol2 directly represses myriad EMT inducers, and its absence switches response to TGF-β from growth arrest to EMT. Furthermore, forced expression of the repressor isoform of Ovol2 is able to reprogram metastatic breast cancer cells from a mesenchymal to an epithelial state. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity in development and cancer

An Ovol2-Zeb1 Mutual Inhibitory Circuit Governs Bidirectional and Multi-step Transition between Epithelial and Mesenchymal States

<div><p>Reversible epithelial-to-mesenchymal transition (EMT) is central to tissue development, epithelial stemness, and cancer metastasis. While many regulatory elements have been identified to induce EMT, the complex process underlying such cellular plasticity remains poorly understood. Utilizing a systems biology approach integrating modeling and experiments, we found multiple intermediate states contributing to EMT and that the robustness of the transitions is modulated by transcriptional factor Ovol2. In particular, we obtained evidence for a mutual inhibition relationship between Ovol2 and EMT inducer Zeb1, and observed that adding this regulation generates a novel four-state system consisting of two distinct intermediate phenotypes that differ in differentiation propensities and are favored in different environmental conditions. We identified epithelial cells that naturally exist in an intermediate state with bidirectional differentiation potential, and found the balance between EMT-promoting and -inhibiting factors to be critical in achieving and selecting between intermediate states. Our analysis suggests a new design principle in controlling cellular plasticity through multiple intermediate cell fates and underscores the critical involvement of Ovol2 and its associated molecular regulations.</p></div

Transcriptional Mechanisms Link Epithelial Plasticity to Adhesion and Differentiation of Epidermal Progenitor Cells

During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such a mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here, we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/Ovol2-deficient epidermal cells fail to undertake α-catenin-driven actin cytoskeletal reorganization and adhesive maturation and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations and defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-α-catenin sequential repression and highlight Ovol1 and Ovol2 as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity

Distinct differentiation propensities of the two intermediate states.

<p><b>A, B, D, E)</b> Stochastic simulations for a population of 5000 cells in four different conditions. Basal parameter set and intermediate external TGF-β concentration (0.5) were used (green star in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004569#pcbi.1004569.g006" target="_blank">Fig 6A</a>). <b>A)</b> Initial condition: I1; small fluctuations. <b>B)</b> Initial condition: I1; large fluctuations. <b>D)</b> Initial condition: I2; small fluctuations. <b>E)</b> Initial condition: I2; large fluctuations. <b>G, H)</b> Stochastic simulations for a population of 5000 cells initially at I1 state. Ovol2 basal production level was reduced by 20% from basal parameter. <b>J, K)</b> Stochastic simulations for 5000 cells initially at I2 state. Ovol2 basal production level was increased by 100% from basal parameter. <b>C, F, I, L)</b> Metaphoric energy landscapes (green curves) for I1 <b>(C)</b>, I2 <b>(F)</b> initial conditions, and reduced <b>(I)</b> or increased <b>(L)</b> Ovol2 basal expression rate. Orange circle represents the initial condition.</p

Roles of the Ovol2-Zeb1 mutual inhibition loop in the four-state EMT system.

<p>Comparison of the basal model (left column), reduced Ovol2-Zeb1 mutual inhibition (middle column), and blocked Ovol2-Zeb1 mutual inhibition (right column) on the four phenotypes. Each subplot is a two-parameter bifurcation diagram similar to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004569#pcbi.1004569.g006" target="_blank">Fig 6A</a>. Subplots in each column highlight the various phenotypes in one condition. Shaded areas are highlighted phenotypes. Colors of the shading correspond to the colored labels on the right.</p

Roles of EMT-promoting and -inhibiting factors in the four-state EMT system.

<p><b>A)</b> Two-parameter bifurcation diagram with respect to external TGF-β and Ovol2 basal production rate. The red curves were computed by extending the saddle-node bifurcation points obtained in one parameter bifurcation analysis (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004569#pcbi.1004569.s007" target="_blank">S4 Fig</a>), and they define different parameter regions that can be mono-stable, bi-stable, tri-stable or tetra-stable depending on the number of possible stable phenotypes (see labels), and each multi-stable region can be viewed as an area where multiple phenotypes co-exist (II-V). The size of each phenotype region is an indication of robustness of the phenotype when the two signals are varied. Green star: a basal parameter set and an intermediate TGF-β concentration that together give rise to four phenotypes. <b>B, C)</b> One-parameter bifurcation diagrams of Ecad with respect to external TGF-β (B) and Ovol2 basal production rate (C). Solid curve: stable steady state. Dashed curve: unstable steady state. A basal parameter set (blue) and a perturbed parameter set (orange) are compared in each plot. Triangles and diamonds denote the conditions under which both I1 and I2 are stable.</p

Time series of Ecad/Vim profile change upon Ovol2 expression in MDA-MB231 cells.

<p>Cells were infected with Ovol2-expressing lentivirus and Ecad/Vim profile was analyzed by flow cytometry at the indicated time points. Empty vector control at day 5 is shown at the top.</p

Roles of the miR34a-Snail and miR200-Zeb1 mutual inhibition loops in the four-state EMT system.

<p>Comparison of removing miR34a-Snail mutual inhibition (left column), miR200-Zeb1 mutual inhibition (middle column), or both (right column) on the four phenotypes. Each subplot is a two-parameter bifurcation diagram similar to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004569#pcbi.1004569.g006" target="_blank">Fig 6A</a>. Subplots in each column highlight the various phenotypes in one condition. Shaded areas are highlighted phenotypes. Colors of the shading correspond to the colored labels on the right.</p