Bigh3 Is Upregulated in Regenerating Zebrafish Fin

Zebrafish is a good model for studying regeneration because of the rapidity with which it occurs. Better understanding of this process may lead in the future to improvement of the regenerating capacity of humans. Signaling factors are the second largest category of genes, regulated during regeneration after the regulators of wound healing. Major developmental signaling pathways play a role in this multistep process, such as Bmp, Fgf, Notch, retinoic acid, Shh, and Wnt. In the present study, we focus on TGF-beta-induced genes, bigh3 and bambia. Bigh3 encodes keratoepithelin, a protein first identified as an extracellular matrix protein reported to play a role in cell adhesion, as well as in cornea formation and osteogenesis. The expression of bigh3 in zebrafish fins has previously been reported. Here we demonstrate that tgf-b1 and tgf-b3 mRNA reacted with delay, first showing no regulation at 3 dpa, followed by upregulation at 4 and 5 dpa. Tgf-b1, tgf-2, and tgf-brII mRNA were back to normal levels at 10 dpa. Only tgf-b3 mRNA was still upregulated at that time. Bigh3 mRNA followed the upregulation of tgf-b1, while bambia mRNA behaved similarly to tgf-b2 mRNA. We show that upregulation of bigh3 and bambia mRNA correlated with the process of fin regeneration and regulation of TGF-b signaling, suggesting a new role for these proteins.

Role of Bcl9 and Bcl9l in Homeostasis, Regeneration and Tumorigenesis of the Gastrointestinal Epithelium

Norbert Wiedemann

Wnt signaling is essential for embryogenesis and adult tissue homeostasis and contributes to cancer development, especially in the colon. A new essential pathway component, legless, was identified in Drosophila melanogaster. Mice with Cre/loxP-mediated conditional ablation of the legless homologues Bcl9 and Bcl9l in the intestinal epithelium were generated to assess the contribution of the genes to Wnt signaling and their potential suitability as therapeutic drug targets. Initial analysis had revealed that both genes were not required for self-renewal of the intestinal epithelium during homeostasis, but for regeneration of experimentally induced colon wounds. Furthermore, formation of chemically induced colon tumors was not impaired and Bcl9/Bcl9-mutant tumors were morphologically indistinguishable from wild-type tumors and displayed hallmarks of active Wnt signaling. This study aimed at further investigating the molecular processes involving Bcl9 and Bcl9l in wound regeneration and tumorigenesis in the colon, and in particular relatedness of the genes to Wnt signaling. To this end, transcriptomes of laser microdissected wound regions were analyzed by microarray-based gene expression profiling and realtime quantitative PCR, but did not reveal significant changes in gene expression at the time point chosen for analysis. However, expression profiling of homeostatic colon epithelium revealed a reduced expression of several intestinal stem cell marker genes in Bcl9/Bcl9l-mutant colon epithelium, suggesting that the regeneration deficit has been caused by functional impairment of intestinal stem cells. This stem cell deficiency did not differ between young and old mice and was also weakly detectable in small intestinal epithelium. To verify that tumors originated from cells in which complete deletion of all four Bcl9 and Bcl9l alleles had occurred and to assess Wnt signaling activity more comprehensively, laser microdissected tumor regions were profiled using microarrays and realtime quantitative PCR. This analysis confirmed that Bcl9 and Bcl9l were not required for colon tumorigenesis and revealed that despite the morphological similarity, transcriptional profiles of Bcl9/Bcl9l-mutant tumors were very different from wild-type tumors. Notably, loss of Bcl9 and Bcl9l caused reduced expression of a subset of direct Wnt target genes, genes related to epithelial-mesenchymal transition (EMT) and intestinal stem cell traits. While wild-type tumor epithelium appeared to be in a metastable state, characterized by co-expression of epithelial and mesenchymal traits, Bcl9/Bcl9l-mutant tumor epithelium predominantly expressed epithelial traits. In addition, a marked reduction in expression of several genes associated with malignant tumor properties was observed in Bcl9/Bcl9l-mutant tumors. This was highlighted by the preservation of an intact basement membrane around Bcl9/Bcl9l-mutant tumor epithelium, which was absent in wild-type tumors, and indicated that loss of Bcl9/Bcl9l decreased tumor aggressiveness. Of note, EMT and stemness traits are correlated to tumor resistance to therapy and thus interfering with Bcl9/Bcl9l function might restore drug responsiveness. As a proof of concept, it was shown that the malignant traits mediated by Bcl9/Bcl9l could be abrogated in wild-type tumors by inducing deletion of the genes. These findings suggest that targeting Bcl9 and Bcl9l with therapeutic drugs has the potential to attenuate the malignancy of colon tumors.

EPFL2011

Inhibition of Fibroblast Growth by Notch1 Signaling Is Mediated by Induction of Wnt11-Dependent WISP-1

Yan Li, Freddy Radtke, Yurong Tan, Jialin Zhang

Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox)) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1(-/-)) MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox) MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

Public Library of Science2012

Novel interventions to recover the regenerative capacity of aged skeletal muscle by targeting the interactions in the stem cell niche

Laura Lukjanenko

The remarkable ability of skeletal muscle to regenerate upon injury is conferred by tissue-resident stem cells called satellite cells. With age, the regenerative capacity of muscle stem cells (MuSCs) dramatically declines. Developing strategies to enhance muscle repair in elderly people is therefore required; in particular to accelerate their recovery from injuries following falls or from surgical interventions affecting muscle tissues. As the causes of MuSC dysfunction with age are multi-systemic, we decided to dissect age-related changes at different levels of the MuSC environment, in order to uncover synergistic ways to restore their regenerative capacities. This thesis describes three major interventions at the extracellular matrix, cell-cell interaction and tissue/systemic level that successfully restored skeletal muscle regeneration in aged mice. Using a proteomic screen, we identified fibronectin as a structural and signaling molecule of the extracellular matrix that is lost in the aged muscle niche. Loss of fibronectin in aged regenerating muscle primarily arises from perturbed cellular turnover of hematopoietic and endothelial cells which were shown to be the major fibronectin producers in muscle upon injury. Our work also uncovered that the function of old MuSCs is impaired by loss of adhesion and cell death by anoikis, and can be rescued by fibronectin treatment both ex vivo and in vivo. At the molecular level, loss of fibronectin is an upstream trigger leading to perturbed MuSC signaling. Fibronectin treatment rescues perturbations of pathways, such as p38 and ERK, that were previously known to be altered in aged MuSCs, as well as the newly discovered age-related perturbations of FAK signaling. Altogether we demonstrated that aging impairs the remodeling of the extracellular matrix of the MuSC niche, and thereby triggers multiple dysfunction of old MuSCs that can be rescued therapeutically. In a second project, we dissected the cellular cross-talk between MuSCs and non-myogenic support cells called Fibro-Adipogenic Progenitors (FAPs). We uncovered that the adipogenic fate of FAPs is tightly correlated to the muscle micro-environment and the myogenic regenerative capacity in different models of regeneration and aging. The function of FAPs and their cross-talk with MuSCs are impaired with age. We identified the secreted protein WISP1 as a paracrine communication factor between FAPs and MuSCs which is perturbed with age. Treating aged mice with WISP1 restored stem cell function and muscle regeneration, highlighting the possibility to target the cross-talk of MuSC with other cells of the niche to ameliorate their function. Our last project identified altered signaling of the small circulating peptide apelin in aged MuSCs through lowered circulating levels of apelin and down-regulation of its receptor APJ in aged MuSCs. We demonstrated that apelin treatment rescued muscle stem cell function and regeneration in aged mice, highlighting that MuSC dysfunction can also be targeted systemically. Taken together, these approaches reveal the multiple possibilities to ameliorate muscle repair by targeting MuSC interactions with their niche. Our results also pave the way to develop integrated therapeutic strategies to boost old MuSC function.

EPFL2016