Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes. Although our understanding of the role of Wnt signaling in regulating development and affecting disease, such as cancer, has been ever increasing, the study of the Wnt proteins themselves has been painstaking and slow moving. Despite advances in the biochemical characterization of Wnt proteins, many mysteries remain unsolved. In contrast to other developmental signaling molecules, such as fibroblast growth factors (FGF), transforming growth factors (TGFβ), and Sonic hedgehog (Shh), Wnt proteins have not conformed to many standard methods of protein production, such as bacterial overexpression, and analysis, such as ligand–receptor binding assays. The reasons for their recalcitrant nature are likely a consequence of the complex set of posttranslational modifications involving several highly specialized and poorly characterized processing enzymes. With the recent description of the first Wnt protein structure, the time is ripe to uncover and possibly resolve many of the remaining issues surrounding Wnt proteins and their interactions. Here we describe the process of maturation of Wnt from its initial translation to its eventual release from a cell and interactions in the extracellular environment.

Significance: Embryonic stem cells (ESCs) are unique in their ability to expand and self-renew indefinitely while retaining the potential to give rise to all mature cell types. The molecular mechanisms underlying these properties remain poorly understood. We investigated the role of the highly conserved WNT signaling pathway in controlling self-renewal and found that the WNT receptor encoded by the frizzled family receptor 7 (FZD7) gene is essential for maintaining human ESCs in an undifferentiated and pluripotent state. Using an FZD7-specific fragment antigen binding protein, as well as knockdown of FZD7 expression, we showed that the FZD7 receptor transduces a WNT/β-catenin signal in human ESCs. These data demonstrate that an endogenous WNT signaling loop is essential for the maintenance of human ESCs in an undifferentiated state.

Many cellular responses during development are regulated by interactions between integrin receptors and extracellular matrix proteins (ECMPs). Although the majority of recent studies in human embryonic stem cell (hESC) differentiation have focused on the role of growth factors, such as FGF, TGFβ, and WNT, relatively little is known about the role of ECMP-integrin signaling in this process. Moreover, current strategies to direct hESC differentiation into various lineages are inefficient and have yet to produce functionally mature cells in vitro. This suggests that additional factors, such as ECMPs, are required for the efficient differentiation of hESCs. Using a high-throughput multifactorial cellular array technology, we investigated the effect of hundreds of ECMP combinations and concentrations on differentiation of several hPSC lines to definitive endoderm (DE), an early embryonic cell population fated to give rise to internal organs such as the lung, liver, pancreas, stomach, and intestine. From this screen we identified fibronectin (FN) and vitronectin (VTN) as ECMP components that promoted DE differentiation. Analysis of integrin expression revealed that differentiation toward DE led to an increase in FN-binding integrin α5 (ITGA5) and VTN-binding integrin αV (ITGAV). Conditional short hairpin RNA-mediated knockdown of ITGA5 and ITGAV disrupted hESC differentiation toward DE. Finally, fluorescence-based cell sorting for ITGA5 and ITGAV significantly enriched cells with gene expression signatures associated with DE, demonstrating that these cell surface proteins permit isolation and enrichment of DE from hESCs. These data provide evidence that FN and VTN promote endoderm differentiation of hESCs through interaction with ITGA5 and ITGAV, and that ECMP-integrin interactions are required for hESC differentiation into functionally mature cells.

The WNT/β-catenin signaling pathway is a prominent player in many developmental processes, including gastrulation, anterior–posterior axis specification, organ and tissue development, and homeostasis. Here, we use human pluripotent stem cells (hPSCs) to study the dynamics of the transcriptional response to exogenous activation of the WNT pathway. We describe a mechanism involving the WNT target gene SP5 that leads to termination of the transcriptional program initiated by WNT signaling. Integration of gene expression profiles of wild-type and SP5 mutant cells with genome-wide SP5 binding events reveals that SP5 acts to diminish expression of genes previously activated by the WNT pathway. Furthermore, we show that activation of SP5 by WNT signaling is most robust in cells with developmental potential, such as stem cells. These findings indicate a mechanism by which the developmental WNT signaling pathway reins in expression of transcriptional programs.