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Molecular Controls of Animal Stem Cells and Differentiation
Summary: Judith Kimble studies the regulation of stem cells and differentiation in animals.
Stem cells generate tissues during the development of multicellular creatures and maintain those tissues in adults. Understanding stem cell regulation is not only fascinating in its own right but also crucial for regenerative medicine. My lab studies how stem cells are controlled to self-renew or differentiate in the small nematode Caenorhabditis elegans. We have identified key regulators of stem cells and differentiation that are conserved in vertebrates, including humans. We have also identified conserved pathways and mechanisms used for fundamental biological regulation more generally.
A Stem Cell Niche, Notch Signaling, and Regulation of a Stem Cell Pool
The concept of a stem cell niche was proposed in 1978 as an "external microenvironment" required to maintain stem cells. In 1981, we discovered that the mesenchymal distal tip cell (DTC) provides the niche for germline stem cells (GSCs) in C. elegans (Figure 1). A major goal of my laboratory has been to delineate the molecular network that regulates GSCs—including the niche signal and the germ cell response to self-renew or differentiate. Our early work revealed that the DTC niche uses Notch signaling to drive GSC self-renewal and that GSCs reside within a stem cell pool. The Notch pathway is broadly conserved among animals; its C. elegans version for GSC regulation is called GLP-1/Notch. More recently, others have found additional mesenchymal cells with niche function; Notch signaling has emerged as a key regulator of stem cells more broadly, albeit with varying roles; and the idea of a stem cell pool has gained traction in vertebrate tissues, including humans.
An RNA Regulatory Network Downstream of Notch Signaling
Over the past decade, we have discovered regulators that act within germ cells to drive self-renewal or differentiation and have forged biochemical links between those regulators to reveal a molecular network of control (for a highly simplified network, see Figure 2). Central to this network is the FBF hub (see below for how FBF works). The FBF/GLD axis of the network regulates whether germ cells self-renew or begin differentiation by entering the meiotic cell cycle; the FBF/FOG axis regulates whether germ cells self-renew or differentiate as sperm or oocyte. One remarkable feature of the network is that its key hubs control gene expression post-transcriptionally. For example, FBF, GLD-1, GLD-3, and FOG-1 are all conserved RNA-binding proteins, and GLD-2 is a conserved poly(A) polymerase. Another striking feature is that no individual component of the network is essential for self-renewal or differentiation; instead, removal of most single components simply shifts the balance between self-renewal and differentiation. Therefore, this RNA regulatory network is both robust and plastic.
FBF, A Conserved RNA-Binding Protein and Stem Cell Regulator
FBF (fem-3 binding factor) is a founding member of the conserved PUF (Pumilio and FBF) family. Indeed, FBF was one of the first PUF proteins shown to bind in a sequence-specific fashion to regulatory elements in the 3'-untranslated region (3'UTR) of its target mRNAs (Figure 3A). (Most FBF analyses have been done in collaboration with Marvin Wickens [University of Wisconsin–Madison].) We showed that FBF is a central regulator of GSC maintenance; others showed that PUF proteins regulate GSCs in fruit flies and neoblasts in planaria, or that puf-encoding RNAs are enriched in vertebrate stem cells. Therefore, PUF proteins appear to be broadly conserved stem cell regulators.
To learn what mRNAs are controlled by PUF proteins to maintain stem cells, we focused on FBF and identified >1,000 target mRNAs. One class of FBF target mRNAs encodes components of the meiotic program, either master regulators of meiotic entry or the meiotic cell cycle machinery. Another class encodes regulators of germ cell differentiation as either sperm or egg, and yet another class encodes more general differentiation regulators (e.g., mpk-1, the nematode homolog of ERK MAP kinase). A comparison of FBF target mRNAs to targets of its human counterparts, which are called PUM, reveals striking parallels—hundreds of mRNAs are shared. The shared targets include human counterparts of the core nematode network shown in Figure 2 (GLD-1, GLD-2, GLD-3, MPK-1, FOG-1, and FOG-3). ERK MAP kinase was validated as a conserved PUF target in both nematodes and human embryonic stem cells (collaboration with James A. Thomson [University of Wisconsin–Madison]). Therefore, not only are the PUF proteins conserved, but some of their regulatory relationships may also be ancient.
Conserved Mechanisms of PUF Repression and Implications for Stem Cell Control
Our early work showed that FBF represses the expression of its target mRNAs. More recently, we found evidence for two distinct molecular mechanisms of FBF repression. One involves recruitment of a deadenylase complex that removes poly(A) from target mRNAs (Figure 3B). This mechanism was discovered first for yeast and human PUF proteins and appears broadly conserved. The second mechanism, we first discovered using FBF and nematode proteins, and then showed conservation with human proteins. Briefly, PUF proteins (C. elegans FBF and human PUM2) form a ternary complex with an Argonaute protein (C. elegans CSR-1 and human AGO1–3) and a core component of the translational machinery, elongation factor eEF1A. Argonautes are best known for binding small RNAs and for their role in microRNA-mediated mRNA repression. The PUF-Ago-eEF1A complex appears to repress mRNAs by stalling ribosomes within the open reading frame at a site roughly 100–140 nucleotides from the AUG start site of translation (Figure 3C). We speculate that PUF-Ago-eEF1A may be the first of many multisubunit complexes that include core components of the translation machinery.
The major idea emerging from our FBF work is that PUF proteins maintain stem cells as broad-spectrum post-transcriptional repressors of differentiation. A challenge for the future is to learn whether the distinct PUF molecular mechanisms play specific roles within stem cells. An attractive model is that PUF repression employs these mechanisms in many organisms, perhaps in many tissues, to complement use of transcriptional regulators for stem cell and progenitor cell control.
Molecular Regulation of the Stem Cell Niche and Insights into the C. elegans Wnt Pathway
Understanding how stem cell niches are themselves regulated will enhance our ability to manipulate stem cells for regenerative medicine and to treat human disease. Yet molecular controls of niche specification and maintenance are largely unexplored. We tackled this fundamental problem in C. elegans, where the DTC niche remains an unusually simple and well-defined niche. First, we learned that the niche fate is specified by Wnt pathway activation of a gene encoding the CEH-22/tinman/Nkx2.5 homeodomain transcription factor. An intriguing but speculative parallel is the role of Nkx2.5 in development of the vasculature, which serves as part of the niche for neural and hematopoietic cells in vertebrates. Second, we discovered a functional β-catenin not recognizable by its amino acid sequence, a finding that expands the definition of β-catenins and suggests that other divergent β-catenins await discovery. Third, our work, together with that of others, revealed that the major C. elegans Wnt pathway is branched—one branch up-regulates β-catenin and the other down-regulates T cell factor (TCF). We next showed that this branched pathway controls the ratio of β-catenin to TCF and that it is this ratio that drives Wnt-dependent transcriptional activation. If vertebrates similarly rely on the ratio of β-catenin to TCF, then pharmaceutical lowering of TCF would activate Wnt signaling, a prediction with important clinical implications.
The Sperm/Egg Decision and Its Chemical Reprogramming
Differentiation of a germ cell as sperm or egg is a fundamental cell fate decision. We have begun to understand, for the first time in any organism, the mechanism that acts within germ cells to control the sperm/egg fate decision. One unexpected outcome of knowing these molecular regulators has been the discovery that adult germ cells are sexually labile—manipulation of key regulators can sexually transform the adult germline from spermatogenesis to oogenesis, or vice versa. Therefore, these regulators must remain active in adults to continue specifying germ cells as sperm or oocyte during the continuous production of gametes.
To provide a potentially general means to understand cell fate reprogramming, we have used small molecules to reprogram germ cells. This work arose from understanding that ERK/MAP kinase is one of the regulators that affects the sperm/egg decision. We added chemical inhibitors of the ERK/MAP kinase pathway to a spermatogenic animal and sexually transformed its germline to make oocytes that appeared normal and could produce viable offspring (Figure 4). This reprogramming of germ cell fates in C. elegans provides a powerful model for analysis of molecular mechanisms of in vivo reprogramming. In addition, our findings provide a paradigm that may facilitate pharmacological approaches to therapeutic cellular reprogramming in other organisms, including humans.
A grant from the National Institutes of Health provided support for some of these studies on germline controls.
As of May 30, 2012
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