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Molecular Controls of Germline Development and Organogenesis
Summary: Judith Kimble studies controls of growth, differentiation, and morphogenesis in animal tissues.
Animal development is a remarkable feat of biological regulation. How are cells, tissues, and organs governed to reach their proper size, shape, and pattern of differentiated cells? We have embarked on an in-depth analysis of a single organ, the gonad, to learn how controls of growth, patterning, and morphogenesis are integrated to generate a complex biological structure. Using the nematode Caenorhabditis elegans, we have uncovered genes, proteins, and pathways fundamental to the development of all animals, including humans.
A Single-Celled Stem Cell Niche and Its Control
Stem cells are specialized for the production of both more stem cells and more differentiated cells. The stem cell niche is an external microenvironment that nurtures and maintains stem cells. Our early work identified a single-celled niche, the distal tip cell (DTC; Figure 1). More recently, we have investigated controls of the DTC itself, as a model for understanding mechanisms governing both niche size and strength.
We have found that the Wnt pathway and a specific Wnt target gene, which encodes the CEH-22/tinman/Nkx2.5 homeodomain transcription factor, specify the DTC niche fate. Ectopic CEH-22 makes extra DTCs and extra niches, while reduced CEH-22 has the opposite effect. We have also found that a helix-loop-helix transcription factor governs maintenance of the DTC niche function. Understanding how the size and strength of a niche is controlled in nematodes may enhance our ability to manipulate stem cells for regenerative medicine and to treat human diseases that result from stem cell defects, including cancer.
A Molecular Network Controls the Balance Between Germline Self-Renewal and Early Differentiation
The balance between self-renewal and differentiation is critical for both growth and maintenance of tissues. Our early work demonstrated that Notch signaling from the DTC is essential for germline self-renewal and prevents early differentiation. More recently, we have investigated factors that act downstream of Notch signaling and within germ cells to control their ability to self-renew or enter meiosis. Our work has delineated a regulatory network that controls this critical balance (Figure 2). In this network, each protein and regulatory link has been analyzed genetically and biochemically, mostly in our lab. The network is robust, plastic, and relies almost entirely on RNA regulators. Our current work explores the dynamics of this network as it transitions from a self-renewal state to a differentiated state, examines the roles of individual components in its balance between self-renewal and differentiation, and investigates network modulations that may provide insight into its evolvability.
FBF, a Conserved Regulator of Stem Cell Maintenance
We originally identified FBF as a regulator of the sperm/oocyte decision but soon discovered that it is also essential for germline self-renewal. (FBF identification and biochemistry were done in collaboration with Marvin Wickens [University of Wisconsin–Madison].) PUF (Pumilio and FBF) RNA-binding proteins are ancient and broadly used regulators of stem cell maintenance. Our recent work focuses on FBF as a model for how PUF proteins control stem cell maintenance. We have found that the fbf-2 gene is a direct target of Notch signaling, a finding that forges a direct molecular link between Notch signaling and the RNA regulatory network that acts within germ cells.
We have also found that FBF proteins promote germline self-renewal by repressing meiotic entry and differentiation. FBF binds specifically to regulatory elements in the 3'-untranslated region (3'UTR) of target mRNAs and represses their translation or stability. Among many FBF target mRNAs identified to date (Figure 2), mpk-1 encodes the nematode ERK/MAP kinase, a conserved regulator of differentiation. Remarkably, human PUM2 also binds specifically to the human ERK/MAP kinase 3'UTR and controls its expression in human embryonic stem (hES) cells. (The hES analyses were done in collaboration with James Thomson [University of Wisconsin–Madison].) The parallels are striking. Both key regulators and regulatory links have been conserved, suggesting that at least some features of the nematode stem cell regulatory network are ancient.
Integration of the Mitosis/Meiosis and Sperm/Oocyte Decisions
Animal germ cells enter the meiotic cell cycle and differentiate as either sperm or oocytes. Controls of meiotic entry and germline sex determination are linked in most animals, including mammals, but the nature of that link has been elusive. Our work demonstrates that, in the nematode, all major regulators of the mitosis/meiosis decision also govern the sperm/oocyte decision, and vice versa. Examples include FOG-1, which we originally identified as a sperm fate regulator, and GLD-2, which we originally identified as a regulator of meiotic entry.
FOG-1 is a homolog of Xenopus CPEB (cytoplasmic polyadenylation element–binding) protein, a conserved regulator of mRNA translation. We have learned that FOG-1 acts in a dose-dependent fashion: low FOG-1 promotes mitotic divisions, and high FOG-1 drives germ cells into the sperm fate. Remarkably, Xenopus CPEB is also dose-dependent, promoting mitotic divisions when scarce and meiotic maturation when abundant. This striking parallel suggests that a FOG-1/CPEB gradient may be used widely for patterning growth and differentiation.
GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that adds poly(A) tails to target mRNAs and activates their translation. We identified two distinct RNA-binding proteins, GLD-3 and RNP-8, that bind GLD-2 and stimulate its PAP activity. GLD-3 and RNP-8 compete with each other for GLD-2 binding and antagonize each other biologically. GLD-3 promotes the sperm fate and meiotic entry, while RNP-8 promotes the oocyte fate. Indeed, GLD-2 appears to be a fate-neutral enzyme that is biased toward directing specific germ cell fates by the RNA-binding specificity of its partner.
New Insights into the C. elegans Wnt Pathway
The Wnt pathway controls asymmetric cell divisions during C. elegans development. Many components are conserved in the C. elegans and canonical Wnt pathways, but when we began our studies, the C. elegans pathway lacked one of the most important pieces. We found that missing bit, the SYS-1 protein, which is a highly divergent β-catenin transcriptional coactivator and essential for the pathway. SYS-1 not only functions genetically and biochemically as a β-catenin but also shares critical structural features with β-catenins. (The SYS-1 structure was solved in collaboration with Wenqing Xu [University of Washington].) Our analysis of SYS-1 expands the definition of β-catenins and suggests that other divergent β-catenins await discovery.
The Wnt pathway activates transcription of target genes via two dedicated transcription factors, SYS-1/β-catenin and POP-1, the C. elegans TCF DNA-binding protein. By manipulating SYS-1 and POP-1, both genetically and molecularly, we learned that the ratio of these two factors determines their transcriptional activity. We also discovered that the Wnt pathway up-regulates SYS-1; others had previously found that the pathway down-regulates POP-1. Putting our work together with previous studies, we have learned that the C. elegans Wnt pathway is forked (Figure 3) and regulates the SYS-1 to POP-1 ratio to activate or repress downstream genes.
Regulation of Organ Sexual Dimorphism and Polarity
Animal gonads are dimorphic organs with distinct structures and tissues in each sex. The study of gonadogenesis, therefore, provides an extraordinary inroad into the problem of how organogenesis controls can be modulated to generate diverse structures. Our focus has been on a sexually dimorphic asymmetric division and its generation of sex-specific regulatory cells that control organ polarities and niches. We have identified transcription factors that promote the male or female mode and a conserved cell cycle regulator, known as cyclin D, that coordinates activities of these transcription factors with the cell cycle at this pivotal juncture of animal development. This work has identified both gender-neutral and sex-specific regulators of the niche and has recently uncovered a sex-specific regulator of organ polarity.
A grant from the National Institutes of Health provided support for some of these studies on germline controls.
Last updated December 03, 2008
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