A higher order PUF complex is central to regulation of C. elegans germline stem cells.

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including gld-1. Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses gld-1 via a pair of adjacent FBF binding elements (FBEs) in its 3'UTR.

Mutation of GLP-1/Notch RAM domain results in a strong Glp-1 phenotype.

The distal tip cell niche uses GLP-1 /Notch signaling to maintain germline stem cells. The RAM domain, which resides within the intracellular portion of the GLP-1 /Notch receptor, is integral to formation of a signaling-dependent transcription activation complex. Here we report the generation of a mutation in the GLP-1 RAM domain, created in a GLP-1 /Notch receptor with a C-terminal V5 tag.

The PUF RNA-binding protein, FBF-2, maintains stem cells without binding to RNA.

Like all canonical PUF proteins, FBF-2 binds to specific RNAs via tripartite recognition motifs (TRMs). Here we report that an FBF-2 mutant protein that cannot bind to RNA, is nonetheless biologically active and maintains stem cells. This unexpected result challenges the conventional wisdom that RBPs must bind to RNAs to achieve biological activity.

A higher order PUF complex is central to regulation of germline stem cells.

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including . Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses via a pair of adjacent FBF-2 binding elements (FBEs) in its 3ÚTR.

PUF partner interactions at a conserved interface shape the RNA-binding landscape and cell fate in Caenorhabditis elegans.

Protein-RNA regulatory networks underpin much of biology. C. elegans FBF-2, a PUF-RNA-binding protein, binds over 1,000 RNAs to govern stem cells and differentiation.

LST-1 is a bifunctional regulator that feeds back on Notch-dependent transcription to regulate germline stem cells.

Notch signaling regulates stem cells across animal phylogeny. Notch signaling activates transcription of two genes, and , that encode potent regulators of germline stem cells. The LST-1 protein regulates stem cells in two distinct ways: It promotes self-renewal posttranscriptionally and also restricts self-renewal by a poorly understood mechanism.

Analysis of the C. elegans Germline Stem Cell Pool.

The Caenorhabditis elegans germline is an excellent model for studying the genetic and molecular regulation of stem cell self-renewal and progression of cells from a stem cell state to a differentiated state. The germline tissue is organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated gametes at the other. A simple mesenchymal niche caps the GSC pool and maintains GSCs in an undifferentiated state by signaling through the conserved Notch pathway.

The in vivo functional significance of PUF hub partnerships in C. elegans germline stem cells.

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of Caenorhabditis elegans germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we previously proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy.

Functional significance of PUF partnerships in germline stem cells.

Unlabelled: PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy.

Image-Based Single-Molecule Analysis of Notch-Dependent Transcription in Its Natural Context.

Notch signaling is crucial to animal development and homeostasis. Notch triggers the transcription of its target genes, which produce diverse outcomes depending on context. The high resolution and spatially precise assessment of Notch-dependent transcription is essential for understanding how Notch operates normally in its native context in vivo and how Notch defects lead to pathogenesis.

C. elegans germ granules require both assembly and localized regulators for mRNA repression.

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function.

Sexual dimorphism of niche architecture and regulation of the germline stem cell pool.

Stem cell maintenance by niche signaling is a common theme across phylogeny. In the gonad, the broad outlines of germline stem cell (GSC) regulation are the same for both sexes: GLP-1/Notch signaling from the mesenchymal distal tip cell niche maintains GSCs in the distal gonad of both sexes and does so via two key stem cell regulators, SYGL-1 and LST-1. Yet most recent analyses of niche signaling and GSC regulation have focused on XX hermaphrodites, an essentially female sex making sperm in larvae and oocytes in adults.

Single-molecule RNA Fluorescence Hybridization (smFISH) in .

Single-molecule RNA fluorescence hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj , 2008 ; Lee , 2016a ). We adapted this technique to visualize RNAs in the whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee , 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for extruded gonads.

Analysis of the C. elegans Germline Stem Cell Pool.

The Caenorhabditis elegans germline is an excellent model for studying the regulation of a pool of stem cells and progression of cells from a stem cell state to a differentiated state. At the tissue level, the germline is organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated cells at the other. A simple mesenchymal niche caps the GSC region of the germline and maintains GSCs in an undifferentiated state by signaling through the conserved Notch pathway.

The PUF binding landscape in metazoan germ cells.

PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells.

A DTC niche plexus surrounds the germline stem cell pool in Caenorhabditis elegans.

The mesenchymal distal tip cell (DTC) provides the niche for Caenorhabditis elegans germline stem cells (GSCs). The DTC has a complex cellular architecture: its cell body caps the distal gonadal end and contacts germ cells extensively, but it also includes multiple cellular processes that extend along the germline tube and intercalate between germ cells. Here we use the lag-2 DTC promoter to drive expression of myristoylated GFP, which highlights DTC membranes and permits a more detailed view of DTC architecture.

Germline stem cells and their regulation in the nematode Caenorhabditis elegans.

C. elegans germline stem cells exist within a stem cell pool that is maintained by a single-celled mesenchymal niche and Notch signaling. Downstream of Notch signaling, a regulatory network governs stem cells and differentiation.

The C. elegans adult male germline: stem cells and sexual dimorphism.

The hermaphrodite Caenorhabditis elegans germline has become a classic model for stem cell regulation, but the male C. elegans germline has been largely neglected. This work provides a cellular analysis of the adult C.

Progression from a stem cell-like state to early differentiation in the C. elegans germ line.

Controls of stem cell maintenance and early differentiation are known in several systems. However, the progression from stem cell self-renewal to overt signs of early differentiation is a poorly understood but important problem in stem cell biology. The Caenorhabditis elegans germ line provides a genetically defined model for studying that progression.

FBF and its dual control of gld-1 expression in the Caenorhabditis elegans germline.

FBF, a PUF RNA-binding protein, is a key regulator of the mitosis/meiosis decision in the Caenorhabditis elegans germline. Genetically, FBF has a dual role in this decision: it maintains germ cells in mitosis, but it also facilitates entry into meiosis. In this article, we explore the molecular basis of that dual role.

Analysis of the C. elegans germline stem cell region.

We present methods for characterizing the mitotic and early meiotic regions of the Caenorhabditis elegans germline. The methods include examination of germlines in living and fixed worms, cell cycle analysis, analysis of markers, and initial characterization of mutants that affect germline proliferation.

Germline proliferation and its control.

The C. elegans germ line proliferates from one primordial germ cell (PGC) set aside in the early embryo to over a thousand cells in the adult. Most germline proliferation is controlled by the somatic distal tip cell, which provides a stem cell niche at the distal end of the adult gonad.

Controls of germline stem cells, entry into meiosis, and the sperm/oocyte decision in Caenorhabditis elegans.

The Caenorhabditis elegans germ line provides an exceptional model for analysis of the molecular controls governing stem cell maintenance, the cell cycle transition from mitosis to meiosis, and the choice of sexual identity-sperm or oocyte. Germline stem cells are maintained in an undifferentiated state within a well-defined niche formed by a single somatic cell, the distal tip cell (DTC). In both sexes, the DTC employs GLP-1/Notch signaling and FBF/PUF RNA-binding proteins to maintain stem cells and promote mitotic divisions, three additional RNA regulators (GLD-1/quaking, GLD-2/poly(A) polymerase, and GLD-3/Bicaudal-C) control entry into meiosis, and FOG-1/CPEB and FOG-3/Tob proteins govern sperm specification.

Cellular analyses of the mitotic region in the Caenorhabditis elegans adult germ line.

The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions.