Haupt KA et al. (2019) Genetics "A PUF Hub Drives Self-Renewal in Caenorhabditis elegans Germline Stem Cells."

Law KT, Kimble J, Haupt KA, Wickens M, Kanzler CR, Enright AL, Shin H

[Genetics, 2019]

Stem cell regulation relies on extrinsic signaling from a niche plus intrinsic factors that respond and drive self-renewal within stem cells. A <i>priori,</i> loss of niche signaling and loss of the intrinsic self-renewal factors might be expected to have equivalent stem cell defects. Yet this simple prediction has not been borne out for most stem cells, including <i>Caenorhabditis elegans</i> germline stem cells (GSCs). The central regulators of <i>C. elegans</i> GSCs include extrinsically-acting GLP-1/Notch signaling from the niche, intrinsically-acting RNA binding proteins in the PUF family, termed FBF-1 and FBF-2 (collectively FBF), and intrinsically-acting PUF partner proteins that are direct Notch targets. Abrogation of either GLP-1/Notch signaling or its targets yields an earlier and more severe GSC defect than loss of FBF-1 and FBF-2, suggesting that additional intrinsic regulators must exist. Here, we report that those missing regulators are two additional PUF proteins, PUF-3 and PUF-11. Remarkably, an <i>fbf-1 fbf-2; puf-3 puf-11</i> quadruple null mutant has a GSC defect virtually identical to that of a <i>glp-1/</i>Notch null mutant. PUF-3 and PUF-11 both affect GSC maintenance; both are expressed in GSCs; and epistasis experiments place them at the same position as FBF within the network. Therefore, action of PUF-3 and PUF-11 explains the milder GSC defect in <i>fbf-1 fbf-2</i> mutants. We conclude that a &quot;PUF hub&quot;, comprising four PUF proteins and two PUF partners, constitutes the intrinsic self-renewal node of the <i>C. elegans</i> GSC RNA regulatory network. Discovery of this hub underscores the significance of PUF RNA-binding proteins as key regulators of stem cell maintenance.

Haupt KA et al. (2019) Development "The molecular basis of LST-1 self-renewal activity and its control of stem cell pool size."

Haupt KA, Wickens M, Ferdous AS, Shin H, Kershner AM, Enright AL, Kimble J

[Development, 2019]

PUF RNA-binding proteins have diverse roles in animal development, with a broadly conserved role in stem cells. Two paradigmatic PUF proteins, FBF-1 and FBF-2, promote both self-renewal and differentiation in the <i>C. elegans</i> germline. The LST-1 protein is a pivotal regulator of self-renewal and oncogenic when mis-expressed. Here we demonstrate that LST-1 self-renewal activity resides within a predicted disordered region that harbors two KXXL motifs. We find that the KXXL motifs mediate LST-1 binding to FBF, and that point mutations of these motifs abrogate LST-1 self-renewal activity. The LST-1-FBF partnership is therefore critical to stem cell maintenance and a key element in the FBF regulatory network. A distinct region within LST-1 determines its spatial expression and size of the GSC pool. Most importantly, the molecular understanding of how an IDR-rich protein works in an essential partnership with a conserved stem cell regulator and RNA-binding protein suggests broad new avenues for combinatorial control.

Haupt, K.A. et al. (2019) International Worm Meeting "A four-part PUF hub is responsible for germline stem cell self-renewal throughout C. elegans development."

Law, K., Wickens, M., Shin, H., Haupt, K.A., Kimble, J.

[International Worm Meeting, 2019]

Genetic redundancy is common in metazoans, especially in developmental processes. Not only do redundant regulators lend robustness to critical pathways, they can also facilitate rapid adjustments to environmental change (Kafri et al, 2009). Using highly redundant pathways, C. elegans germline stem cells (GSCs) generate the germline tissue during larval development and maintain it during adulthood and reproduction. Our work reports additional redundancy underlying the control of GSCs. C. elegans GSC maintenance requires glp-1/Notch signaling from the stem cell niche. In the absence of niche signaling both sexes make only 4-8 germ cells, called the Glp-1 phenotype (Austin & Kimble, 1987). Downstream of glp-1/Notch signaling, two PUF RNA-binding proteins, fbf-1 and fbf-2, are also important for GSCs. However, this cannot be the whole story: fbf-1 fbf-2 mutants make ~100 germ cells (Crittenden et al, 2002). We hypothesized that FBF might function redundantly with other PUF proteins such as the nearly identical puf-3 and puf-11 genes. We asked whether these genes could enhance fbf-1 fbf-2 GSC defects. Strikingly, an fbf-1 fbf-2; puf-3 puf-11 quadruple mutant has an essentially Glp-1 phenotype in both hermaphrodites and males. Subsequent genetic epistasis experiments place puf-3 puf-11 in the self-renewal pathway parallel to fbf-1 fbf-2, downstream of glp-1/Notch signaling and upstream of the gld genes. In addition, we epitope tagged PUF-3 and PUF-11 and found them expressed in GSCs. We conclude that puf-3 and puf-11 are GSC regulators that together with fbf-1 fbf-2 comprise a four-part PUF hub responsible for GSC maintenance in both sexes. Finally, we considered the biological implication of this redundancy for GSC regulation. While the fbf-1 fbf-2 GSC phenotype is temperature-sensitive (most severe at 15 deg C and least severe at 25 deg C) (Merritt & Seydoux, 2010), we find that puf-3 puf-11 enhance fbf-1 fbf-2 to essentially Glp-1 at 15, 20 and 25 deg C. We conclude that fbf-1 fbf-2 are more important at 15 deg C while puf-3 puf-11 have a larger role at 25 deg C, and that this redundancy serves to buffer the GSCs against environmental changes.

Wright KA et al. (1981) Canadian Journal of Zoology "The buccal capsule of C. elegans (Nematoda: Rhabditoidea): An ultrastructural study."

Wright KA, Thomson JN

[Canadian Journal of Zoology, 1981]

The buccal cavity of the free-living nematode Caenorhabditis elegans has been analysed by serial section electron microscopy. Whereas the regions classically identified in the rhabditid buccal capsule can be distinguished, the cuticle lining does not constitute separate cuticular plates, but rather, structural-functional differentiations within a cuticle continuous with that of the esophagus. Only the lip region (cheilostom) is lined by body wall cuticle. The prostom cuticle is underlain by two rings of syncytial arcade cytoplasm connected to nine cell bodies. The mesostom cuticle is underlain by the nonmuscular epithelial cells of the esophagus, whereas the cuticle of the metastom and telostom is underlain by esophageal muscle cells m1 and m2. During moulting, buccal cuticle is produced later than body cuticle and its formation is characterized by accumulation of dense granules in both arcade and esophageal cytoplasm. It is concluded that the buccal capsule should be considered as "astomatous" in the terminology of K.A.

Cole DG et al. (1998) J Cell Biol "Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons."

Cole DG, Rosenbaum JL, Himelblau AL, Diener DR, Beech PL, Fuster JC

[J Cell Biol, 1998]

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979-992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.

Hall, David H. et al. (2009) International Worm Meeting "Electron tomography: a new tool for exploring C. elegans cellular anatomy."

Fisher, Kevin, Nguyen, Ken C.Q., Hall, David H., Crocker, Chris, Derr, KD, Rice, William J., Politi, Kristin A., Gunther, Leslie

[International Worm Meeting, 2009]

C. elegans has been studied intensively using serial section reconstruction for several decades, so that every cell type is now known in considerable detail. Nevertheless, limitations of the serial section technique (notably the poor resolution of detail within the depth of each 50 nm "thin" section) have made it difficult to model the shapes of fine details in many organelles, such as the cristae of a mitochondrion, the canaliculi of the excretory canal, membrane ruffles in many cell types, or the constituents at a chemical synapse. High pressure freeze fixation and freeze substitution (1) are a required element in preserving smaller structures that generally escaped our notice in conventional TEM imaging. Modern electron microscopes using higher energy electrons now offer much better resolution by collecting multiple images in a tilting series through comparatively thick sections (150 nm) using the SerialEM program (2). The Protomo software package (3) is used to compute a 3D tomographic reconstruction that offers the same level of detail in any dimension (roughly 2 nm resolution). Annotation and modeling is done using IMOD (4) and/or Amira. This permits us to take a new look at many familiar objects in the anatomy of C. elegans, to identify missing parts of the whole anatomy, and potentially, to detect smaller anatomical defects in a variety of mutant backgrounds. Here we will introduce the tomographic procedure, and share finished 3D models of some typical intracellular organelles at high resolution. Supported by NIH RR 12596 to DHH. 1.Weimer, R.M. (2006) Methods Mol. Biol. 351: 203-221. 2.Mastronarde, D.N. (2005) J. Struct. Biol. 152: 36-51 3.Winkler, H. and Taylor, K.A. (2006) Ultramicroscopy 106: 240-254. 4.Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) J. Struct. Biol. 116: 71-76.

Chris R Gissendanner et al. (2002) Mid-west Worm Meeting "NHR-6: a nuclear receptor transcription factor required for ovulation."

Tri Q Nguyen, Claude V Maina, Chris R Gissendanner, Marius Hoener, Jianping Xiao, Ann E Sluder

[Mid-west Worm Meeting, 2002]

The C. elegans genome encodes greater than 270 nuclear receptor (NR) genes (Sluder et al, 1999). Among these are fifteen genes that, based on comparative sequence analysis, encode nuclear receptor proteins conserved across metazoan phyla (Sluder et al, 1999). One of these conserved NR genes is nhr-6, which encodes the only C. elegans member of the NGFI-B NR sub-family (Wilson et al, 1992; Kostrouch et al, 1995; Sluder et al, 1999). We have initiated a functional analysis of nhr-6. Data from RNAi experiments and analysis of the deletion mutant lg6001 have established a role for nhr-6 in gonad development and/or function. Specifically, nhr-6(RNAi) and nhr-6(lg6001) animals have extremely low brood sizes and lay abnormally shaped eggs. Further analysis demonstrated that these phenotypes were due to an ovulation defect. Oocytes undergoing ovulation in lg6001 hermaphrodites frequently fragment entering the spermatheca. Only a fraction of the oocyte fragments become fertilized and many of these fail to develop properly. Oocyte fragments that remain in the gonad can become endomitotic (Emo phenotype). An nhr-6::GFP transgene is expressed in the spermatheca, suggesting a role for nhr-6 in spermathecal development and/or function. Currently, our analysis of nhr-6 is focused on the following: 1) a detailed characterization of nhr-6 expression and the ovulation defect observed in lg6001 mutants; 2) the in vivo significance of a conserved Akt/PKB phosphorylation site within the DNA binding domain of NHR-6; and 3) a comparative biochemical analysis of NHR-6 and an NGFI-B homolog (DiNHR-2) from the filarial nematode Dirofilaria immitis. The results of these investigations will be presented. Wilson, T.E., Paulsen, R.E., Padgett, K.A., and Milbrandt, J. (1992). Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors. Science, Vol. 256(5053), 107-10.

Crittenden, S.L. et al. (2019) International Worm Meeting "Sexual dimorphism of niche architecture and regulation of the C. elegans germline stem cell pool."

Knobel, K., Lee, C., Battula, S., Mohanty, I., Crittenden, S.L., Kimble, J.

[International Worm Meeting, 2019]

The fundamentals of C. elegans germline stem cell (GSC) molecular regulation are the same in both sexes: GLP-1/Notch signaling from the mesenchymal niche, called the Distal Tip Cell (DTC), maintains GSCs in the distal gonad via transcriptional activation of two genes that encode key stem cell regulators, SYGL-1 and LST-1 (1, 2). Detailed analyses of niche architecture and GSC regulation have been done in hermaphrodites and revealed the extent of cellular contacts between the hermaphrodite DTC (hDTC) and GSCs (3,4), size of the hermaphrodite GSC pool (5), extent and shape of Notch-dependent transcriptional activation in hermaphrodite GSCs (4), and extent of key stem cell regulatory proteins expressed in response to niche signaling in hermaphrodite GSCs (6). We analyzed the male DTCs, GSC pool and the Notch response in the male gonad to ask whether niche architecture and GSC pool characteristics are shared between the sexes. Males have two DTCs which are positioned asymmetrically and we find that they lack the extensive plexus found in the single hDTC and, as a result, do not contact all GSCs. We find that GSC pools are of comparable size despite this sexual dimorphism. Consistent with similar GSC pool sizes, the molecular response to GLP-1/Notch signaling is nearly identical in the two sexes: using smFISH, we find that Notch-dependent transcriptional activation is graded through the GSC pool in males as in hermaphrodites, and LST-1 and SYGL-1 proteins are similarly restricted to the GSC pool region. We found, unexpectedly, that the transcriptional response to GLP-1/Notch signaling can occur in GSCs that do not have detectable contact with the signaling mDTC. In sum, this work demonstrates that niches with distinct architectures can drive similar molecular responses and maintain a similar number of GSCs in their stem cell pools. (1) Austin and Kimble, 1987 (2) Kershner et al., 2014 (3) Byrd et al., 2014 (4) Lee et al., 2016 (5) Cinquin et al., 2010 (6) Shin, Haupt et al., 2017.

Ferdous, Ahlan et al. (2021) International Worm Meeting "Regulation and function of the "PUF hub" governing C. elegans germline stem cells"

Czerniak, Cazza, Kanzler, Charlotte, Kimble, Judith, Shin, Heaji, Lynch, Tina, Ferdous, Ahlan

[International Worm Meeting, 2021]

The "PUF hub" is responsible for self-renewal of germline stem cells (GSCs)1. This hub consists of four PUF (Pumilio and FBF) RNA-binding proteins - FBF-1, FBF-2, PUF-3 &amp; PUF-11 - and two novel proteins, LST-1 and SYGL-1. The molecular elucidation of both regulation and function of the PUF hub is essential to understand how GSCs are maintained in the face of challenges from physiology and the environment. GLP-1/Notch signaling activates lst-1 and sygl-1 transcription to launch and maintain hub activity2,3. We now find that point mutations at one or more of three LAG-1 binding sites (LBS) in the sygl-1 promoter tune its response to niche signaling. Single LBS mutants substantially lower the number of sygl-1 active transcription sites (ATS) and double mutants nearly eliminate them; importantly, the GSC pool shrinks in single mutants and is lost in double mutants, when assayed without LST-1. We also find that LST-1 has an unexpected role in sygl-1 transcription: sygl-1 ATS number increases in lst-1(&oslash;) mutants with a corresponding increase in SYGl-1 protein. The converse is not true: lst-1 expression is unaffected in sygl-1(&oslash;) mutants. Therefore, LST-1, but not SYGL-1, affects transcription. The primary PUF hub function is regulation of target RNAs. We proposed earlier that each hub PUF protein forms a complex with either LST-1 or SYGL-1 to repress differentiation RNAs4. We have investigated this model. We first used co-immunoprecipitation (coIP) to explore the proposed battery of complexes. Though the matrix is only about half done, all coIPs so far support the model. We next identified "PUF-interaction sites" (PIS) in both LST-1 and SYGL-1. Intriguingly, each harbors two redundant PIS: single PIS mutants of either protein retain self-renewal activity but double mutants abolish it. We finally tethered LST-1 and SYGL-1 to a reporter RNA with the lambdaN-boxB system, and found that both lambdaN-LST-1 and lambdaN-SYGL-1 repressed a GFP::H2B reporter with boxB sites in its 3'UTR. These results provide strong evidence that PUF:LST-1 and PUF:SYGL-1 partnerships are essential for GSC maintenance and that LST-1 and SYGL-1 are RNA repressors. In sum, both hub regulation and function are intensively buffered, and our mutants provide new tools for analyses of the hub when poised in an intermediate state. 1 Haupt et al 2020 Genetics 214,147; 2 Kershner et al 2014 PNAS 111, 3739; 3 Lee et al 2016 eLife 5, e18370; 4 Shin et al 2017 PLoS genetics 13, e1007121.

Pearson RD (1996) Worm Breeder's Gazette "Is there Regenerative Capacity in the Enoplida (Nematoda)?"

Pearson RD

[Worm Breeder's Gazette, 1996]

There is precious little in the literature regarding the regenerative ability of nematodes (Poinar, 1988). The received wisdom has been that the determinate cleavage in these pseudocoelomates precludes any possibility of epimorphic regeneration (K.A. Wright pers. comm., 1988). Although Filipjev (1921) stated that regeneration is "completely absent" in nematodes, other reports by Micoletzky and Kreis (1930) and Allgen (1959) present another picture which seemingly contradicts such a blanket generalization. Last year it was reported that a gastrotrich (undescribed species of Turbanella) of the genera Macrodasyidae, a primitive sister group of the Nematoda, restored epidermis with complete wound closure following transection; restructuring of Y organ and intestine; and caudal adhesive tubes forming anew (Manylov, 1995). This is the first report of regeneration in this group. In another recent paper from the former Soviet Union, Voronov and Panchin (1995a) report that a nematode, of the order Enoplida (E.brevis), has a process of gastrulation which contradicts the patterns of cleavage formerly ascribed to the Enoplida (Malakhov,1994). They observed that up to the sixteenth cell stage cleavage is usually (though this can vary) equal and synchronous, producing blastomeres of equal appearance; elsewhere they observe that the primordia which gives rise to all the endoderm can be derived from either the anterior or posterior at the two-cell stage (Voronov and Panchin,1995b). This variability, they offer, makes the Enoplida different from other nematodes studied. Malakhov believes that this variability "can even engender the idea that the cleavage among members of marine Enoplida is indeterminate, but this is not so." (p.166). However, the cleavage of the Enoplida may be indeterminate enough to allow for the regenerative phenomena recently witnessed in a gastrotrich. In sum, Enoplid cleavage patterns would appear to be similar to the more primitive patterns seen in the Macrodasyidae, which is consistent with the notion that equal cleavage is ancestral and determination of early blastomere fate derived (Baguna and Boyer,1990). Also, it should be remembered that in addition to the single species reported by Micoletzky and Kreis, all nine of the species which Allgen found evidence of regeneration were marine Enoplids. Allgen,C.A.(1959)Free living marine nematodes. Further Zool. Results Swed. Antarct. Exp. 1901-03 vol.5 no.2: 1-293. Baguna,J.,B.C.Boyer(1990)Descriptive and experimental embryology of the Turbellaria: Present knowledge, open questions and future trends. In Marthy, H.(ed), Experimental Embryology in Aquatic Plants and Animals. NATO ASI 195; 95-128. Filipjev,I.N.(1921)Free living nematodes in the vicinity of Sevastapol. (in Russian), Akad. Nauk SSSR. Trudy osob. zool. lab. ser 2 41: 351-614. Malakhov,V.V.(1994)Nematodes: Structure, Development, Classification and Phylogeny. Smithsonian Inst. Press. Manylov,O.G.(1995)Regeneration in Gastrotricha - I. Light microscopical observations on the regeneration in Turbanella sp. . Acta Zool. 76:1-6. Micoletzky,H.,H.A.Kreis(1930)Freilbende marine Nematoden von den sunda-Inseln. Dansk natur. Foren. Vid. medd. Bd 87: 243-339. Poinar,G.O.(1992)Immune responses and wound repair. In Diseases of Nematodes. vol 1, p.133-40, CRC Press, Boca Raton, Florida. Voronov,D.A.,Y.V.Panchin(1995a)The early-stage of the cleavage in the free-living marine nematode Enoplus brevis (Enoplida, Enoplidae) in the normal and experimental conditions. Zool. Zhurn. 74(6): 31-38. Voronov,D.A.,Y.V.Panchin(1995b)Gastrulation in the free-living marine nematode Enoplus brevis and the localization of endodermal material at the stage of 2 blastomeres in the nematodes of the order Enoplida. Zool. Zhurn. 74(10): 10-18.