Chen, P. et al. (2019) International Worm Meeting "Investigating Centriole Duplication during Spermatogenesis in C.elegans."

Chen, P., Wu, J.

[International Worm Meeting, 2019]

The number of centrosome is important for correctly generating bipolar spindle. Centrosome would duplicate and separate once for every mitotic cell cycle. However, at the end of male meiosis, there are four centrosomes for four haploid spermatids indicating there is an additional duplication. The precise timing of two centrosome duplication events in male meiosis and the regulation mechanism of the additional duplication have not been investigated in detail. To examine when two duplication happening, we compared the levels of centriole structure protein SAS-4 in different meiotic stages. Quantification of immunofluorescence showed a two-folded increase in SAS-4 levels through the long meiotic prophase, implying the first centriole duplication. In addition, we found SAS-4 levels of a centrosome were comparable in primary and secondary spermatocytes. Because the number of centrosomes doubles from primary to secondary spermatocyte, this indicates the second centriole duplication event takes place during the transition between two male meiotic divisions. We confirmed the growth of SAS-4 level after primary meiotic division through live-imaging with GFP-SAS-4. By tracing intensity dynamics of GFP-SAS-4 during meiotic division, we surprisingly noticed the second centriole duplication finished in about ten minutes between two divisions. We conducted FRAP and bleached one of the two GFP-SAS-4 foci during first division, and found rapid fluorescence recovery of GFP-SAS-4 at the transition between two divisions. Taken together, our results reveal that the centrosomes exhibit two very distinct duplication kinetics during spermatogenesis: a long first duplication event during meiotic prophase, and a rapid second duplication event at the transition of meiotic divisions. It is intriguing whether both events are distinctly regulated. We are currently investigating the kinetics of the two duplication events in mutants defective in the canonical centrosome duplication pathway.

Chen P-J et al. (1998) Midwest Worm Meeting "fog-3 encodes a novel protein that is regulated by TRA-1A"

Chen P-J, Ellis RE

[Midwest Worm Meeting, 1998]

Both fog-1 and fog-3 are required to specify that germ cells develop as sperm rather than as oocytes. However, mutations in these genes do not affect any somatic cell fates. Because epistasis tests indicate that fog-1 and fog-3 act downstream of other sex-determination genes, we suspect they might directly control sexual fate in germ cells. The fog-3 gene produces a single transcript of approximately 1200 nucleotides, which is trans-spliced to the SL1 leader sequence. Computer analysis predicts that fog-3 encodes a novel protein of 263 amino acids. This protein does not contain obvious transmembrane domains or other known motifs. However, the amino-terminal 116 amino acids of FOG-3 resemble those of the vertebrate Tob proteins (Matsuda et al. 1996, Oncogene 12: 705-713). Within this domain, 33% of the FOG-3 residues are identical to those found in the Tob proteins, and an additional 21% are similar; there is a single gap of two nucleotides in the alignment. To identify essential residues of FOG-3, we located and sequenced the lesions caused by nine fog-3 mutations. Two alleles are deletions that cause a shift in the reading frame. One of these, q520, removes nucleotides 17-20 of the coding region, and is thus likely to be a null mutation. The other seven alleles are missense mutations. Six of these alter one of the first 116 amino acids. This clustering of mutations suggests that the region with similarity to the Tob proteins is crucial for FOG-3 activity. The Tob proteins can bind the erbB-2 receptor tyrosine kinase, and over-expression of these proteins inhibits cell proliferation, but their biochemical function remains unknown. Extrachromosomal arrays encoding truncated FOG-3 cause all germ cells to differentiate as oocytes. By contrast, control arrays that contain the promotor but lack the coding region have no effect. This dominant negative phenotype suggests that FOG-3 normally binds either itself or another protein, and that truncated FOG-3 still binds its target, but cannot promote spermatogenesis. To identify proteins that interact with FOG-3, we are screening a yeast two hybrid library using FOG-3 as bait. The promotor region on the smallest clone that rescues fog-3 is less than 1400 base pairs long. It contains three perfect TRA-1A binding sites, as predicted by Zarkower and Hodgkin, and two imperfect sites. Northern analysis shows that the level of fog-3 transcripts in hermaphrodites reaches its highest point in L4 larvae, and declines dramatically during adulthood. However, this level remains high in fem-3(gf) adults, which continue producing sperm. These results are consistent with the hypothesis that TRA-1A binds to the fog-3 promotor and represses transcription in adult hermaphrodites. If this model is correct, then fog-3 is the first identified target of TRA-1A. To test this hypothesis, we are preparing and studying mutant versions of the fog-3 promotor, to determine (1) their ability to bind TRA-1A in vitro, and (2) how they regulate transcription of fog-3 in transgenic animals.

Chen P-J et al. (1997) International C. elegans Meeting "fog-3 ENCODES A PROTEIN TYROSINE PHOSPHATASE REGULATED BY TRA-1A"

Ellis RE, Kimble JE, Chen P-J

[International C. elegans Meeting, 1997]

Previous studies showed that fog-3 specifies germ cells to form sperm rather than oocytes. By using a variety of cosmids for transformation rescue, we defined a small region likely to contain fog-3. Extrachromosomal arrays containing a 5.6 kb subclone from this region restore self-fertility to more than 90% of fog-3(q504) hermaphrodites. Analysis of the genomic sequence predicts that this fragment encodes a single protein of 577 amino acids. The carboxy-terminal half consists of a protein tyrosine phosphatase catalytic domain, with a perfectly conserved active site. The predicted protein does not appear to be a receptor. We are designing mutations in the active site to see if it is required for rescue. Extrachromosomal arrays that contain only the promotor region and 5' half of the fog-3 gene cause all germ cells to differentiate as oocytes. This dominant negative phenotype suggests that the amino-terminal half of FOG-3 might bind to another protein that specifies spermatogenesis. The promotor region on the 5.6 kb subclone is less than 1400 base pairs in extent. It contains three perfect TRA-1A binding sites, as predicted by Zarkower and Hodgkin. The sequence of each site is TTTCnnnnTGGGTGGTC. Only four additional sites with this sequence occur in the 63 Mb of sequenced C. elegans DNA. We propose that TRA-1A binds to these sites to repress transcription of fog-3. When the three FEM proteins are active, they prevent TRA-1A from functioning, allowing transcription of fog-3. However, because mutations in the fem genes are epistatic to mutations in tra-1, the FEM proteins must also directly activate FOG-3 or FOG-1. There are many potential serine and threonine phosphorylation sites in FOG-3; perhaps some of these are targets of the FEM-2 phosphatase.

Chen, P. et al. (2009) International Worm Meeting "The Early-Onset Torsion Dystonia Gene Product, torsinA, is a Mediator of ER Stress and Protein Homeostasis."

Roberts, N., Porter, J.C., Burdette, A.J., Berkowitz, L.A, Ricketts, J.C., Caldwell, K.A., Caldwell, G.A., Chen, P., Fox, S.A.

[International Worm Meeting, 2009]

The capacity of cells to carry out their various functions is wholly dependent upon efficient protein synthesis, processing, trafficking, and degradation. In this context, dysfunction or deficit of proteins that monitor and ensure the proper maintenance of cellular homeostasis can have serious consequences in terms of disease onset, penetrance, or progression. A single codon deletion (GAG = DE) in the gene encoding human torsinA causes a non-degenerative neurological disorder termed early-onset torsion dystonia. TorsinA, an endoplasmic reticulum (ER) resident protein, belongs to the diverse AAA+ (ATPases Associated with a variety of cellular Activities) family. Here we use a well established ER stress model in the nematode C. elegans (hsp-4::GFP) to investigate the role of torsinA in maintaining protein homeostasis in the ER. Using GFP as an ER stress reporter, we found that transgenic nematodes expressing wild type (WT) human torsinA exhibited a striking reduction in ER stress caused by tunicamycin, while animals with either DE or a mixture of WT/DE torsinA failed to do so. Interestingly, DE and WT/DE torsinA worms exhibited higher ER stress than WT torsinA animals, even in the absence of tunicamycin treatment. In addition, mutations within torsinA ER signal sequence and N-terminal hydrophobic region abolished its capacity to maintain an ER stress threshold against cellular stressors, indicating the importance of its localization to the ER for this activity. Furthermore, although torsinA possesses very low ATPase activity, the ATPase domain appeared important for ER stress suppression, as two different mutations within the ATPase domain, K108A and E171Q, resulted in loss of stress suppression. A single nucleotide polymorphism (SNP), D216H, diminishes the penetrance of dystonia from 30-40% (WT/DE) to 3% (D216H/DE) in patients. In C. elegans, coexpression of DE torsinA with D216H torsinA significantly protected worms from ER stress caused by tunicamycin, thereby recapitulating the effect on penetrance observed with patients. In order to find genetic interacting factors of torsinA, a small scale of RNAi screen for altered ER stress levels in DE torsinA worms, was carried out. Positives from the screen included glutaredoxin, H3 histones, and 13 core components and regulators of Ras signaling. Taken together, our data indicate that torsinA serves to maintain protein homeostasis in the ER, shedding new light on the pathophysiology of torsion dystonia.

Chen, Sway P. et al. (2011) International Worm Meeting "How larvae wiggle: functional analysis of the L1 larva locomotory circuit."

Tang, Anji, Chen, Sway P., Wen, Quan, Samuel, Aravinthan D. T.

[International Worm Meeting, 2011]

At all developmental stages, C. elegans navigates its environment by generating and propagating sinusoidal bending waves along its body. The neural circuit controlling the locomotory behavior, however, changes significantly at the late L1 larval stage [1]. Neuroanatomy shows that almost no cholinergic motor neurons innervate the ventral body wall muscles in the L1 larva, raising the question of how the larva is capable of normal locomotion. Here we hypothesize that ventral body wall muscles in the L1 larva contract by default. During the dorsal muscle contracting phase, DB cholinergic motor neurons activate DD GABAergic motor neurons and cause periodic relaxation of the ventral muscles. This hypothesis is supported by two experimental observations. First, we examined the swimming behavior of GABA-deficient mutant unc-25 in the L1 stage and found that the tail consistently curved to one side. Second, we found that wild-type worms expressing GFP in VNC neurons consistently curved to the ventral side after being paralyzed by ivermectin, a drug that silences the motor circuit but has no effect on muscles. Further optogenetic and calcium imaging experiments will be carried out to quantify and relate motor neuron and muscle activity in the free-moving larva. Our approach will build toward a detailed mechanistic model of L1 locomotion. Hopefully, comparison of L1 and adult worm locomotion will shed light on conserved principles of rhythmic motion in general. 1.White, J.G., et al., The structure of the ventral nerve cord of Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 1976. 275(938): p. 327-48.

Nadeem Moghal et al. (2001) International C. elegans Meeting "Modulation of inductive signaling during C. elegans vulval development"

Nadeem Moghal, Paul W Sternberg, L Rene Garcia, Chieh Chang

[International C. elegans Meeting, 2001]

In C. elegans hermaphrodites, the vulval precursor cells (VPCs) P3.p-P8.p can adopt vulval or hypodermal fates. In wild-type animals, the anchor cell secretes LIN-3, which activates its receptor, LET-23, and causes P5.p-P7.p to adopt vulval fates. Although P3.p, P4.p, and P8.p also express LET-23, it is thought that their distance from the anchor cell prevents levels of activated LET-23 from accumulating necessary to overcome default hypodermal fates. We have used two approaches to study modulation of responses to this inductive signal. In one approach, we tested candidate loci for interaction with this pathway. We find that a loss of function mutation in the egl-15 negative regulator, clr-1 , a receptor tyrosine phosphatase, increases responsiveness of all 6 VPCs to the inductive signal. In contrast, an activated allele of egl-30 Gq alpha facilitates responsiveness of P5.p-P7.p, but not P3.p, P4.p, and P8.p. In a second approach, we identified EMS-induced mutations that enhance the choice of vulval fates by P3.p, P4.p, and P8.p in the presence of a gain-of-function allele of let-23 . Some of these mutations, such as sy622 , strongly enhance the responsiveness of P3.p, P4.p, and P8.p, while only weakly affecting P5.p-P7.p. These results indicate that there are both pan-Pn.p, as well as Pn.p-specific mechanisms operating to modulate vulval induction. Pan-Pn.p mechanisms might function as buffers to lower the overall noise inappropriately contributing to vulval induction. In conjunction with pathways defined by egl-30(gf) and sy622 which increase responsiveness of P5.p-P7.p and decrease responsiveness of P3.p, P4.p, and P8.p, respectively, a robust response by only a subset of cells might be guaranteed during inductive signaling.

Min Zheng et al. (2001) International C. elegans Meeting "Cell fate specification during vulva formation in Pristionchus pacificus"

Ralf J Sommer, Min Zheng

[International C. elegans Meeting, 2001]

We compare vulva development between Caenorhabditis elegans and Pristionchus pacificus to study the evolution of developmental processes. Cell fate specification during vulva development differs in these two nematodes. In C.elegans , P8.p is part of the vulva equivalence group and adopts a 3deg fate under wild-type conditions. In contrast, several experiments indicate that P8.p in P.pacificus represents a new cell type. First, P8.p is unable to respond to an inductive signal from the gonad and fuses with the epidermis, but can respond to lateral signal from P6.p and form vulval tissue. Second, P8.p provides a lateral inhibitory signal that prevents P5.p and P7.p to adopt a 1deg fate. Third, P8.p provides a negative signal that inhibits gonad-independent vulva formation of P(5-7).p. To study P8.p specification we screened for mutants with P8.p specification defects and identified three candidate mutants in which P8.p behaves like a normal VPC. In one of the mutants, named tu151 , the nucleus of P8.p has an abnormal size. Cell ablation experiments in tu151 revealed that P8.p is able to respond to the inductive signal from the gonad to form vulval tissue. Also, P8.p in this mutant cannot provide the lateral inhibitory signal so that after P6.p ablation, P5.p and P7.p can adopt the 1deg fate. In addtion, P8.p loses the function of providing negative signal in tu151 mutant animals since gonad-independent vulva formation occurs. Positional cloning of tu151 is currently in progress.

Min Zheng et al. (2002) European Worm Meeting "Genetic and molecular analysis of the vulval patterning mutants ped-7 and ped-17 in Pristionchus pacificus"

Min Zheng, Ralf J Sommer

[European Worm Meeting, 2002]

We compare vulva development between Caenorhaditis elegans and Pristionchus pacificus to study the evolution of developmental processes. Cell fate specification during vulva development differs in these two nematodes. In C. elegans, P8.p is a member of the vulva equivalence group and adopts a 3 fate under wild-type conditions. In contrast, several experiments indicate that P8.p in P. pacificus represents a novel cell type. First, P8.p is unable to respond to an inductive signal to form vulval tissue, but it can respond to lateral signal from the neighboring 1 cell P6.p. Second, P8.p provides a lateral inhibitory signal that prevents P5.p and P7.p, but not P6.p, from adopting a 1 fate. Third, P8.p provides a negative signal that inhibits gonad-independent vulva formation of P(5-7).p. To study P8.p specification, we screened for mutants with P8.p specification defects and identified three mutants in which P8.p behaves like a normal VPC. In one of these mutants, namely ped-17, the nucleus of P8.p has an abnormal size. Cell ablation experiments in ped-17 revealed that P8.p is able to respond to inductive signal from the gonad to form vulval tissue. Also, P8.p in this mutant cannot provide the lateral inhibitory signal. After P6.p ablation in these animals, either P5.p or P7.p can adopt a 1 fate. In addition, P8.p loses the function of providing negative signal in ped-17 mutant animals since gonad- independent vulva formation occurs. In another mutant, ped-7, P7.p migrates towards P8.p and forms an ectopic invagination. Genetic analysis revealed that ped-7 represents a multivulva mutation. We have mapped both mutants to the genetic linkage map of P. pacificus. ped-7 maps to chromosome V and is located between the markers S130 and S13. ped-17 is on chromosome II and is located between S111 and S24. Positional cloning of ped-7 and ped-17 is currently ongoing.

Alper SD et al. (1997) International C. elegans Meeting "Regulation of Pn.p Cell fusion in C. elegans"

Kenyon CJ, Alper SD

[International C. elegans Meeting, 1997]

Cell fusion is a ubiquitous process in C. elegans, with approximately one third of all somatic nuclei in adults found in variably sized multinucleate syncytia. In hermaphrodites, the hypodermal cells P1.p, P2.p, and P(9-11).p all fuse with hyp7 during late L1 while P(3-8).p remain unfused and become members of the vulval equivalence group. In males, P(3-6).p and P(9-11).p remain unfused; P(9-11).p subsequently become the male equivalence group. The Pn.p fusion decision is regulated by the Hox genes lin-39 and mab-5. lin-39 is expressed in P(3-8).p and in hermaphrodites prevents fusion of these cells. mab-5 is expressed in P(7-11).p. In males, lin-39 and mab-5 each individually prevent Pn.p cell fusion in P(3-6).P and P(9-11).p, respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they somehow neutralize one another's effects, so that P7.p and P8.p fuse with hyp7. We are carrying out a screen to identify additional genes that affect Pn.p cell fusion. In a lin-12(d) background, those Pn.p cells that remain unfused all generate ectopic pseudovulvae or hooks that protrude from the ventral surface of the worm. We are screening for mutations that change the pattern of ventral protrusion in lin-12(n137); him-5(e1467); thus far, we have identified five mutants with extra ventral protrusions in the hermaphrodite and one mutant with extra protrusions in the male. MH27, a monoclonal antibody that stains adherins junctions, has been used to verify that extra Pn.p cells fail to fuse with hyp7 in some of these mutants. Preliminary experiments suggest that some of these mutations may affect upstream regulators of the Hox genes. We also hope to find genes that integrate the Hox signals as well as genes required for the actual process of cell fusion.

Simske JS et al. (1995) International C. elegans Meeting "SEQUENTIAL SIGNALING during vulval development."

Kim SK, Simske JS

[International C. elegans Meeting, 1995]

During the induction of the C. elegans vulva, cell signaling causes initially equipotent cells to express a reproducible pattern of cell fates. The position of the anchor cell determines the pattern of vulval precursor cell fates, such that the closest precursor cell (P6.p) expresses the primary cell fate, the next closest cells (P5.p and P7.p) both express the secondary cell fate, and each of the precursor cells located at a distance (P3.p, P4.p, and P8.p) express the uninduced, tertiary cell fate. We present data indicating that this stereotypical pattern of cell fates can be generated by sequential signals. We identified genetic mosaic animals in which P5.p and P7.p were defective in the anchor cell signal transduction pathway and observed that these cells adopted the secondary cell fate, indicating that anchor cell signal transduction in P5.p and P7.p is not required for the expression of the secondary cell fate. These results suggest that the anchor cell induces P6.p to express the primary cell fate, and that P6.p subsequently induces P5.p and P7.p to express the secondary cell fate.