Wynne, David et al. (2009) International Worm Meeting "Motor-Driven Motion Associated with Meiotic Chromosome Pairing."

Carlton, Pete, Wynne, David, Dernburg, Abby

[International Worm Meeting, 2009]

Accurate segregation of homologous chromosomes during meiosis is necessary for the faithful transmission of the genome from parent to progeny. To segregate properly, homologs must first undergo pairing, synapsis, and recombination. The mechanism of homologous chromosome pairing remains a major mystery of meiosis. In C. elegans, as in many other eukaryotes, pairing is accompanied by a global rearrangement of chromosomes. We have found that this rearrangement is driven through the association of special chromosome regions known as Pairing Centers (PC) with nuclear envelope proteins and cytoskeletal motors (Phillips et al. 2005, Sato A. unpublished). Using fluorescent markers for nuclear envelope attachment sites and Pairing Centers, we are analyzing chromosome dynamics through real-time imaging and quantitative motion tracking. Our results reveal a dramatic increase in chromosome motion at the onset of chromosome pairing that persists after homologous loci are paired. We show that this increased mobility is dependent on the conserved cell cycle checkpoint kinase, CHK-2, and that motion slows following knockdown of cytoplasmic dynein. Our data supports a model in which homolog pairing is promoted by a small number of fast, motor-driven movements that augment the smaller, Brownian motions seen prior to meiosis. The observation that fast motions persist well after pairing is completed suggests additional roles in chromosome synapsis or recombination.

Wynne, David et al. (2021) International Worm Meeting "Characterizing the function of the histone H3 kinase HASP-1 in the germline"

Moore, Amy, Macaraeg, Jommel, Reinhard, Isaac, Wynne, David, Ward, Matthew, Carmeci, Danielle

[International Worm Meeting, 2021]

Haspin is a protein kinase that phosphorylates histone H3 at threonine 3 during mitosis and meiosis. Together with the activity of another histone kinase Bub1, haspin-dependent histone phosphorylation (H3T3ph) promotes recruitment of the Chromosomal Passenger Complex (CPC) to chromosomes, which activates the Aurora B kinase subunit of the CPC. The function and regulation of haspin outside of mitosis remains less well characterized. It is not known whether the mechanism by which haspin is activated that was established in vertebrate systems is used in a wide variety of cell types and it is unclear how the activities of Bub1 and haspin are coordinated in the context of meiotic chromosome and kinetochore structure. Haspin inhibition in mouse oocytes has been shown to reduce the recruitment of the CPC to chromosome axes but not kinetochores, suggesting that there are two pools of CPC recruited to meiotic chromosomes by distinct mechanisms (Nguyen et al. 2014). In C. elegans, depletion of the haspin homolog hasp-1 has been shown to reduce the CPC to undetectable levels in diakinesis oocytes (Ferrandiz et al. 2018), suggesting that C. elegans BUB-1 activity may play a less important role in CPC recruitment to meiotic chromosomes in oocytes. To better understand the coordination between Bub1 and haspin, we are investigating the roles of hasp-1 in CPC recruitment in the different types of cell divisions in the C. elegans germline. We generated a conditional hasp-1 allele using the auxin-inducible degron system (Zhang et al. 2015), which allows us to distinguish the effects of hasp-1 depletion during mitosis in germline stem cells, oocyte meiosis, and spermatogenesis. We found that phenotypes caused by hasp-1 depletion during oocyte meiosis are much more severe than during spermatogenesis or stem cell proliferation, suggesting that bub-1 is able to compensate for hasp-1 loss in mitosis and in spermatocyte meiosis. We are testing this hypothesis by combining hasp-1 depletion with mutations expected to reduce bub-1 function and monitoring the effects on CPC recruitment and chromosome segregation in the germline.

David Wynne (2006) Development & Evolution Meeting "Characterizing the Role of SUMO Conjugation in Meiosis"

David Wynne

[Development & Evolution Meeting, 2006]

The small ubiquitin-like modifier SUMO is a good candidate for a regulator of meiotic chromatin dynamics based on its established roles in cell cycle progression, chromatin structure and nuclear organization in multiple organims. SUMO modification plays a role in meiotic chromosome segregation in Drosophila and there are many essential meiotic proteins in C. elegans with high-probability SUMOylation sites. Worms contain a single SUMO gene, smo-1, and complete loss of function of smo-1 has been shown to function in embryonic development and development of the somatic gonad (Broday et al. 2004). In mutant hermaphrodites the progression of oocytes through the pachytene stage appears normal by chromatin staining but my initial characterization has shown that the structure of bivalents at diakinesis is abnormal. Intriguingly, transgenic expression of smo-1 in the C. elegans soma restores some fertility but also reveals chromosome segregation defects (L. Broday, personal communication). These results suggest that smo-1 loss of function in the germline causes specific defects in meiotic progression in C. elegans that could be revealed by more sensitive assays. I will present a cytological analysis of meiotic defects associated with loss of smo-1 activity in the germline.

David Wynne et al. (2001) International C. elegans Meeting "Approaches to Studying the Function of APH-1"

Valerie Hale, Katie Scangos, Caroline Goutte, Murline Gelin, David Wynne

[International C. elegans Meeting, 2001]

The aph-1 gene is involved in Notch-mediated signaling events in the C. elegans embryo. Mutations in aph-1 were isolated in the Priess lab by virtue of their embryonic lethal phenotype which showed a striking resemblance to the phenotype caused by mutations in the glp-1 gene. Further analysis of the aph-1 mutant phenotype demonstrated that the aph-1 gene product was necessary for GLP-1-mediated events at both the 4-cell and 12-cell stages of embryogenesis, thus identifying a new component of the GLP-1 signaling pathway in the early embryo. We have cloned the aph-1 gene and found that it encodes a novel protein with unknown function. Several lines of experimentation are underway to determine what is the specific function of the APH-1 protein. One approach we have taken is to build an aph-1::GFP fusion construct to look at the cellular and subcellular localization of the APH-1 protein in embryos. We have established transgenic lines with these constructs and shown that the fusion protein can rescue the aph-1 phenotype, albeit with low penetrance. Given that the fusion has some wild type activity, we are now analyzing the fluorescence pattern obtained from these constructs. A second approach we have taken is to search for genes that interact with aph-1. We have used a leaky allele of aph-1 to isolate extragenic suppressor mutations. Six independent mutations were found to suppress the aph-1 embryonic lethal phenotype. Two of these are dominant mutations in two different genes, and the other four mutations are recessive and identify two additional genes. Mapping data and further characterization of these four genes will be presented.

David Wynne et al. (2008) Development & Evolution Meeting "Real-Time Imaging of Meiotic Chromosome Dynamics"

Pete Carlton, David Wynne, Abby Dernburg

[Development & Evolution Meeting, 2008]

Accurate segregation of chromosomes during meiosis is necessary for the faithful transmission of the genome from parent to progeny. Conversely, meiotic missegregation leads to impaired viability and fertility, as well as aneuploid progeny that can have dramatic developmental defects, such as Down syndrome in humans. To segregate properly, homologs must first undergo pairing, synapsis, and recombination. Despite its central role in meiosis, pairing of homologous chromosomes remains poorly understood. In C. elegans, as in many other eukaryotes, this process is accompanied by a global rearrangement of chromosomes. We have found that this rearrangement is driven through the association of special chromosome regions, known as Homolog Recognition Regions or Pairing Centers, with the nuclear envelope. In other organisms, telomeres establish similar attachments to the NE during "bouquet stage" of meiosis, but the function of this configuration has not been established in any system. Chromosomes are not merely tethered at the nuclear envelope, but instead associate with specific nuclear envelope proteins and cytoskeletal motors (Phillips et al. 2005, Sato A. unpublished). Using ZYG-12:GFP (Malone et al. 2003) as a marker for the chromosome attachment sites, we have observed that they undergo rapid movements throughout the pairing process. Disruption of cytoplasmic dynein causes a dramatic decrease in patch motion, supporting a model in which pairing dynamics are driven by cytoskeletal components outside the nucleus. These data suggest that meiotic chromosome pairing is an active process rather than being purely a result of a diffusion-based mechanism.

Schein JE et al. (1997) International C. elegans Meeting "POSITIONING OF ESSENTIAL GENES IN THE lin-3 - let-60 INTERVAL"

Franz NW, Rose AM, Janke DL, Bilkhu N, Baillie DL, Schein JE

[International C. elegans Meeting, 1997]

The lin-3 - let-60 interval of the right arm of chromosome IV contains thirteen essential gene mutations and 22 overlapping sequenced cosmids. The Sanger Center has nearly finished sequencing the entire interval. Analysis of cosmid sequences shows over one hundred potential coding regions. Of the coding regions with proposed protein function, more than 50% have strong homology to human genes. Most notably, the putative genes include three phosphatases, four protein kinases, a kinesin, and a cluster of histones. C. elegans strains carrying sequenced cosmids (in extrachromosomal arrays) have been constructed (see abstract for D. Janke et al., this meeting) and will be used in rescue experiments involving the essential genes in this interval. lin-3 and let-60(ras), the two genes bracketing this interval, were previously placed on the physical map by others. One of the remaining eleven essential genes has been already positioned to a cosmid: let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido). By the time of the meeting, we anticipate that several of the remaining mutations will have been positioned to cosmids. This work is being funded by a grant from the Medical Research Council to Ann Rose and David Baillie and by a grant from the Natural Sciences & Engineering Research Council to David Baillie.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

O'Neil NJ et al. (1996) West Coast Worm Meeting "SYSTEMATIC HIGH RESOLUTION MAPPING OF ESSENTIAL GENES IN THE sDf125 REGION BY TRANSGENIC RESCUE."

Kuervers LM, Baillie DL, Vatcher GP, O'Neil NJ

[West Coast Worm Meeting, 1996]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the left end of the central cluster into manageable regions which are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints (see N.Franz et al. abstract, this meeting). Into these regions, we have mapped a set of EMS induced lethals and maternal effect lethals. (see abstracts by H. Stewart et al. and G. Vatcher and D. Baillie, this meeting). One such region is defined by sDf125 and sDp8. This region spans 340 000 base pairs and is completely covered by nine cosmids. Within this region there are 75 putative genes predicted by Genefinder. We have used a sytematic approach to rescue essential genes within the sDf125 region with transgenic cosmids from the library created by J. Schein, D. Janke and The Ha as part of a CGAT grant to Ann Rose and David Baillie (see abstracts by J.Schein et al. and D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region by this approach. These transgenic rescues map these genes to 40 kB (one cosmid) resolution as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of ORFs which may be responsible for the gene function. The next step will be the subcloning of cosmids with which we have rescued essential genes. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate ORF. We have already begun the subcloning of cosmid C05D11 (see G.Vatcher and D.Baillie abstract, this meeting). This project is funded by a CGAT grant to Ann Rose and David Baillie.

O'Neil NJ et al. (1997) International C. elegans Meeting "ANALYSIS OF THE sDf127 REGION BY TRANSGENIC RESCUE."

O'Neil NJ, Vatcher GP, Kuervers LM, Baillie DL

[International C. elegans Meeting, 1997]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the region between dpy-17 and unc-36 into manageable regions that are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints. Into these regions, we have mapped a set of EMS induced lethal and maternal effect lethal mutations (See abstract H.Stewart et al., this meeting). One such region is defined by sDf127 and sDp8. This region spans 340 000 base pairs and is completely covered by ten cosmids. Within this region there are 75 putative genes predicted by Genefinder. To date, we have mapped 14 essential genes into this region. We have used a sytematic approach to rescue these essential genes with cosmid transgenics from the library created as part of a CGAT grant to Ann Rose and David Baillie (See abstract by D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region utilizing this approach. These transgenic rescues map these genes to 40 kB (one cosmid) as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of coding sequences which may be responsible for the gene function. The next step will be to subclone the cosmids with which we have rescued essential gene mutations. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate coding sequence. We have already begun the subcloning of the cosmids C05D11 (see abstract by G.Vatcher et al., this meeting), C31H11 and C16A3. This project is funded by a grant from the Medical Research Council of Canada to Ann Rose and David Baillie.

David R. Bell et al. (2006) European Worm Meeting "The Function of Latrophilin in C. elegans"

S. van der Poel, P.N.R. Usherwood, D. Kendall, J. Davy, A. Ademola, I.R. Duce, D. de Pomerai, David R. Bell, I. R. Mellor

[European Worm Meeting, 2006]

David R. Bell, A. Ademola, J. Davy, S. van der Poel, D. Kendall, P.N.R. Usherwood, I.R. Duce, I. R. Mellor and D. de Pomerai. Black Widow spider venom (BWSV) contains latrotoxins that induce catastrophic neurotransmitter release in mammals, and is known to bind with high affinity to three neural proteins in mammals, including latrophilin. We have investigated C. elegans as a model system for studying the function of these proteins, and their role in regulating neurotransmitter release by latrotoxins.. We have shown that latrophilin is required for the lethality of BWSV in C. elegans by RNAi experiments. We now present data on the characterisation of null mutants of the C. elegans latrophilin, lat-1, illustrating the function of this gene in C. elegans, and show that lat-1 nulls are resistant to emodepside. The expression of latrophilin-GFP constructs is examined, and compared with the phenotypes seen in null mutants. Genetic interactions between endogenous signaling pathways and the lat-1 nulls have been examined by the construction of double mutant worms, revealing novel facets of lat-1 function.