Naomi Phillips et al. (2008) Development & Evolution Meeting "Direct Estimate of the Rate and Spectrum of Microsatellite Mutation in Rhabditid Nematodes"

Andy Custer, Matthew Salomon, Gigi Ostrow, Naomi Phillips, Charles Baer

[Development & Evolution Meeting, 2008]

Simple sequence repeats (SSR, or microsatellites) are one of the most widely used classes of genetic markers, from genetic mapping and association studies to investigations of population structure and conservation. Despite their utilization, many unresolved questions concerning the mutational processes shaping SSR evolution remain. Here we describe a study aimed to provide unbiased estimates of the rate and spectrum of new SSR mutations in a novel mutation accumulation (MA) model system. The mutational properties of dinucleotide SSRs were characterized in two strains (= genotypes) of two species in the genus Caenorhabditis, C. briggsae and C.elegans after 250 generations of MA. Mutations have been allowed to accumulate in the (relative) absence of natural selection, thus allowing us to genotype paired control and generation 250 MA lines at approximately 40 dinucleotide loci of perfect, imperfect, and compound SSRs. Furthermore, we investigate how the mutational properties of SSRs are correlated with the genomic mutation rate for fitness in these two species.

Salomon, Matthew et al. (2009) International Worm Meeting "Persistence time of mutations affecting fitness and body size in Caenorhabditis briggsae and C. elegans."

Upadhyay, Ambuj, Salomon, Matthew, Baer, Charles, Levy, Laura, Keller, Thomas, Phillips, Naomi, Blanton, Dustin, Ostrow, Dejerianne, Bour, Whitney, Sylvestre, Thamar

[International Worm Meeting, 2009]

The level of genetic variation present in a population is a composite function of mutation, population size, and natural selection. Historically, efforts to understand differences (or similarities) between groups in levels of genetic variation have focused on the interplay between population size and natural selection. However, much less attention has been paid to the alternative possibility that differences among groups are due to systematic differences in the underlying rate of mutation. Much of the difficulty in interpreting the role of mutation stems from the fact that most of what is known about genomic mutational properties, for quantitative traits in multicellular eukaryotes, comes from a handful of phylogenetically distant and biologically dissimilar model organisms, making meaningful comparisons difficult. Over the past several years our lab has been investigating the properties of new mutations in a model nematode system within a comparative phylogenetic framework. Mutations have been allowed to accumulate in the (relative) absence of natural selection, thus allowing us to estimate the genetic variance introduced by new mutation (VM) for two species of rhabditid nematodes, Caenorhabditis elegans and C. briggsae. Previous work in this system suggests that the mutation rate in C. briggsae is on the order of twice that of C. elegans for quantitative traits and dinucleotide repeats. Here we report the standing genetic variance (VG) for two quantitative traits, lifetime reproduction and body size, in worldwide collections of C. briggsae and C. elegans natural isolates. Comparisons of VG to VM between the natural isolates and our mutation accumulation lines allow us to infer the magnitude and pattern of constraint on phenotypic evolution in these two species. Taking the results from the two species together, the persistence time (VG/VM) of new mutations affecting fitness is on the order of tens to perhaps hundreds of generations, with an average selection coefficient against homozygotes of a few per-cent. Furthermore, the pattern of persistence time for mutations affecting adult body size is onsistent with that of fitness in both species. These results suggest that idiosyncratic selection, perhaps due to random hitchhiking - "genetic draft" - is paramount in shaping the standing genetic variance of these traits in these species.

Meneely, Philip M. et al. (2009) International Worm Meeting "HIM-8 interacts with SUN-1."

Daou, Maral, Thompson, Scott, Meneely, Philip M., Burch, Lauren

[International Worm Meeting, 2009]

During homolog recognition and pairing in meiosis, pairing centers at or near one end of each meiotic chromosome are localized to the nuclear envelope. SUN-1 is an evolutionarily conserved protein located in the inner nuclear membrane that appears to be directly involved in mediating telomere attachment in mice and yeast; in worms, a direct interaction between pairing center proteins and SUN-1 at the nuclear envelope has not yet been proved (1, 2). In sun-1 null mutants, none of the meiotic chromosomes are paired but the pairing centers are localized to the nuclear envelope (2). HIM-8 is a C2H2 zinc finger protein that binds specifically to the pairing center on the X chromosome and is necessary for the pairing of the X chromosome and for synapsis (3). When a zinc finger is mutated in him-8, such as in e1489 or the tm611 deletion, the protein does not bind to the X pairing center, the X chromosomes do not pair, but the chromosomes are still located at the nuclear envelope. Thus, pairing and nuclear envelope attachment may be separable activities for the pairing center proteins. The autosomes are paired normally in him-8 mutants. The three ZIM proteins, which are highly related to HIM-8 in sequence, mediate pairing of the autosomes (4). We find that SUN-1 and HIM-8 physically associate with each other in a yeast two-hybrid assay. Preliminary evidence suggests a similar physical interaction occurs between SUN-1 and at least one of the ZIM proteins. This suggests a model whereby meiotic chromosomes are tethered to the nuclear envelope for pairing via a direct interaction between SUN-1 in the inner membrane and HIM-8 or one of the ZIM proteins on each specific chromosome. 1.Penkner et al. 2007. Develop. Cell 12: 873-885 2.Fridkin, et al., 2009. Cell Mol. Life Sci. 66: (in press) 3.Phillips et al. 2005 Cell 123: 1051-1063 4.Phillips and Dernburg 2006 Develop. Cell 11: 817-829.

Yuriko Harigaya et al. (2007) International Worm Meeting "Assessing the ability of C.elegans spermatocytes to segregate achiasmate chromosomes."

Yuriko Harigaya, Anne Villeneuve

[International Worm Meeting, 2007]

Accurate segregation of homologous chromosomes at meiosis I is an essential step in sexual reproduction. This is usually achieved by a mechanism that relies on chiasmata, which are recombination-based linkages that temporarily connect homologs and allow them to orient toward opposite spindle pole. However, certain meiotic systems, such as those in Drosophila females and males, possess the ability to segregate homologs without chiasmata (achiasmate chromosome segregation) (Hawley et al. 1993). It has been proposed that this type of mechanism may operate in humans as a back up system in case of recombination failure (Koehler & Hassold 1998). Indirect genetic evidence has suggested that C.elegans spermatocytes may also have mechanisms that allow segregation of (at least) a single pair of achiasmate chromosomes (Albertson et al. 1997, for review). We have set out to revisit this issue, taking advantage of recent progress in the field of C. elegans meiosis. Phillips & Dernburg (2006) have identified a family of C2H2 zinc-finger proteins (ZIMs), which play a pivotal role in meiotic pairing and recombination. They have demonstrated that each of the three ZIM proteins act on specific autosome pairs in oocyte meiosis, that is, the zim mutations provide situations where one or two specific homologous pair(s) is/are consistently achiasmate. Results from the following analyses will be presented: 1) investigation of pairing and synapsis states in zim male germlines to confirm that ZIMs function similarly in spermatocyte meiosis; 2) testing cross progeny from the zim males to investigate whether achiasmate chromosome pairs, if any, are successfully inherited during meiosis in zim mutant spermatocytes; and 3) cytological observation of chromosomal placement at metaphase I and anaphase I in mutant spermatocytes using FISH analysis, to gain insights into the mechanistic bases for the chromosome inheritance pattern. Albertson et al. (1997) in C. ELEGANS II:p47; Hawley et al. (1993) Dev Genet 13:440; Koehler & Hassold (1998) Annu Hum Genet 62:467; Phillips & Dernburg (2006) Dev Cell 11:817.

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.

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.

Haag, E.S. et al. (2019) International Worm Meeting "Evolution of sex ratio through gene loss."

Haag, E.S., Yin, D.

[International Worm Meeting, 2019]

The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species of Caenorhabditis nematodes have evolved the androdioecious mating system, where selfing hermaphrodites and males coexist. They reproduce mostly through self-fertilization, but can also outcross. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio [1]. Here, we focus on the contribution of the male secreted short (mss) gene family to male mating success, sex ratio, and population growth. The mss family is essential for full sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species [2]. Using a transgene to restore mss function to the androdioecious C. briggsae, we examined how mating system and population subdivision influence the fitness of the mss+ genotype. Consistent with theoretical expectations, mss+ is sufficient to increase male frequency and depress population growth in genetically homogenous androdioecious populations. When mss+ and mss-null (i.e. wild-type) genotypes compete, mss+ is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness of mss+, it is insufficient to explain its parallel loss. We propose that the lack of inbreeding depression [3] and the strong subdivision that characterize natural Caenorhabditis populations [4] impose selection on sex ratio that makes loss of mss adaptive. By reducing, but not completely eliminating outcrossing, loss of the mss genes tunes the sex ratio to its new optimum after self-fertility is established. 1. Stewart AD, Phillips PC (2002) Genetics 160: 975 2. Yin et al. (2018) Science 359: 55 3. Dolgin et al. (2007) Evolution 61: 1339 4. Kiontke KC, et al. (2011) BMC Evol Biol 11: 339

Sym M et al. (1996) West Coast Worm Meeting "mig-13 AND CELL MIGRATION"

Kenyon CJ, Sym M

[West Coast Worm Meeting, 1996]

The Q cells are born as left/right homologs (QL and QR) and undergo identical cell lineages. However, QL and QR migrate in opposite directions: QL and its descendants migrate posteriorly; whereas, QR and its descendants migrate anteriorly. The mig-13(mu31) mutation was identified in a screen for mutations affecting Q cell migration (Naomi Robinson, Ph. D. thesis, 1995). The initial migration of QR itself is not affected in mig-13(mu31), but the subsequent anterior migrations of its descendants are significantly shortened. Thus, these cells are found substantially posterior to their wild-type positions. The posterior migration of QL and its descendants is not affected in this mutant, suggesting that migration per se does not require mig-13(+). However, in mutants where the descendants of QL migrate anteriorly instead of posteriorly (e.g., mab-5, egl-20), mig-13(+) is required for anterior migration of both the QR and QL descendants. Molecular characterization of mig-13 has shown that the gene is SL1-spliced and contains 8 exons, which encode a predicted protein of 362 amino acids. Sequence comparisons revealed an N-terminal signal sequence followed by two potential protein interaction domains and a single-pass transmembrane domain. The predicted cytoplasmic C-terminus bears no homology to anything currently in the database. A C-terminal GFP fusion was constructed that exhibits full mig-13-rescuing activity. Worms bearing this mig-13-GFP fusion show reproducible expression in a number of cells, most of which are neurons. The fusion protein is localized to the periphery of each expressing cell, as predicted from the transmembrane domain within MIG-13. During the time of Q cell migration (L1), mig-13-GFP is expressed in the pharygeal-intestinal valve cells and in approximately 10 ventral cord neurons, but is not detected in the Q cell lineage. Most of the ventral cord neurons show staining in the ventral and/or dorsal axon bundles as well as commissural axons, which project into the dorsal cord. Interestingly, the ventral cord neurons that express the fusion protein are located in the anterior two-thirds of the body axis, through which QR and its descendants migrate. Ventral cord neurons in the posterior third of the body do not express mig-13-GFP. Thus, the expression pattern suggests that mig-13 acts non-autonomously to influence Q cell migration. Mosaic analysis is in progress, which should determine whether this is indeed the case. Perhaps MIG-13 acts as an attractive or permissive cue along the migratory pathway to promote anterior Q cell migration.

Lester Laddaran et al. (2004) West Coast Worm Meeting "Genetic mapping of tba-1(ju89) suppressors"

Whitney Benz, Lester Laddaran, Yishi Jin, Luciana Yoshiyasu, Renee Baran

[West Coast Worm Meeting, 2004]

Regulation of microtubule dynamics is important for multiple aspects of neuronal development and function including neuron outgrowth, axonal transport, synapse growth and axon stability. The neuronal functions of many microtubule associated proteins (MAPs) have been difficult to confirm and study in detail because of functional redundancy or an essential role for these proteins during early embryogenesis. We are using a gain-of-function mutation ( ju89 ) of the alpha-tubulin gene tba-1 to identify and study genes that regulate microtubules in synapse development and stabilization. tba-1 is one of nine alpha-tubulin genes in C. elegans , and is widely expressed in the C. elegans nervous system (1). ju89 was isolated originally from an EMS screen for mutants with abnormal expression patterns of the synaptic vesicle marker synaptobrevin::GFP (SNB::GFP). ju89 mutants are uncoordinated, and the number of GABAergic motor neuron synapses is reduced by over 30%. Using a SNB::GFP marker that is specifically expressed by the C. elegans DD motor neurons under the control of the flp-13 promoter (J. Meir and Y. Jin ), we have shown that synapse numbers are reduced in mutant worms when axon outgrowth appears to be normal. Recently, RNAi has been used to show that tba-1 is redundant with a second alpha-tubulin during early embryonic dvisions (2), suggesting the null phenotype of tba-1 is likely wild-type or animals with later stage differentiation defects. The ju89 mutation is located in the tba-1 C- terminus and would alter a residue predicted to lie on the external face of microtubules (3). The location of the mutation and the mutant phenotypes suggest that ju89 alters interactions between MAP(s) and the TBA-1 C terminal region. To understand how ju89 disrupts microtubule regulation we have begun to map and characterize five extragenic suppressor mutants. The ju89 suppressors were identified in an EMS mutagenesis screen for mutations that reverse the uncoordinated and SNB::GFP phenotypes of ju89 . The homozygous suppressors appear to have no observable neuronal phenotype on their own. Progress in mapping and molecular characterization of ju116 , ju130 , ju131 , ju132 and ju287 will be presented. (1) Fukushige et al., J. Mol. Biol.1993; Biochim.Biophys.Acta 1995. (2) Nogales et all., Nature 1998. (3) Phillips et al., Cell Motility and Cytoskeleton, 2004.

Hingwing, Kyla et al. (2011) International Worm Meeting "Identification of genes that function with dsh-2 to regulate asymmetric cell division."

Hingwing, Kyla, Wong, Tammy, Chen, Jack, Hawkins, Nancy

[International Worm Meeting, 2011]

Asymmetric cell division is essential to generate cell diversity during development. In C. elegans, Wnt signaling regulates many asymmetric cell divisions. We have focused our analysis on DSH-2, a key Wnt signaling pathway component. Loss of both maternal and zygotic dsh-2 function results in asymmetric neuroblast division defects and embryonic/early larval lethality, while loss of zygotic dsh-2 function disrupts asymmetric cell division of the somatic gonadal precursor cells, Z1 and Z4. To identify genes that function with dsh-2 in asymmetric division, we undertook a genetic screen to isolate suppressors of dsh-2 lethality. This screen was highly successful and we isolated over 60 suppressors, all of which are dominant. These suppressor mutations could be activating mutations in downstream pathway components or in parallel signaling pathways that function in concert with DSH-2. We have focused our characterization on Sup245, Sup305 and Sup327, which suppress all dsh-2 defects examined thus far. We have further characterized suppressor function in Z1/Z4 division. Asymmetric division of Z1/Z4 involves the reciprocal asymmetric localization of POP-1/TCF and SYS-1/b-catenin in the proximal and distal daughters resulting in a high ratio of SYS-1 to POP-1 ratio in the distal daughter and the specification of distal cell fate. dsh-2 mutants disrupt both POP-1 and SYS-1 asymmetry (1, 2). To determine if Sup245, Sup305 and Sup327 suppress dsh-2 by restoring asymmetric localization of POP-1 and/or SYS-1 we have analyzed the expression of two GFP reporters, Psys-1::SYS-1::GFP (qIs95) and Psys-1::POP-1::GFP (qIs74) in the dsh-2; Sup strains. All three suppressors partially re-establish both POP-1 and SYS-1 asymmetry. Thus, a sufficient SYS-1 to POP-1 ratio is likely restored to specify distal cell fate. Genetic mapping experiments have placed both Sup305 and Sup327 on chromosome I to the left of lin-17. Based on their genetic map position, and similar phenotypes they are likely to be allelic. Sup245 has been mapped to the left of unc-54 on the right arm of chromosome I. To determine the molecular identity of the suppressor mutations, two strains were sent for whole genome sequencing; one containing Sup305 and a second strain containing both Sup327 and Sup245. We are currently analyzing the resulting sequence and testing potential candidates. These studies will identify novel genes that function with the Wnt signaling pathway to control asymmetric cell division. 1. Chang et al., (2005) Mech. Dev. 122:781-789 2. Phillips et al., (2007) PNAS 104:3231-3236.