Petrella, Lisa N. et al. (2013) International Worm Meeting "Natural Variants of C. elegans demonstrate defects in both sperm function and oogenesis at elevated temperatures."

Petrella, Lisa N., Strome, Susan, Buskuskie, Thomas

[International Worm Meeting, 2013]

The temperature sensitivity of the germ line is conserved from nematodes to mammals. Previous studies in C. briggsae and Drosophila showed that strains originating from temperate latitudes lose fertility at a lower temperature than strains originating from tropical latitudes. Through analysis of 22 different wild-type isolates of C. elegans originating from both temperate and tropical locations, we are investigating mechanisms underlying maintenance of fertility at high temperature. We determined the fertility of temperate and tropical strains of C. elegans and observed no correlation between latitude of strain origin and fertility at high temperature. We also observed a wide distribution of population fertility among wild-type isolates at 27 deg C, ranging from 7% to 56%. To better understand the causes of high temperature sterility, temperature shift experiments were performed. Males up-shifted to high temperature as L4/young adults maintain fertility, while males raised at high temperature lose fertility. Sterile animals contain a wild-type-appearing germ line. Down-shifting males raised at high temperature does not restore fertility. This result differs from that observed in Drosophila and suggests that in C. elegans spermatogeneis is irreversibly impaired in males that develop at high temperature. Mating and down-shift experiments with hermaphrodites were performed to investigate the relative contributions of spermatogenic and oogenic defects to high temperature loss of fertility. We identified isolates that demonstrate predominantly spermatogenic defects, strains that show a mixture of spermatogenic and oogenic defects, and one strain that shows predominantly oogenic defects. Our studies have uncovered unexpectedly high variation in both the loss of fertility and problems with sperm function in natural variants of C. elegans at high temperature. One variant provides a novel example of oogenic defects underlying loss of fertility. These variants can now be used to investigate the molecular mechanisms that underlie the buffering of fertility in the face of environmental change.

Petrella, Lisa N. et al. (2011) International Worm Meeting "Antagonizing germline fate in somatic cells: opposing roles of the MES and synMuv B chromatin regulators."

Strome, Susan, Rechtsteiner, Andreas, Spike, Caroline A., Petrella, Lisa N., Wang, Wenchao

[International Worm Meeting, 2011]

The newly fertilized embryo inherits a chromatin state with germline characteristics. As development proceeds, those cells destined to generate somatic tissues must be reprogrammed to abolish this germline chromatin state. Previous studies demonstrated that a subset of synMuv B mutants ectopically express germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision (Unhavaithaya et al. 2002 Cell 111:991; Wang et al. 2005 Nature 436:593). Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. We have shown that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage (Petrella et al. 2011 Development 138:1069). High temperature arrest (HTA) is preceded by the expression of germline genes in somatic cells starting in late embryogenesis. This leads to somatic expression of P-granule proteins, components of the synaptonemal complex, and other meiosis proteins. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae. HTA is suppressed by loss of global regulators of germline chromatin, including the MES proteins, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. Consistent with this, the somatic cells of synMuv B mutants display elevated levels of chromatin marks that are normally enriched in the primordial germ cells. We are currently exploring how chromatin states are affected in synMuv B mutants and by elevated temperature, using nuclear spot assays.

Petrella, Lisa N. et al. (2009) International Worm Meeting "Numerous synMuv B genes show temperature sensitive early larval arrest and ectopic germline gene expression that is suppressible by germline chromatin modifiers."

Rechsteiner, Andreas, Spike, Caroline A., Wang, Wenchao, Petrella, Lisa N., Strome, Susan

[International Worm Meeting, 2009]

In many animals the primordial germ cells are separated from the somatic lineages very early in development. In C. elegans this separation begins as early as the first embryonic cell division and is complete by the 24-cell stage. Newly hatched L1 larvae contain only two primordial germ cells, Z2 and Z3, which can be distinguished from somatic cells by the presence of P granules. Recent studies have revealed that mutants in several of the synMuv B class of chromatin regulators display phenotypes that suggest a partial transformation of soma to germ line. These phenotypes include ectopic expression of P-granule components in somatic cells of L1 larvae and hypersensitivity of worms to RNAi. In addition, the synMuv B gene mep-1 shows an early larval arrest phenotype (Unhavaithaya et al., 2002; Wang et al., 2005). These phenotypes can be suppressed by mutations in the germline chromatin regulators mes-2, mes-3, mes-6 and mes-4. Here we show that 9 of 15 synMuv genes tested arrest as L1 larvae at elevated temperature. The synMuv B mutants that arrest are those that express P-granule components in somatic cells. The high temperature L1 arrest phenotype is suppressed by mutations or RNAi of the mes genes and a subset of other known synMuv B suppressors. We determined that the synMuv B L1 arrest phenotype requires loss of both maternal and zygotic synMuv B gene function, is not dependent on the daf-2 insulin signaling pathway, and is irreversible. Microarray analysis showed that, in addition to P-granule genes, several other classes of germline-specific genes are also over-expressed in lin-35 and lin-15B arrested L1s. This over-expression is suppressed by RNAi of mes-4. Immunostaining for two over-expressed meiosis factors revealed ectopic somatic expression in arrested L1s, similar to that of P-granule components. We are currently performing a genome-wide RNAi screen to identify additional genes that can suppress the L1 arrest phenotype, with the goal of identifying chromatin regulators that participate along with the MES proteins in the crucial germ versus soma decision.

Matthews LR et al. (1994) Worm Breeder's Gazette "A Potential C. elegans E2F Homolog: Is it zyg-9?"

Galas S, Matthews LR

[Worm Breeder's Gazette, 1994]

A potential C. elegans E2F homolog: Is it zyg-9? Lisa Matthews and Simon Galas, CRBM, CNRS Montpellier, France

Fealey, Meghan E et al. (2015) International Worm Meeting "Loss of synMuv B proteins resuls in extended open chromatin environments during early development."

Fealey, Meghan E, Petrella, Lisa N

[International Worm Meeting, 2015]

The development of an organism requires cell fate decisions that are dictated by changes in gene expression and chromatin environment. As development proceeds, gene products not needed in a particular cell type are packaged into compact chromatin environments whereas genes important in cell identity remain primed for gene expression in an open chromatin state. synMuv B proteins are a group of chromatin regulators important for correct deployment of the epigenome throughout development. synMuv B proteins include LIN-15B and members of the DRM complex, such as LIN-35 and LIN-54. Mutations in many synMuv B genes result in a high temperature larval arrest phenotype due to ectopic expression of germline genes in somatic cells (Petrella et al., 2011). This phenotype can be rescued by loss of germline chromatin modifiers, suggesting that changes in chromatin states underlie the temperature-sensitive gene misexpression in synMuv B mutants. To investigate changes in chromatin in synMuv B mutants we are using the lacO/LacI system and FISH techniques to visualize chromatin compaction at elevated temperatures. We analyzed the level of compaction of lacO containing extrachromosomal arrays in two ways: visually scoring arrays as either open or closed and by using Metamorph software to quantify the volume of the array contained in the nuclei. We analyzed wild-type, lin-15B, lin-35, and lin-54 mutant intestinal cells at 8E, 16E and L1 developmental stages at 20degC and 26degC. Open chromatin found in early development is closed and compacted in wild-type embryos by the 8E stage. We found that chromatin compaction is delayed in synMuv B mutants. At 20degC many cells at the 8E stage in synMuv B mutants contain open arrays, but the majority of these arrays are closed by the 16E stage. Increased temperature enhances this phenotype, as synMuv B mutant embryos exhibit prolonged open arrays at both 8E and 16E stages at 26degC. synMuv B mutants at high temperature exhibit open chromatin during a developmental window where ectopic expression of germline genes in somatic tissue begins to occur in these mutants. Open chromatin during this crucial developmental time point may be the reason synMuv B mutants incorrectly express germline genes in somatic tissue. Genes located on improperly open chromatin may be poised for ectopic expression during development and lead to the high temperature larval arrest demonstrated by synMuv B mutants. FISH experiments to look at the native genomic loci are underway in order to investigate the chromatin environment at synMuv B regulated genes. .

Uttal, Katherine et al. (2021) International Worm Meeting "Temperature stress effects cytoplasmic streaming during oogenesis"

Uttal, Katherine, Petrella, Lisa N

[International Worm Meeting, 2021]

Apoptosis during oogenesis is highly conserved in animals. During Caenorhabditis elegans oogenesis, roughly fifty percent of germ cells undergo apoptosis. While C. elegans oogenesis is widely studied, it is unclear why this mass cell death occurs. Under moderate temperature stress, there is a decrease in worm brood size but an increase germline apoptosis. There are two proposed hypotheses for the function of increased germline apoptosis that would aid in developing fit progeny under temperature stress. The first is that apoptotic cells serve as nurse cells, donating their cytoplasmic materials to developing oocytes. An increase in apoptosis during temperature stress may help ensure that developing oocytes receive sufficient cytoplasmic materials when an embryo is born under stress conditions. The second hypothesis is that apoptosis serves to remove defective cells that are made due to problems with oogenesis under moderate temperature stress. If the first hypothesis is correct and apoptotic cells act as nurse cells, an increase in apoptosis and thus an increase in cytoplasmic donations under temperature stress would also increase the rate of cytoplasmic streaming in the germline. Additionally, in mutants that have reduced apoptosis compared to wild type worms, the rate of cytoplasmic streaming would decrease compared to wild type animals at the same temperature. To test if increased apoptosis contributes increased amounts of cytoplasmic materials to developing oocytes, DIC microscopy was used to video tape live anesthetized young adult N2 worms raised at 20°C as well as worms upshifted to 26°C for 24 hours. Six worms were taped under both temperature conditions. Using ImageJ, five particles within the rachis of the germline were tracked in each worm over a two-minute period and each particles average velocity was calculated (n=30). Our analysis showed a significant increase in the rate of cytoplasmic streaming in worms upshifted to 26°C compared to worms raised at 20°C. Additional imaging is currently being performed on lin-35 and lin-54 mutants, which have been shown to have decreased apoptosis at high temperatures. Because these mutants do not show an increase in apoptosis under temperature stress, it is expected that their analysis will not show a significant increase in rate of cytoplasmic streaming as seen in the wildtype.

Nett, Emily M. et al. (2017) International Worm Meeting "Decreased mating ability may play a role in C. elegans male low fertility at high temperature."

Petrella, Lisa N., Nett, Emily M.

[International Worm Meeting, 2017]

The germline, especially the spermatogenic germline, is temperature-sensitive, such that as the temperature increases, germline function and fertility drop rapidly. Our previous work has shown wild type strains of C. elegans males raised at 27 deg C are almost all sterile, while males raised at 20 deg C but upshifted to 27 deg C for mating are almost all fertile. Additionally, there are significant differences between wild type strains in the ability of males to regain fertility upon downshifting from 27 deg C to 20 deg C (Petrella, 2014). High temperature loss of male fertility could be due to loss of sperm function, reduced sperm number, changes in mating ability, or all three. To test sperm function, we have analyzed the rates of in vitro sperm activation in males that were raised at 27 deg C. We found that activation rates are reduced by approximately 10-15%. This level of decreased activation is not sufficient to explain the almost complete loss of fertility seen at this temperature. Experiments are currently underway to quantify the number of sperm produced by males at 27 deg C. However, as the number of sperm does not decrease with temperature in hermaphrodites, we anticipate there may not be a significant difference in males. Finally, male mating interest and execution were tested with males raised at 27 deg C. All wild type strains show declines in multiple steps of male interest and mating execution during timed assays. However, the decrease in total successful mating events at 27 deg C differed significantly between strains. Our data indicate that reduced sperm activation is not the main cause of low fertility at 27 deg C but that decreased mating ability may play an important role. As loss of fertility at high temperature is conserved across phyla, our research may uncover conserved temperature-sensitive germline pathways that account for the germline temperature sensitivity.

Mikeworth, Brian P. et al. (2015) International Worm Meeting "LIN-35 is Necessary for Germ Line Development and Function When Faced With High Temperature Stress."

Petrella, Lisa N., Mikeworth, Brian P.

[International Worm Meeting, 2015]

It is necessary for all organisms to protect themselves from ever changing environmental conditions to maintain viability and fertility. It has recently been observed that members of the nematode DRM complex, such as lin-35 and lin-54, play a role in germline gene regulation in the both the soma and germ line. It has been shown that loss of LIN-35 leads to a high temperature larval arrest (HTA) phenotype when grown at 26degC but not at 20degC. This is due to ectopic expression of germline genes within the intestine, leading to failure of intestine cell function and starvation of the organism (Petrella et al. 2011). Rescue of lin-35 via extrachromosomal array expression in the intestinal cells (elt-2p::lin-35) or all somatic tissues (let-858p::lin-35) allows for lin-35 mutants raised at 26degC to develop to adulthood with germ lines that lack LIN-35. Although both lin-35 transgenic strains can develop germ lines when raised at 26degC, these animals display a weak fertile or sterile phenotype. Conversely, both transgenic strains are fertile when raised at 20degC. We found that upshifting either transgenic line at the L4 larval stage from 20degC to 26degC resulted a significant decrease in fecundity, thus suggesting that LIN-35 plays a functional role in maintaining proper germ line function when faced with high temperature stress. Temperature sensitive sterility was not rescued in transgenic hermaphrodites raised at 26degC and mated with wild-type (N2) males, suggesting that oocyte development or fertilization may require LIN-35 to function properly. However, when assessing the ability for male sperm to localize in both transgenic strains, it was observed that the sperm were not properly localized to the spermatheca of mutants with elt-2p::lin-35 raised at 26degC, where male sperm was found in the spermatheca of mutants with let-858p::lin-35 raised at 26degC. This data suggests that LIN-35 may be necessary for the proper development or function of the spermatheca, or for the expression of proper cues for sperm localization under high temperature stress conditions. Upon investigating germline development via DIC microscopy, oocytes were not observed beyond the bend in the gonad arm of mutants with elt-2p::lin-35 raised at 26degC, where they were observed in mutants with let-858p::lin-35 raised at 26degC. This suggests that the LIN-35 may aid in cross talk between the somatic gonad and germ cells to initiate oocyte development in high temperatures situations. Taken together, our lab has found that LIN-35 plays a multitude of roles in the development and function of the germ line and the somatic gonad in C. elegans when faced with high temperature stress.

Mikeworth, Brian P. et al. (2013) International Worm Meeting "Temperature Sensitive Fertility of lin-35 Mutants."

Petrella, Lisa N., Mikeworth, Brian P.

[International Worm Meeting, 2013]

Temperature plays a critical role in the ability for an organism to become fertile. High environmental temperatures cause infertility in organisms from nematodes to mammals. One mechanism that may play an important role in high temperature infertility is improper buffering of gene expression levels during germline development, spermatogenesis, or oogenesis. SynMuv B proteins are transcriptional repressors that act to suppress expression of germ line-specific genes within somatic cells. Loss of synMuv B proteins cause high temperature larval arrest (HTA) phenotype resulting in synMuv B mutants that arrest at the L1 stage of development at 26 deg C. While investigating the HTA phenotype we found that lin-35 mutant L1s display defects in P-granule morphology and changes in histone modifications in primordial germ cells (PGCs). It has been shown that lin-35 mutants have decreased fertility at moderate temperatures although the mechanism is not understood. We are investigating temperature sensitivity of the germ line of lin-35 mutant. In order to test the temperature sensitivity of fertility in lin-35 mutants we are taking advantage of a strain that rescues the HTA phenotype by expressing LIN-35(+) in the intestine but leaves the germ line deficient for LIN-35. Initial experiments demonstrate that lin-35 mutant L1s raised at 26 deg C displayed a range of abnormal P-granule protein localization when compared with mutants raised at 20 deg C or wild type PGCs at either temperature. To determine the degree of this phenotype, we are scoring the frequency of P-granule defects in the PGCs of lin-35 mutant L1s raised at 20 deg C and 26 deg C. Additionally, to determine if lin-35 mutants show high temperature loss of fertility, we are scoring the fecundity of lin-35 mutants raised at 20 deg C and 26 deg C. If a high penetrance of P-granule defects and low fecundity are scored and correlated in lin-35 mutants raised at 26 deg C, it could suggest that defects in P-granules in the PGCs could lead to a defective germ line in adult mutants and cause infertility at high temperatures. Finally, we are assessing all stages of germline development to determine at what stages germline defects begin and if they extend throughout larval development.

Sepulveda, Nicholas B et al. (2021) MicroPubl Biol

Petrella, Lisa N, Sepulveda, Nicholas B

[MicroPubl Biol, 2021]

Exposure to moderate temperature stress can have profoundly negative effects on an organism's reproductive capacity at temperatures where there are minimal or indiscernible effects on the organism as a whole. These negative effects are often more pronounced in males of the species that produce sperm. Previously we showed that few males of Caenorhabditis elegans wild type strains are able to successfully produce any cross progeny after experiencing temperature stress. However, these experiments did not assess the number of progeny from temperature stressed males. To understand if temperature stress can reduce the number of progeny a male sires, we crossed temperature stressed males of three wild type strains of C. elegans: JU1171, LKC34, and N2, to strain matched hermaphrodites of their own genetic background or to uncoordinated hermaphrodites in the N2 background. We found that significantly fewer males exposed to moderate temperature stress can successfully mate and that the small number of males in the population that do successfully mate produce significantly fewer viable cross progeny than unstressed controls. Our results suggest that exposure to moderate temperature stress significantly reduces male C. elegans chances at reproducing similar to what is seen in other organisms.