Baker, Emily et al. (2021) International Worm Meeting "T-box radiation: A window into evolution in real time"

Baker, Emily, Woollard, Alison

[International Worm Meeting, 2021]

Little is known about the genetic changes that catalyse speciation events. While diverging populations are known to accumulate genomic variation over time, examples of causative loci that lead to speciation have been seldom characterised. This is due, in large part, to how speciation can only be studied retrospectively in most systems. However, naturally occurring populations of C. elegans, or wild isolates, diverging for only thousands of years, present a unique opportunity to capture processes which may precede new species divergence, thereby uncovering the genetic hallmarks associated with its onset. T-box transcription factors are key players in metazoan development and exhibit minimal copy number variation throughout the animal kingdom. However, in the Caenorhabditis genus, T-box genes are gained and lost at an unprecedented scale, suggestive of their rapid evolution. Among the suite of ten C. elegans specific T-box genes not found in the rest of the genus are three paralogue pairs all expressed during embryogenesis. In contrast with N2 which retains both functional paralogues, we show that these three pairs display remarkable patterns of mutation accumulation in wild isolates, with one or other genes in the pair accumulating loss-of-function mutations, but never both. The phenotypic consequences of this reciprocal pattern of mutation accumulation could have far-reaching implications for our understanding of speciation if, for example, such patterns led to hybrid inviability. To investigate this, we have characterised the roles of the tbx-35/tbx-36 gene pair. The reciprocal nature of deleterious mutation accumulation in wild isolates in the tbx-35/tbx-36 gene pair suggests that they act redundantly. However, tbx-35 has been shown to be involved in muscle specification during mid-embryogenesis, distinct from the role and expression of tbx-36 in early embryos that we describe here. Strikingly however, we find that overlapping functionality is indeed revealed when the environmental conditions are changed, suggesting that tbx-36 has in fact retained a role in muscle specification that is not necessarily seen under standard laboratory conditions. If the early embryonic role of tbx-36 is N2-specific, this could be a lab adaptation, raising the possibility that N2 is on the road to becoming a new species due to the lab environment. Thus, the rapidly evolving T-box gene family provides a paradigm for investigating evolution and speciation in action in C. elegans populations today.

Kathleen Estes et al. (2007) International Worm Meeting "C. elegans vulva development as a model for determining the role of UNC-6 Netrin in tissue morphogenesis."

Kathleen Estes, Wendy Hanna-Rose

[International Worm Meeting, 2007]

UNC-6 Netrin is a well-studied axon guidance molecule. Recently, this protein has been observed acting in morphogenesis of different tissues1. C. elegans unc-6 mutants have a well documented egg-laying defect2. Interestingly, a vulva morphology defect has also been reported but not well-characterized2. We are currently conducting an in-depth phenotypic analysis of the vulva morphology defect in various unc-6 alleles. We are also examining unc-40, the only one of the two well-characterized unc-6 receptors to exhibit an egg-laying defect2. In our initial analysis using an apical junction marker, we have found that the dorsal vulva toroid of unc-6 mutants is severely misshapen and does not seem to display this marker. We also have preliminary data suggesting the anchor cell may not invade the basement membrane, which may contribute to the vulva morphology defect. By studying the phenotype of unc-6 and unc-40 mutants in C. elegans vulva morphogenesis, we can examine this new role of netrin in tissue morphogenesis. 1. reviewed in Baker, et. al. 2006. Curr Opin Neurobiol 16: 529-534 2. Hedgecock, et. al. 1990. Neuron 2: 61-85.

Kao, Aimee Wen Yi et al. (2009) International Worm Meeting "Loss of the C. elegans progranulin homolog, PGRN-1, results in decreased programmed cell death and an age-dependent response to cellular stress."

Farese, Robert, Bagley, Joshua, Kao, Aimee Wen Yi, Kenyon, Cynthia, Nakamura, Ayumi, Eisenhut, Robin

[International Worm Meeting, 2009]

Frontotemporal lobar degeneration (FTLD) is the second most common cause of dementia in those under the age of 65. Mutations in the human progranulin (PGRN) gene have recently been shown to be causal for both inherited and sporadic forms of FTLD (Baker et al., 2006, Hutton et al., 2006). Progranulin is a complex, multifunctional, secreted trophic factor that is expressed in a variety of tissues including neurons and astrocytes (Ahmed et al., 2007). Although PGRN is involved in development, wound healing, inflammation and tumor growth, its function in the nervous system is poorly understood (Eriksen and Mackenzie, 2008). In order to better understand the biological function of progranulin, we decided to study its homolog in C. elegans. Similar to the mammalian protein, the C. elegans progranulin homolog, PGRN-1, is expressed by the intestine and a subset of neurons. pgrn-1 mutant worms appear grossly normal and live a normal lifespan compared to control worms. However, we have found that pgrn-1 mutants have a decreased number of programmed cell deaths during development. In addition, young adult pgrn-1 mutants display resistance to osmotic, thermal and ER stress compared to wild-type worms while older worms are sensitive to osmotic stress. The stress resistance phenotype can be rescued by expression of either worm or human progranulin. We are currently examining whether these constructs also rescue the cell death phenotype. Unlike mutations in the insulin-IGF-1 receptor gene, daf-2, pgrn-1 mutations do not cause resistance to genotoxic or oxidative stress, suggesting a novel pathway for selective stress resistance. This dichotomous response to certain stressors-resistance while young and increased susceptibility with age-may shed light on why individuals with progranulin mutations develop FTLD symptoms late in adulthood and may explain the variable age of disease onset seen with identical mutations. Interestingly, our findings suggest that susceptibility to neurodegeneration may be determined during a developmental period when excess or unneeded neurons undergo programmed cell death.

Wang, A. et al. (2019) International Worm Meeting "Progranulin Polymorphisms in Aging and Stress Resistance."

Kao, A, Cortopassi, W, Butler, V, Jacobson, M, Wang, A.

[International Worm Meeting, 2019]

The cysteine-rich protein progranulin (PGRN) has been implicated in the pathobiology of several diseases including neurodegeneration and lysosomal storage related diseases (Baker, Nature 2006; Cruts, Nature 2006; Neumann, Science 2006; Smith, Am J Hum Genet 2012). Progranulin can be processed into bioactive granulin peptides, the number of which vary from species to species. C. elegans progranulin contains 3 granulins. Despite the variance in number of domains, the granulins share a stable, compact structure of beta-pleated sheets connected by disulfide bonds(Palfree, Plos One 2015). Studies have shown that loss of progranulin leads to features of accelerated aging, such as lipofuscin deposits and decreased lysosome protease activity (Ahmed, The American Journal of Pathology 2010; Ward, Science Translational Medicine 2017). In killifish, progranulin polymorphisms segregate with a short-lived aging phenotype (H151Q, C449W, R158H) (Valenzano, Cell 2015). The cysteine to tryptophan polymorphism is particularly interesting due to the cysteine's structural contribution to disulfide bonds. In addition, the histidine to glutamine polymorphism could potentially alter pH sensitivity. We have shown that pgrn-1(-) animals demonstrate a robust, stress-resistant phenotype that interestingly, can be decoupled from lifespan extension (Butler, J Neurosci 2019; Meredith, Plos Genetics 2013). Furthermore, progranulin acts in the lysosome and directly increases the activity of cathepsin-D (CTSD), a crucial lysosomal protease (Butler, Human Molecular Genetics 2018). We hypothesize that while complete absence of progranulin and granulin does not affect lifespan, that the polymorphisms in the granulin domains will impact aging and lifespan as well as stress response via regulation of lysosomal protease activity. To address this, we have created single copy insertions using MosSCI of these progranulin SNPs into C. elegans. We plan to test lifespan of these worms. We will also measure markers of aging and test stress response. To better understand potential molecular mechanisms, we will use molecular modeling to elucidate changes to progranulin and CTSD/ASP-3 interactions and test these in vivo. We anticipate that certain progranulin polymorphisms will increase lifespan and healthspan. We also predict that stress resistance will improve. We anticipate these will be a result of progranulin polymorphisms enhancing proteostasis via improved lysosomal protease activity.

Stephen D Fields et al. (2003) International Worm Meeting "HUM-2, a myosin V homologue, is expressed in head neurons and affects the basal slowing response to food."

John R McManus, Stephen D Fields, Jim Rand, Greg Mullen

[International Worm Meeting, 2003]

Myosin V, an unconventional myosin, seems to be common to most organisms but takes part in rather diverse processes, including localization of mRNA, recycling plasma membrane receptors, and positioning melanosomes and synaptic vesicles. Myosin V is expressed in mammalian nervous systems, and mutations in human myosin V are associated with at least one neurological disease. Accordingly, we are interested in whether HUM-2, the C. elegans homologue of myosin V (Baker and Titus, 1997), is expressed in neurons. HUM-2 is predicted to have several alternatively spliced variants, the longest of which is HUM-2b. We performed 5' RACE for the hum-2b transcript and found that the actual start codon does not correspond to the site predicted by genefinder. We used the PCR-based method of Hobert et al (2002) to fuse the upstream promoter region and first exon of hum-2b to GFP. This product, along with pBX, was injected into Pha-1 worms, and seven GFP-expressing transgenics were obtained. Most of the transgenic lines express GFP in six to eight head neurons with cell bodies in the anterior and lateral ganglia. At least four of these neurons send processes to the nose as well as into the nerve ring and occasionally into the ventral nerve cord. Those worms showing the strongest expression also have one or two GFP-positive cell bodies in the pre-anal ganglion, as well as the anal sphincter muscle. In addition, all lines express the promoter driven GFP in the pharyngeal-intestinal valve, and several lines weakly express GFP in the excretory system. The C. elegans knockout consortium has isolated a 2.1 kb deletion in the hum-2 gene (OK596) that begins in the region corresponding to the motor domain of the predicted protein. This deletion is expected to result in a truncated, null protein. The outcrossed hum-2 mutants do not have an obvious phenotype although they do not exhibit the typical slowing response upon entering food, suggesting that there is either a mechano- or chemosensory defect. We are in the process of identifying the relevant neurons, but the GFP expression pattern and the modulation defect in the mutant suggets that the dopaminergic neurons are involved. Finally, we are determining the expression pattern of the predicted splice variants of HUM-2, including HUM-2c, which is a truncated version of HUM-2b lacking the motor domain.

Anne Hart (2001) International C. elegans Meeting "Using C elegans to teach genetics: SNP mapping in three weeks!"

Anne Hart

[International C. elegans Meeting, 2001]

C. elegans has been used to the teach fundamentals of genetic mapping for the last three years in the Neurobiology summer course at the Marine Biological Laboratories (MBL). The nine week course is primarily aimed at graduate students and postdoctoral fellows. The molecular section of the course I help teach lasts three weeks including lectures in the morning and lab work in the afternoons and evenings. Two students work with each of the six faculty members in the molecular section of the course. The two students assigned to me spend about 120 hours each working in the lab spread out over a three week period. Consistently, the students have had no experience in C. elegans and, often, little or no prior experience in molecular biology. Given the short duration the molecular section, and the desire to accomplish something of substance, the project has to be chosen with care. The short life cycle of C. elegans makes it an ideal organism to teach the fundamentals of genetic mapping in a limited amount of time. The project chosen last year was recombinant mapping of an interesting mutation, rt70 , using single nucleotide polymorphisms (SNPs) from the Hawaiian strain, CB4856. rt70 causes ASH neuron degeneration and is described in an abstract/poster by Emily Bates, et al. Prior to the start of the course last year, a lon-2(e678) rt70 egl-15(n484) chromosome was generated. All mutations are recessive. Heterozygous lon-2 rt70 egl-15 / CB4856 animals were generated and picked one per plate. These heterozygous animals were transported to MBL along with hundreds of seeded plates. The students practiced picking C. elegans , then singled animals carrying recombinant chromosomes/ lon-2 rt70 egl-15 for the first few days (putative recombinant lines). We attempted to generate homozygous recombinant lines by picking single 9 animals in the next generation and were successful about 80%of the time; the remaining 20% were repicked for analysis at a later date. The homozygous recombinant lines were 1) frozen in a 96 well format using Michael Koelle's protocol and 2) allowed to starve and lysed for PCR analysis using previously defined SNPs identified by Stephen Wicks. To facilitate analysis, only previously characterized SNPs creating a restriction site polymorphism were utilized. Screening more than 100 lines by PCR was not a problem and we were able to refine the rt70 map position. The biggest difficulty we had was scoring egl-15(e484) . In retrospect, I should have chosen marker mutations with very obvious phenotypes so they could be scored unambiguously and easily. PCR reactions were optimized while we were waiting for the recombinant strains to grow. The MBL students also had time to learn how to generate transgenic C. elegans by microinjection. PCR machines for the course were generously loaned by MJ Research, Taq was kindly provided by Fisher and NEB generously donated restriction enzymes. Thanks to all who donated their time, reagents and help- especially MBL students Michael Long and Ryohei Yasuda for their unflagging enthusiasm!