Osborne, Jennifer F et al. (2021) MicroPubl Biol

Hart, Anne C, Osborne, Jennifer F, Yanagi, Katherine S

[MicroPubl Biol, 2021]

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. While the mechanisms underlying motor neuron death in ALS are not well understood, mutations in over 25 genes can cause this disease (Marangi and Traynor 2015). It remains unclear which, if any, of these genes act in the same disease-associated pathway(s), or if they act in the same pathway(s) as genes associated with the related disorder, frontotemporal dementia (FTD) (Ling et al. 2013). The first ALS-causing gene to be identified was superoxide dismutase 1 (SOD1), a regulator of cytoplasmic redox homeostasis (Rosen et al. 1993). We can begin to construct a pathway for neurodegeneration through SOD1 by identifying genes whose loss of function (LOF) modifies the level of degeneration in a C. elegans SOD1 ALS model. This will contribute to our understanding of whether ALS/FTD genes act in a single or multiple pathways to cause disease.

Osborne BA (1996) Trends Genet "Cell death in vertebrates: lessons from the worm."

Osborne BA

[Trends Genet, 1996]

Several years ago, Horvitz and Sulston demonstrated that it is the fate of 131 cells to die during development of the small nematode, Caenorhabditis elegans. The recognition that these particular cells are destined to die at exactly the same time in each and every worm suggested that the control and, perhaps, even the elements of this program were genetically determined. From these beginnings, the still-unfolding genetics of cell death has emerged. During the past 10 years, classical genetic approaches have identified loci in worms that either positively or negatively regulate cell death during development. Recently, some of the genes encoded by these loci have been cloned and sequenced. The results from these studies, coupled with experiments directed at identifying the events that regulate mammalian cell death, have led to the important observation that, in many instances, genes that control cell death in worms are conserved structurally and functionally between nematodes and vertebrates.

Osborne Nishimura, Erin et al. (2015) International Worm Meeting "Cell-specific and sub-cellular mRNA patterning in early embryogenesis."

Lieb, Jason D., Wertz, Adam D., Goldstein, Bob, Tintori, Sophie, Zhang, Jay C., Osborne Nishimura, Erin

[International Worm Meeting, 2015]

We are interested in understanding how the transcriptomes of embryonic cells are regulated at single-cell and sub-cellular levels and how key mRNA transcripts promote lineage-specific development. Using RNA-seq on pooled, hand-dissected AB and P1 blastomeres, we identified over 200 transcripts that were asymmetrically patterned after the first cell division in the C. elegans zygote. We confirmed asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of some transcripts including chs-1 and bpl-1 that were granularly distributed in cells of the P lineage. We screened asymmetric transcripts for embryonic defects using RNAi and identified the gene neg-1 as an AB-enriched (anterior) transcript that was required for proper morphology of anterior tissues. In addition, we identified sequence motifs and features that were over-represented in asymmetric transcripts, giving us clues to regulatory mechanisms. Current research includes efforts to unveil mechanisms responsible for cellular and subcellular patterns of mRNA abundance. Further, we are using single-cell transcriptome profiling to identify blastomere-specific transcripts in the 4-cell to 16-cell stages of embryogenesis.

Osborne Nishimura, Erin et al. (2013) International Worm Meeting "Single-blastomere transcriptome profiling after the first embryonic division."

Osborne Nishimura, Erin, Lieb, Jason D., Goldstein, Bob, Werts, Adam, Zhang, Jay C.

[International Worm Meeting, 2013]

We are interested in understanding how reproducible patterns of RNA inheritance and abundance arise in dividing cells, and for what purpose. The C. elegans early embryo represents a unique biological system to study this problem because the lack of zygotic transcription removes the potential complication of transcriptional fluctuations. Following fertilization and prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres. To identify asymmetrically distributed transcripts, we dissected the AB and P1 blastomeres and performed low-input RNA-seq. Our results are consistent with a recently published study with a similar experimental design (Hashimshony et al, 2012 Cell Reports). We developed a scoring method for ranking transcripts, verified asymmetric patterns using qRT-PCR and in situ hybridization, and narrowed our analysis to a subset of transcripts that represent the most asymmetric RNAs in each cell at the 2-cell stage. AB and P1 are the founders of two lineages with distinct fates. We found that transcripts enriched in AB tend to encode proteins that function in mitosis and epithelium development, whereas transcripts segregated to the P1 are associated with translational regulation. To test whether any of the genes in our AB-enriched or P1-enriched subsets play a functional role in asymmetric cell division or downstream embryonic patterning, we performed controlled RNAi phenotyping and video microscopy to categorize embryological defects. Using these methods, we identified F32D1.6, an AB-enriched (anterior) transcript with a role in anterior-specific morphological events. In addition, two posterior enriched transcripts, T04A8.7 and puf-3, were found to exhibit noteworthy early embryological defects. We are currently identifying sequence elements and RNA binding proteins that are required for this process with the goal of elucidating the mechanisms behind asymmetric RNA partitioning.

Osborne, Erin A et al. (2011) International Worm Meeting "Single-blastomere transcriptome profiling reveals asymmetric segregation of maternal transcripts in the first embryonic division."

Lieb, Jason D, Osborne, Erin A

[International Worm Meeting, 2011]

By coupling blastomere dissection with RNA-seq, we have characterized the identity and abundance of each RNA transcript in the AB and P1 cells at the 2-cell stage of development. Our results confirm the asymmetric segregation of many previously identified transcripts such as mex-3, which is enriched in the AB cell, and cey-2, enriched in the P1 cell. In addition, our study significantly expands the list of asymmetrically patterned transcripts. Our method of linear RNA amplification followed by RNA-seq has generated transcriptome profiles that are highly correlated with traditional RNA-seq protocols, and the technical reproducibility is high. We plan to expand this study to discover factors important for cell fate specification and to gain a mechanistic understanding of how a single pool of maternal RNA in the oocyte gives rise to distinct populations of RNA and proteins in individual cells during early development.

Osborne N et al. (2021) Biochem Pharmacol "CYP4V2 fatty acid omega hydroxylase, a druggable target for the treatment of metabolic associated fatty liver disease (MAFLD)."

Leahy C, Song BJ, Hardwick JP, Osborne N, Kwang Lee Y

[Biochem Pharmacol, 2021]

Fatty acids are essential in maintaining cellular homeostasis by providing lipids for energy production, cell membrane integrity, protein modification, and the structural demands of proliferating cells. Fatty acids and their derivatives are critical bioactive signaling molecules that influence many cellular processes, including metabolism, cell survival, proliferation, migration, angiogenesis, and cell barrier function. The CYP4 Omega hydroxylase gene family hydroxylate various short, medium, long, and very-long-chain saturated, unsaturated and polyunsaturated fatty acids. Selective members of the CYP4 family metabolize vitamins and biochemicals with long alkyl side chains and bioactive prostaglandins, leukotrienes, and arachidonic acids. It is uncertain of the physiological role of different members of the CYP4 omega hydroxylase gene family in the metabolic control of physiological and pathological processes in the liver. CYP4V2 is a unique member of the CYP4 family. CYP4V2 inactivation in retinal pigment epithelial cells leads to cholesterol accumulation and Bietti's Crystalline Dystrophy (BCD) pathogenesis. This commentary provides information on the role CYP4V2 has in metabolic syndrome and nonalcoholic fatty liver disease progression. This is accomplished by identifying its role in BCD, its control of cholesterol synthesis and lipid droplet formation in c. elegans, and the putative function in cardiovascular disease and gastrointestinal/hepatic pathologies.

Osborne Nishimura E et al. (2015) PLoS Genet "Asymmetric transcript discovery by RNA-seq in C. elegans blastomeres identifies neg-1, a gene important for anterior morphogenesis."

Zhang JC, Goldstein B, Lieb JD, Werts AD, Osborne Nishimura E

[PLoS Genet, 2015]

After fertilization but prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres, each of which goes on to generate distinct cell lineages. To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division, we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq. Our approach identified over 200 asymmetrically abundant mRNA transcripts. We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1. We screened transcripts enriched in a given blastomere for embryonic defects using RNAi. The gene neg-1 (F32D1.6) encoded an AB-enriched (anterior) transcript and was required for proper morphology of anterior tissues. In addition, analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions, which are common in developing organisms.

Osborn, Gregory et al. (2013) International Worm Meeting "Genomic analysis of the duct and pore cells reveals novel effectors and regulators of morphogenesis."

Walton, Travis, Osborn, Gregory, Sundaram, Meera, Murray, John

[International Worm Meeting, 2013]

Organogenesis involves complex cellular processes such as migration, morphological changes and differentiation, which are frequently orchestrated by the integration of regulatory networks. To understand how these cellular behaviors are coordinated at single cell resolution, we are studying the C. elegans excretory system, which contains three tubular cells: the duct, pore and canal cell. During embryogenesis, the excretory duct and pore cell first exhibit mesenchymal characteristics as they migrate from disparate locations to meet the canal cell at the ventral midline, where they change shape, differentiate into epithelia, and convert into a contiguous three cell tubular organ. To identify molecular factors involved in these complex processes, we are characterizing the transcriptomes of these cells during development. To isolate the duct and pore, we have taken a fluorescence-activated cell sorting (FACS) strategy, which allows for isolation of cells expressing a fluorescent reporter of interest. Specific early markers for the duct and pore during organogenesis are not known; thus we have taken an intersectional approach, sorting for cells expressing both ceh-6::GFP and hlh-16p::mCherry in C. elegans embryos. The expression of these reporters overlaps exclusively in the duct and pore, as well as their sisters, the DB1/3 motor neurons. RNA-seq of these cells shows an enrichment of factors known to be important in the formation of the excretory system, such as EGF signaling components (let-23, lin-1) and genes involved in proper tube formation and function (aff-1, let-4, lpr-1). Interestingly, novel factors were also enriched, including many HOX transcription factors and proteins predicted to be secreted or membrane-localized. This dataset could be augmented in the future by using additional available markers to separate the duct/pore from the DB1/3 motor neurons by FACS and determining the expression signatures unique and shared between these cells. This work will provide a framework to identify novel candidates to functionally test as regulators and effectors of the complex cellular behaviors of the developing excretory system.

Sokolowski MB (1998) Neuron "Genes for normal behavioral variation: recent clues from flies and worms."

Sokolowski MB

[Neuron, 1998]

The question of how genes contribute to normal individual differences in behavior has captured our imagination for more than a century. Several fundamental questions come to mind. How do genes and their proteins act in the nervous system and in response to the environment in order to cause individual differences in behavior? Do genetic differences between natural variants arise from alterations in the structural or regulatory region of a gene? Can we predict which genes for behavior, identified by mutant analysis in the laboratory, will have natural allelic variation? Three groundbreaking studies (Osborne et al., 1997; Sawyer et al., 1997; de Bono and Bargmann, 1998) published in the past year demonstrate that we now have the knowledge and technological capability to address these questions empirically. Each study has successfully identified a single major gene for a given behavior and, with the aid of transgenic animals, shown that its gene product is responsible for naturally occurring individual differences

Negga R et al. (2012) Neurotox Res "Exposure to glyphosate- and/or Mn/Zn-ethylene-bis-dithiocarbamate-containing pesticides leads to degeneration of -aminobutyric acid and dopamine neurons in Caenorhabditis elegans."

Richardson SJ, Negga R, McVey KA, Fitsanakis VA, Lizek AJ, Stuart JA, Salva J, Machen ML, Osborne AS, Mirallas O

[Neurotox Res, 2012]

Previous studies demonstrate a positive correlation between pesticide usage and Parkinson's disease (PD), which preferentially targets dopaminergic (DAergic) neurons. In order to examine the potential relationship between two common pesticides and specific neurodegeneration, we chronically (24h) or acutely (30min) exposed two Caenorhabditis elegans (C. elegans) strains to varying concentrations (LC(25), LC(50) or LC(75)) of TouchDown() (TD) as percent active ingredient (glyphosate), or Mancozeb() (MZ) as percent active ingredient (manganese/zinc ethylene-bis-dithiocarbamate). Furthermore, to more precisely model environmental exposure, worms were also exposed to TD for 30min, followed by 30-min incubation with varying MZ concentrations. Previous data from out lab suggested general neuronal degeneration using the worm strain NW1229 (pan-neuronal//green fluorescent protein (GFP) construct). To determine whether distinct neuronal groups were preferentially affected, we specifically used EG1285 (GABAergic neurons//GFP construct) and BZ555 (DAergic neurons//GFP construct) worms to verify GABAergic and DAergic neurodegeneration, respectively. Results indicated a statistically significant decrease, when compared to controls (CN), in number of green pixels associated with GABAergic neurons in both chronic (*P<0.05) and acute (*P<0.05) treatment paradigms. Analysis of the BZ555 worms indicated a statistically significant decrease (*P<0.05) in number of green pixels associated with DAergic neurons in both treatment paradigms (chronic and acute) when compared to CN. Taken together, our data suggest that exposure to TD and/or MZ promotes neurodegeneration in both GABAergic and DAergic neurons in the model organism C. elegans.