3K Papers found (0.014 seconds)

Kroetz, Mary B. et al. (2011) International Worm Meeting "Sex-specific regulation of gonadal development in C. elegans."

McWhirter, Rebecca D., Zarkower, David, Berkseth, Matthew R., Kroetz, Mary B., Spencer, W. Clay, Miller, III, David M.

[

International Worm Meeting,

2011]

The gonad of C. elegans originates as a four-cell primordium in the embryo. Initially it is morphologically identical in both sexes, but soon after the animal completes embryogenesis the gonad begins to develop into one of two distinct sex-specific organ structures. Despite the extensive sexual dimorphism and previously defined cell lineages of the gonad, the genetic pathways that direct the sex-specific development of this organ remain largely unknown. All sexual dimorphism is under the control of the TRA-1 transcriptional regulator, but no gonadal direct targets of TRA-1 are known. The overall aim of this work is to define the genetic network of sex-specific gonadal development, focusing on the direct targets of TRA-1. To identify the sex-specific gonadal regulators, cell-specific microarray-based mRNA expression profiling was conducted. Single sex populations of animals were generated by employing sex determination pathway mutants. The somatic gonadal precursor cells (Z1/Z4) expressed a gfp reporter allowing for their identification by FACS from dissociated embryonic cells. Z1/Z4-enriched transcripts were identified for both sexes. The majority of proteins with known Z1/Z4-enriched expression were identified by this method, and a number of novel Z1/Z4-enriched transcripts have subsequently been validated by reporter analysis, confirming the effectiveness of this approach. Work is currently underway to validate several of the sex-enriched transcripts. Recently our laboratory has conducted TRA-1 chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Comparison of the sex-specific gonadal transcriptome with the direct targets of TRA-1 as identified by ChIP-seq should identify which sex-specific transcripts are directly regulated by TRA-1 and presumably at the top of the hierarchy of gonadal regulators.

Kroetz, Mary B. et al. (2015) International Worm Meeting "Transcriptome analysis of the sex-specific differences in the somatic gonadal precursor cells in Caenorhabditis elegans."

Zarkower, David, Kroetz, Mary B.

[

International Worm Meeting,

2015]

The C. elegans somatic gonad was the first organ to have its cell lineage determined and the gonadal lineages of the two sexes differ greatly in their pattern of cell divisions, cell migration and cell types. Despite much study, the genetic pathways that direct early gonadal development and establish its sexual dimorphism remain largely unknown, with just a handful of regulatory genes identified from genetic screens. To help define the genetic networks that regulate gonadal development in both sexes, I employed cell-specific RNA-seq. Single sex populations of animals were generated using sex determination pathway mutants and a highly specific GFP reporter was used to isolate the somatic gonadal precursor cells, Z1/Z4 or the Z1/Z4 daughter cells, by FACS of dissociated larval cells from synchronized animals harvested just before and just after the division of Z1/Z4, when somatic gonad sexual differentiation begins. I identified transcripts present in each sex in Z1/Z4 or Z1/Z4-daughter cells and for comparison I also identified the transcripts in each sex in whole animals at both time points. Pairwise comparisons of samples identified several hundred gonad-enriched transcripts including most known Z1/Z4-enriched mRNAs, and reporter analysis has confirmed the effectiveness of this approach. Prior to the Z1/Z4 division only about 25 sex-enriched Z1/Z4 transcripts were detectable, but less than six hours later during development, I identified about 250 sex-enriched transcripts in the Z1/Z4-daughters, of which about a third also were enriched in the somatic gonad cells compared to whole animal. This blossoming of sex-specific gene expression indicates that a robust sex-specific developmental program, some of it gonad-specific, initiates in these cells around the time of the first Z1/Z4 division. Transcripts that have similar patterns in timing and expression are likely to be regulated by a similar mechanism. Sex-enriched gonadal transcripts were categorized based on similar enrichment patterns from the aforementioned pairwise comparisons. Current work is underway to identify and experimentally test shared motifs in conserved regions of promoters of transcripts that belong to the same expression category. This method of cell-specific RNA-seq should not only help define the regulatory networks controlling gonadal development but will also provide an effective general approach to identify genetic networks driving development of other cell lineages in C. elegans.

Kroetz, Mary B. et al. (2013) International Worm Meeting "Identifying regulators of sex-specific gonadal development in C. elegans by cell-specific RNA-seq."

Zarkower, David, Kroetz, Mary B.

[

International Worm Meeting,

2013]

The C. elegans gonad originates in the embryo with the coalescence of a four-cell primordium composed of two somatic precursor cells (Z1/Z4) that flank two germ line precursor cells (Z2/Z3). The gonadal primordium is morphologically identical in the two sexes until the division of Z1/Z4. With the division of these cells, the gonadal primordium begins to develop via one of two distinct sex-specific programs of organogenesis. Despite the extensive sexual dimorphism and previously defined cell lineages of the gonad, the genetic pathways that direct the development of this organ, including its sex-specific development, remain largely unknown. To define the genetic networks that regulate gonadal development in both sexes, we employed cell-specific transcriptional profiling of Z1/Z4 using RNA-seq to identify the early gonadal regulators. Single sex populations of animals were generated by employing sex determination pathway mutants. A Z1/Z4-specific gfp reporter was used to isolate these cells by FACS from dissociated mid-L1 larval cells, just prior to the division of Z1/Z4 when the first sex-specific differences of the gonad arise. A few hundred Z1/Z4-enriched transcripts were identified for both hermaphrodites and fully masculinized XX-pseudomales, and a much smaller number of sex-specific Z1/Z4-enriched transcripts were also identified. The majority of transcripts with known Z1/Z4-enriched expression were identified by this method, including fkh-6, gem-4, and pes-1, and a number of novel Z1/Z4-enriched transcripts, including previously unannotated isoforms, have subsequently been validated by reporter analysis, confirming the effectiveness of this approach. Loss of function phenotypes are being determined by RNAi depletion and mutant analysis, with emphasis on transcripts that are sex-specifically Z1/Z4-enriched. Taking advantage of the recent innovations in larval dissociation and cell-specific RNA-seq, this work, which focuses on identifying the genetic networks responsible for the sex-specific developmental of the C. elegans gonad from a biopotential promordium, should advance our understanding of the regulatory logic and mechanisms underlying sexually dimorphic organogenesis and cell fate determination.

Crosby, Aaron et al. MicroPubl Biol

Kroetz, Mary B, Crosby, Aaron

[

MicroPubl Biol,

2018]

Torres, Edwin et al. (2019) International Worm Meeting "Characterizing the gonad-enriched transcripts in Caenorhabditis elegans ."

Torres, Edwin, Escobar, Giulianna, Kroetz, Mary B.

[

International Worm Meeting,

2019]

Previous forward and reverse genetic screens have identified a handful of genes that when their expression is abrogated lead to defects in the development of the somatic gonad in C. elegans. Many genes that are important for gonadal development could be genetically redundant; alternatively, they could be essential for the viability of the organism. Either case would cause the genes not to have been easily isolated in previous screens. To help identify additional genes responsible for promoting gonadal development, a cell-specific transcriptional approach was conducted that identified transcripts enriched for expression in the developing somatic gonad compared to the whole animal in each sex. We are now working to further study how the gonad-enriched transcripts are driving the development of the somatic gonad in either sex. We are using CRISPR-Cas to N-terminally tag the endogenous loci of gonad-enriched genes of interest with GFP. To generate the GFP-tagged strains, we are using a self-excising cassette (Dickinson et al. 2015). Prior to the eviction of the self-excising cassette, the self-excising cassette is inserted between the promoter and open reading, disrupting the gene expression, and allowing for us to test the loss-of-function phenotype. After excision, the GFP-tagged protein is generated for expression studies. Of the gonad-enriched transcripts, approximately 15% are encoded by essential genes, and therefore the loss-of-function gonadal phenotype has not been possible to study. To determine the role that these essential genes are playing within the gonad, we are modifying a tool that has been shown to lead to tissue-specific degradation of genes (Wang et al. 2017). We are constructing a strain that allows us to degrade GFP-tagged proteins exclusively in the gonad, allowing us to test the gonadal function of essential genes. Finally, as part of a Course-based Undergraduate Research Experience, we are using CRISPR-Cas to create serial deletions in the upstream regulatory promoter of the fkh-6 gene, which is important for gonadal development of both sexes. We have identified a region in the regulatory promoter that leads to feminization of male gonads but does not drastically change the expression pattern of FKH-6 in either sex.

Lake, Menesha et al. (2017) International Worm Meeting "Characterization of the transcriptional network that promotes gonadal development in Caenorhabditis elegans ."

Braun, Caleb J., Swann, Sellers, Lake, Menesha, Kreitz, Naden, Strickland, Kayman, Kroetz, Mary B.

[

International Worm Meeting,

2017]

During embryogenesis, the C. elegans gonad originates as a primordium of cells consisting of two somatic gonadal cells that flank two germ line precursor cells. The gonadal primordium is morphologically identical in the two sexes. Midway through the first larval stage, the somatic gonadal cells divide and differentiate, and during the course of development they will ultimately form one of two dramatically different organs: either a single-armed testis in the male or two bilaterally symmetric ovotestes connected to a central uterus in the hermaphrodite. To help define genes responsible for promoting gonadal development, we identified transcripts enriched for expression in the developing somatic gonad compared to the whole animal using cell-specific RNA-seq (Kroetz and Zarkower, 2015). Additionally, transcripts that were expressed in a sex-biased manner in the gonad have also been identified. Sex-biased transcripts were defined as transcripts that were enriched in one sex of gonadal cells compared to the expression in the gonadal cells of the other sex. Of the more than 900 gonad-enriched transcripts, approximately 80 encode for proteins that are transcriptional regulators. Seventeen transcription factors are expressed in a male-biased manner, and nine transcription factors are expressed in a hermaphrodite-biased manner in the developing gonad. To better understand if and how these transcription factors are controlling expression of the gonad-enriched transcripts, we are characterizing the expression patterns of proteins whose transcripts show the strongest gonadal enrichment. We are using CRISPR-Cas to fuse GFP to the C-terminus of the endogenously expressed gonadal proteins. We then use loss-of-function mutations or RNAi against the sex-biased or gonad-enriched transcription factors to determine if the transcription factors are important for expression of the GFP-tagged genes. This work will better define the transcriptional networks controlling gonad development and give insights into how key genetic regulators promote sex-specific organogenesis.

Kalis, Andrea K. et al. (2009) International Worm Meeting "Regulation of male gonadal cell fates."

Kalis, Andrea K., Zarkower, David, Larson, Kathleen M., Kroetz, Mary B.

[

International Worm Meeting,

2009]

Male and hermaphrodite C. elegans gonads develop from apparently identical primordia, but are very different organs as a result of sex specific differences in axis formation, cell lineages, leader cell migrations and terminal differentiation. Axis formation in both sexes requires a Wnt pathway, and this is overlaid with sex-specific regulation involving the global sex determination pathway, acting through tra-1, and the organ-specific sexual regulator fkh-6. We performed a genome-wide RNAi screen using sex-specific gonadal GFP reporters as sensitive indicators of disrupted gonadogenesis in animals treated with RNAi as larvae. This screen identified genes required for gonadal differentiation in both sexes. Strikingly, although we identified a number of genes whose RNAi feminizes the male gonad, no genes were identified whose RNAi masculinizes the hermaphrodite gonad. This suggests that a shared developmental program is present in both sexes and must be modified in XO animals to permit male gonadal differentiation. Genes whose RNAi feminizes the male gonad include fkh-6, the Wnt pathway members pop-1, lit-1, sys-1, and bar-1, the cell-cycle regulators cdk-1 and cyb-3, the matrix metalloprotease gon-1, and the Hox gene egl-5. For some of these genes early gonadal development and cell fate specification appeared normal but terminal differentiation was feminized, indicating that disruption of the mid-larval male gonadal program can cause adoption of a more hermaphrodite-like program. However, most knockdowns with male gonadal defects did not cause feminization. It is notable that during the L3 proliferative stage male gonadal cells undergo a number of asymmetric divisions while the hermaphrodite divisions are mostly symmetrical. The prevalence of Wnt pathway genes and cell cycle regulators among those causing feminization during this period suggests that disruption of cell division asymmetry can transform male cell fates to more female-like fates. egl-5, the Abdominal B homolog, is among several transcription factors identified in the screen, and gonads of egl-5 mutant males are extensively feminized, consistent with previous phenotypic analysis by Andrew Chisholm (Development, 1991). A small regulatory element ~15kb upstream of the egl-5 start codon is sufficient for male-specific gonadal expression of reporter genes and is regulated positively by fkh-6 and negatively by tra-1. Ectopic expression of egl-5 in hermaphrodites masculinizes gonadal tissue, indicating that EGL-5 plays an instructive role in male gonadal fate determination.

Neupane RP et al. (2019) Mar Drugs "Cytotoxic Sesquiterpenoid Quinones and Quinols, and an 11-Membered Heterocycle, Kauamide, from the Hawaiian Marine Sponge Dactylospongia elegans."

Williams PG, Neupane JB, Yip MLR, Turkson J, Parrish SM, Yoshida WY, Head JD, Harper MK, Neupane RP

[

Mar Drugs,

2019]

Several known sesquiterpenoid quinones and quinols (1-9), and kauamide (10), a new polyketide-peptide containing an 11-membered heterocycle, were isolated from the extracts of the Hawaiian marine sponge Dactylospongiaelegans. The planar structure of 10 was determined from spectroscopic analyses, and its relative and absolute configurations were established from density functional theory (DFT) calculations of the GIAO NMR shielding tensors, and advanced Marfey's analysis of the N-MeLeu residue, respectively. Compounds 1 and 3 showed moderate inhibition of -secretase 1 (BACE1), whereas 1-9 exhibited moderate to potent inhibition of growth of human glioma (U251) cells. Compounds 1-2 and 4-7 were also active against human pancreatic carcinoma (Panc-1) cells.

Chen CH et al. (2024) J Agric Food Chem "Novel Flavonol Alkaloids in Green Tea: Synthesis, Detection, and Anti-Alzheimer's Disease Effect in a Transgenic Caenorhabditis elegans CL4176 Model."

Ke JP, Liu Z, Yang Z, Liu G, Yang Y, Chen CH, Bao GH, Li JY, Yao G, Zhang YX

[

J Agric Food Chem,

2024]

Novel N-ethy-2-pyrrolidinone-substituted flavonols, myricetin alkaloids A-C (1-3), quercetin alkaloids A-C (4a, 4b, and 5), and kaempferol alkaloids A and B (6 and 7), were prepared from thermal reaction products of myricetin, quercetin, kaempferol─l-theanine, respectively. We used HPLC-ESI-HRMS/MS to detect 1-7 in 14 cultivars of green tea and found that they were all present in "Shuchazao," "Longjing 43", "Fudingdabai", and "Zhongcha 108" green teas. The structures of 1-4 and 6 were determined by extensive 1D and 2D NMR spectroscopies. These flavonol alkaloids along with their skeletal flavonols were assessed for anti-Alzheimer's disease effect based on molecular docking, acetylcholinesterase inhibition, and the transgenic Caenorhabditis elegans CL4176 model. Compound 7 strongly binds to the protein amyloid β (Aβ1-42) through hydrogen bonds (BE: -9.5 kcal/mol, Ki: 114.3 nM). Compound 3 (100 μM) is the strongest one in significantly extending the mean lifespan (13.4 ± 0.5 d, 43.0% promotion), delaying the Aβ1-42-induced paralysis (PT50: 40.7 ± 1.9 h, 17.1% promotion), enhancing the locomotion (140.0% promotion at 48 h), and alleviating glutamic acid (Glu)-induced neurotoxicity (153.5% promotion at 48 h) of CL4176 worms (p < 0.0001).

Phukhamsakda C et al. (2019) J Nat Prod "Sparticolins A-G, Biologically Active Oxidized Spirodioxynaphthalene Derivatives from the Ascomycete Sparticola junci."

Hyde KD, Phukhamsakda C, Cheng T, Macabeo APG, Stadler M, Huch V

[

J Nat Prod,

2019]

To explore the chemical diversity of metabolites from new species of Dothideomycetes, the ex-type strain of Sparticola junci was investigated. Seven highly oxygenated and functionalized spirodioxynaphthalene natural products incorporating carboxyalkylidene-cyclopentanoid (1-4), carboxyl-functionalized oxabicyclo[3.3.0]octane (5-6), and annelated 2-cyclopentenone/-lactone (7) units, sparticolins A-G, were isolated from submerged cultures of the fungus. Their chemical structures including their relative (and absolute) configurations were established through spectroscopic and X-ray crystallographic analyses. Sparticolin B (2) exhibited inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus, while sparticolin G (7) showed antifungal activities against Schizosaccharomyces pombe and Mucor hiemalis. All other sparticolins were only weakly active against S. aureus and also showed weak activities against the nematode Caenorhabditis elegans. Compounds 2 and 7 also showed moderate cytotoxic activities against seven mammalian cell lines.

load 10 more results