Matthew Eroglu

Columbia University; New York NY, United States of America

Eroglu, Matthew et al. (2021) International Worm Meeting "Transgenerational regulation of sex determination"

Eroglu, Matthew, Fraser, Andrew, Yu, Bin, Derry, Brent, Reinke, Aaron, Mok, Calvin, Schertzberg, Michael, Xiao, Maggie

[International Worm Meeting, 2021]

Multicellular organisms transmit genetic information through an immortal germ line which must undergo self-renewal at each generation. Failure of germ cells to maintain telomeres, small RNA expression, or repair DNA leads to a progressive loss of fertility as germ cells become unable to proliferate or enter diapause from accumulation of stress, a phenomenon known as germline mortality. We have identified a novel class of mutants that display transgenerational sterility which is unrelated to accumulation of stress. Ablation of the ZFAND3/5/6 homologs F22D6.2 and F56F3.4, which we propose to name mstr-1 and mstr-2 (multigenerational sterility, temperature regulated) leads to a progressive transformation of the nematode male germline to female over multiple generations thus causing sterility. Unlike canonical mortal germline mutants, sterile mstr-1; mstr-2 mutants are near-fully fertile when outcrossed to wild-type males. In these mutants, the commitment of germ cells to spermatogenesis can be rescued by novel mutations in the proteasome or the terminal sex regulating transcription factor GLI/tra-1. We then show that MSTR-1 coimmunoprecipitates with the mRNA of QKI/GLD-1, a key sex determinant upstream of tra-1, and spatiotemporally restricts its expression from proximal germ cells fated for spermatogenesis. In mstr-1; mstr-2 mutants, each generation of maintenance at 25oC shows accumulation of ectopic GLD-1 expression in spermatogenic cells, which is rescued by the proteasomal suppressors. Perplexlingly, gld-1 mRNA expression remains constant over multiple generations while its protein product increases. Therefore, we propose a model where MSTR-1 binds to mRNA and targets nascent proteins for proteasomal degradation to ensure proper spatial and temporal-generational expression of cell fate determinants. We are currently investigating the mechanistic basis of this novel post-transcriptional transgenerational regulation of gene expression. Overall, our work shows that in sexually reproducing organisms, germline immortality not only requires the clearance of stress but also renewal of germ cell commitment to spermatogenesis every generation.

JU1615

Caenorhabditis elegans

SSM289

Caenorhabditis elegans

Sathaseevan, Anson et al. (2021) International Worm Meeting "Investigating the germline function of the RNA-binding protein cfim-1"

Sathaseevan, Anson, Subramanian, Aishwarya, Yu, Bin, Derry, William Brent, Eroglu, Matthew, Fluke, Stacey

[International Worm Meeting, 2021]

Post-transcriptional regulation is crucial to proper organismal and tissue-specific development. An abundance of work has uncovered the essentiality of the core repertoire of factors and complexes constituting the cleavage and polyadenylation (CPA) machinery in post-embryonic development. Despite these findings, there is a need to define the precise relationship between these factors and tissue-specific development. Here, we demonstrate a novel role for the RNA-binding protein cfim-1 in germline function. cfim-1 functions in the CFIm complex, which is associated with preferential utilization of distal cleavage sites of pre-mRNAs bound for maturation by the core CPA machinery. Ablation of cfim-1 function results in reduced brood sizes. This effect is exacerbated at higher temperatures where a completely penetrant sterility phenotype is observed. We employ a combination of genetic and imaging analysis tools to characterize the organization and morphology of cfim-1 ablated germlines, demonstrating a precocious organization of meiotic-proliferating cells along the distal-proximal axis of the germline at the sterility-inducing temperature. We further provide genetic evidence to suggest that these effects are specific to the CFIm complex and not a general effect of antagonizing the function of the core CPA machinery. Data mining of worm 3'-sequencing data reveals an enrichment of the UGUA motif that is canonically bound by the CFIm complex upstream of the proximal cleavage sites of genes undergoing preferential transcript isoform utilization in response to ablation of cfim-1. These data, in conjunction with our previous findings, may suggest a novel model of regulation in the worm whereby recruitment of the CFIm complex to proximal cleavage sites in cfim-1 regulated genes antagonizes usage of those sites by the core CPA machinery.

EG8041

Caenorhabditis elegans

EG8040

Caenorhabditis elegans

SSM264

Caenorhabditis elegans

Buechner M et al. (1994) Worm Breeder's Gazette "Mutations Causing Cysts in the Excretory Canals"

Buechner M, Hedgecock EM

[Worm Breeder's Gazette, 1994]

Mutations Causing Cysts in the Excretory Canals. Matthew Buechner & Ed Hedgecoclc, Johns Hopkins University, Baltimore, MD, 21218. We have been studying mutants in which the structure or development of the epithelially-derived excretory canal cell processes is abnormal. One discrete set of mutants shows normal canal development through most of embryogenesis, followed by slow partial or complete collapse of the canals into a series of large cysts. The terminal phenotype varies greatly from one exc gene to the next; it is unlikely that simple blockage of the duct, through which the canal empties, can cause these vaned effects. Defects of the extracellular matrix over which the canal grows are not involved, as mutants known to be defective in these structures form canals which do not grow out properly, but are of normal width. We believe that the defect in these mutants is in the structure and placement of filaments intemal to the canal cytoplasm, possibly including filaments that guide myriad vesicles to the apical surface of this cell. Such structural members are necessary for any hypotonic cell to form a shape other than a sphere, and are critical for the maintenance of long processes such as axons or canals. Other observations support this hypothesis. During canal collapse, the tubule sometimes assumes a helical shape, consistent with the presence of filaments placed non-perpendicularly under the apical surface; a thick density is seen at this surface in electron nicrographs. In addition, some mutants show other phenotypes consistent with abnormal maintenance of filaments. Mutants in exc-5(IV) are slightly Him; mutants in exc-2(X) have abnormal tailspikes, and in exc-8(X) mutants, the pharynx sometimes detaches from the buccal hypoderm. In addition, all alleles of the gene sma-1(V) exhibit very large cysts. sma-1 mutants are especially short as larvae and have rounded heads, consistent with abnormal formation of cuticular filaments. Eight viable exc genes have been characterized and mapped to positions on all chromosomes. Six genes have been placed over deficiencies; this causes no increase in severity of canal defects. Several let genes also exhibit cystic excretory canals. These include let4(X), let-51(IV), and the let- 653(IV) gene characterized by Steven Jones and Dave Baillie. These animals die as late embryos or early larvae (a few survive until L2) vith extremely large cysts at the excretory -cell body underneath the posterior pharyngeal bulb. It is not clear if the canal defect is the cause of lethality in these animals, although this seems likely. Defects in the strongest viable exc genes, exc4(I) and exc-2(X), cause similar large cysts at the excretory cell body, and viability and growth rate are reduced. Construction of animals doubly mutant for genes with milder cyst phenotypes causes both lethality and large cell body cysts. A survey of other kt genes that arrest at these stages may reveal a large number of additional genes that encode products required for excretory canal cell structure and function. The exc defects appear formally analogous to the genetic polycystic kidney diseases (PKD) of both man and mouse. The exc-3(X) gene may be similar to the most frequent human defect, autosomal dominant PKD. Alleles of exc-3 are semi- dominant, showing a strong effect as a homozygote, with less common defects when hemizygous; hemizygotes for the deficiency nDf19 also exhibit infrequent (-5% of animals) defects in one or more of the canals. Woo et al. recently reported (Nature 368, 750-753, '94) that the microtubule- stabilizing drug taxol delayed onset of PKD in mice, while DNA replication and protein-synthesis inhibitors had no effect. This further suggests that control of filamentous structure is defective in the exc genes; we are currently testing drugs that affect both actin and tubulin filaments for effects on excretory canal structure.