Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation

Abstract

Summary Here we describe an in vitro primary culture system for C. elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner

“…We utilized germ cells isolated from the tumorous mutant glp-1(ar202); cki-2(ok2105); daf-16(mu86) , which contains a gain-of-function (gf) temperature sensitive (ts) glp-1 allele; hereafter referred to as glp-1(gf); cki-2; daf-16 . This strain survives longer in culture than other tested tumorous mutant strains (Chaudhari et al, 2016). We observed that the addition of physiological concentrations of Δ 7 -DA (Motola et al, 2006) had deleterious effects on the isolated germ cells, causing them to die more rapidly with increasing concentration (Fig.…”

“…To test the effect of DA on an essentially pure population of germ cells, we utilized an in vitro primary culture system that can maintain viable germ cells from diverse germline tumorous mutants in culture, but without a net increase in germ cell numbers (Chaudhari et al, 2016). We utilized germ cells isolated from the tumorous mutant glp-1(ar202); cki-2(ok2105); daf-16(mu86) , which contains a gain-of-function (gf) temperature sensitive (ts) glp-1 allele; hereafter referred to as glp-1(gf); cki-2; daf-16 .…”

“…As a measure of DNA replication, we determined the percentage of cells incorporating the thymidine analog EdU. Bacterial extract or purified stimulatory folate (5,10-methenyl-tetrahydrofolate-Glu 6 ) were added to increase the rate of EdU incorporation in the isolated germ cells (Chaudhari et al, 2016), and the effect of DA was assessed. It has been reported that Δ 4 -DA is more abundant than Δ 7 -DA in cells, and therefore, we utilized Δ 4 -DA for in vivo studies (Antebi, 2013).…”

“…Bacterial folates derived from ingested bacteria act on germ cells to stimulate their proliferation (Chaudhari et al, 2016). Insulin-like peptides and TGF-β levels are increased in response to food availability.…”

“…Δ 7 -DA or Δ 4 -DA (both from Cayman Chemical Co.) or DMSO or ethanol carrier controls, respectively, were included in the CeM1 medium. For live cell counts, aliquots of cells from 0.5 ml 24-well plate cultures were incubated with the live-cell stain calcein-AM (Biotium; 1 μM) and the deadcell stain ethidium homodimer (Biotium; 0.1 μM) (Chaudhari et al, 2016). Three counts were made for each sample using a cellometer counting grid (CP2, Nexcelom Bioscience LLC) that was analyzed using an inverted fluorescence microscope (Zeiss Axio Observer.Al); cell count variation is presented as SEM.…”