(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71

['Matthew J. Wirick', 'Biochemistry', 'Cell', 'Molecular Biology Program', 'Central Michigan University', 'Mt Pleasant', 'Michigan', 'United States Of America', 'Allison R. Cale', 'Department Of Biology']

Date: 2022-01 

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

In adults and juveniles, tissue-specific stem cells divide as needed to replace cells that are lost due to injury or normal wear and tear. Many stem cells spend long periods of time in cellular quiescence, or non-division. During quiescence, stem cells remain multipotent, where they retain the ability to produce all cell types within their tissue. In this study, we define a new role for the FOXO protein DAF-16 in promoting multipotency during the quiescent C. elegans dauer larva stage. C. elegans larvae enter dauer midway through development in response to adverse environmental conditions. Epidermal stem cells are multipotent in C. elegans larvae but differentiate at adulthood, a process controlled by the “heterochronic” genes. We found that daf-16 blocks the expression of adult cell fate specifically in dauer larvae by promoting the expression of the heterochronic gene lin-41. lin-41 normally blocks adult fate by repressing the expression of another heterochronic gene, lin-29, but surprisingly, lin-29 is not needed for the expression of adult cell fate in this context. These findings may be relevant to mammals where the orthologs of daf-16 and lin-41 are important in stem cell maintenance and opposing differentiation.

Funding: This work was supported by R01GM118875 to JKK from the National Institutes of Health, https://www.nih.gov ; R01GM129301 to JKK from the National Institutes of Health, https://www.nih.gov ; R15GM117568 to XK from the National Institutes of Health, https://www.nih.gov ; CAREER 1652283 to XK from the National Science Foundation https://www.nsf.gov . The funders had nobrole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the manuscript and its Supporting Information files except that mRNA-seq data have been deposited in NCBI under GEO accession number GSE179166. Processed data and scripts used for analysis are available at https://github.com/starostikm/DAF-16 .

Here, we examine the role of daf-16/FOXO in maintaining multipotent seam cell fate during dauer. We find that daf-16 is required to block the expression of multiple adult-enriched collagens during dauer, including col-19, defining a novel role for daf-16 as a dauer-specific heterochronic gene. Using the adult cell fate marker col-19p::gfp as a readout, we find that daf-16 acts via lin-41 to regulate adult cell fate. Surprisingly, lin-29 plays at most a minor role in the regulation of col-19p::gfp during dauer. mRNA-seq experiments identified 3603 genes that are regulated by daf-16 during dauer, including 112 transcription factors that may mediate repression of adult-enriched collagens during dauer downstream of daf-16.

Another group of progenitor cells that must remain quiescent and multipotent during dauer is the lateral hypodermal seam cells that make up part of the worm skin. Seam cells undergo self-renewing divisions at each larval stage and then terminally differentiate at adulthood [ 23 ]. Larval vs. adult seam cell fate is regulated by a network of heterochronic genes [ 24 ]. In general, heterochronic transcription factors and RNA-binding proteins that specify early cell fates are expressed early in development. These early cell fate-promoting factors are then downregulated by microRNAs in order to allow progression to later cell fates [ 25 ]. This pathway culminates with the expression of the LIN-29 transcription factor. LIN-29 is the most downstream regulator of adult cell fate and directly activates the expression of adult-specific collagens such as col-19 [ 26 – 29 ]. LIN-29 protein is not expressed in the hypodermis until late in larval development due to the combined action of the early-promoting heterochronic genes lin-41 and hbl-1 [ 27 , 30 , 31 ]. lin-41 encodes an RNA-binding protein that binds to the lin-29 mRNA and blocks translation of the lin-29a isoform [ 30 , 32 ]. lin-29 is also required for adult cell fate in post-dauer animals. However, many genes that act earlier in the heterochronic pathway are dispensable for post-dauer development, suggesting that the regulation of adult cell fate differs in continuous and dauer life histories [ 3 ].

The role of the DAF-16/FOXO transcription factor in promoting dauer formation is analogous to the role of the FOXO proteins in promoting quiescence in mammalian stem cells [ 17 ]. In addition, DAF-16/FOXO is required for stem cell maintenance and the ability of stem/progenitor cells to differentiate into the correct cell types in both systems [ 5 , 10 , 18 – 22 ]. For example, in C. elegans dauer larvae, daf-16 is required in a set of multipotent vulval precursor cells to block EGFR/Ras and LIN-12/Notch signaling from prematurely specifying vulval cell fates [ 5 ].

Dauer formation is regulated by three major signaling pathways [ 6 , 7 ]. One of these pathways is insulin/IGF signaling, where favorable environmental cues lead to the production of multiple insulin-like peptides in sensory neurons. [ 8 – 13 ]. These signals are released and then received in multiple tissues where insulin signaling blocks the activity of the downstream DAF-16/FOXO transcription factor [ 14 – 16 ]. In adverse environments, DAF-16 is active and regulates the expression of genes that promote dauer formation [ 6 , 10 ].

Tissue-specific stem cells divide as needed to replenish cells lost due to injury or normal wear and tear. Many stem cell types retain the capacity to produce multiple cell types during lengthy periods of quiescence, or non-division, and increased cell division can lead to compromised multipotency and stem cell maintenance [ 1 ]. However, the connections between quiescence and multipotency are incompletely understood. Caenorhabditis elegans (C. elegans) development can be interrupted by a quiescent and stress-resistant stage called dauer in response to adverse environmental conditions [ 2 ]. Stem cell-like progenitor cells maintain or re-establish multipotency during dauer, a situation analogous to mammalian stem cells [ 3 – 5 ].

Results

lin-29 is not required for col-19p::gfp expression in lin-41(-) dauer larvae During continuous development, lin-41 opposes col-19p::gfp and adult cell fate by directly repressing the translation of the LIN-29 transcription factor [30,32]. LIN-29 is in turn a direct activator of col-19 transcription and is thought to be the most downstream regulator of other aspects of adult cell fate [26,27,31]. If the col-19p::gfp expression observed in lin-41(RNAi) dauer larvae is due to misexpression of LIN-29, then loss of lin-29 should prevent col-19p::gfp expression. To ask this question, we used the lin-29(xe37) deletion allele that removes all but 27 amino acids of LIN-29 [45]. Surprisingly, we saw no effect of the loss of lin-29 on the expression of col-19p::gfp in lin-41(RNAi) dauer larvae (Fig 4A). As a control, we established that our strain produced the expected lin-29(0) phenotypes in adults: a drastic reduction of col-19p::gfp and a complete lack of adult alae formation (S9 Fig). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. lin-41 regulates col-19p::gfp independently of lin-29 in dauer larvae. (A) lin-29 is not required for col-19p::gfp expression in lin-41(RNAi) dauers. Fluorescence levels were determined as described in S7 Fig. ns = not significant (p = 0.4856, two-tailed Mann-Whitney Test). n = 33–41; see S7 Table for complete underlying numerical data. (B) lin-41(0); lin-29(0) dauer larvae expressed col-19p::gfp. nIs408 is a transgene used to balance lin-41(0) (see text). lin-41(0) larvae are dauer-defective [47]. Numbers indicate the number of dauers expressing col-19p::gfp over total dauers scored. (C, D) lin-29::gfp expression imaged on a compound microscope (C) or a confocal microscope (D). Images were taken with a 63x objective. Arrowheads indicate hypodermal nuclei with visible lin-29::gfp expression. Numbers indicate the number of larvae expressing any detectable lin-29::gfp over total number of larvae. lin-41(RNAi) produced substantial precocious expression of lin-29::gfp in continuously developing L3 staged larvae, easily discernable under the compound microscope. However, lin-41(RNAi) produced only very dim expression in dauer larvae. Dauer larvae produce substantial autofluorescence at the microscope settings required to visualize lin-29::gfp. https://doi.org/10.1371/journal.pgen.1009881.g004 We next confirmed the lin-41 RNAi results using the null allele lin-41(n2914). Since lin-41(0) hermaphrodites are sterile [30], we used an available transgene that is integrated close to the lin-41 locus as a balancer: nIs408[lin-29::mCherry, ttx-3p::gfp] [46]. This transgene had the added advantage of rescuing some of the lin-29(0) defects, making the strain easier to maintain (see Methods). When we induced dauer formation in the progeny of lin-41(0)/nIs408 and lin-41(0)/nIs408; lin-29(0) mothers, we found that lin-41(0); lin-29(+) homozygous larvae, recognized by the lack of ttx-3p::gfp expression, were largely dauer defective (see [47]). Of the two lin-41(0) dauer larvae we recovered, one expressed col-19p::gfp (Fig 4B). Interestingly, 62% of larvae expressing ttx-3p::gfp displayed col-19p::gfp expression. This percentage is close to the 2/3 of transgene-containing larvae segregating from the heterozygous parent that would be predicted to be lin-41(0)/nIs408 heterozygotes, suggesting that lin-41 is haploinsufficient in its role in blocking col-19p::gfp during dauer. To bolster this supposition, we confirmed that dauer larvae homozygous for nIs408 do not express col-19p::gfp (Fig 4B). Although lin-41(0) homozygous larvae display a dauer-defective phenotype, lin-41(0); lin-29(0) larvae do not [47]. We were therefore able to test lin-41(0); lin-29(0) dauer larvae for col-19p::gfp expression. Approximately half of these larvae expressed col-19p::gfp (Fig 4B). Although the dauer-defective phenotype of lin-41(0) single mutants prevented us from determining the extent to which loss of lin-29 suppresses the col-19p::gfp phenotype in lin-41(0) homozygous mutant dauer larvae, these results do confirm that col-19p::gfp can be misexpressed in lin-41(0) dauer larvae even in the absence of lin-29. To better understand the relationship between lin-41 and lin-29 during dauer, we asked whether lin-41 is required to block LIN-29 expression in dauer larvae. During continuous development, lin-41 mutants display precocious hypodermal expression of LIN-29 [30,32]. We used lin-29(xe61[lin-29::gfp]), which tags both isoforms of lin-29 [32] to assess the effect of lin-41 RNAi during dauer. Using settings that allow unambiguous visualization of hypodermal lin-29::gfp during L4 and adult stages, we saw only extremely dim lin-29::gfp expression in lin-41(RNAi) dauer larvae, just at the edge of detection (Fig 4C). We used confocal microscopy as a more sensitive assay to confirm that lin-29::gfp is expressed, albeit at very low levels in lin-41(RNAi) but not control dauer larvae (Fig 4D). In control RNAi experiments run in parallel, lin-41(RNAi) was able to cause strong col-19p::gfp expression in dauer larvae (24/27 larvae examined), demonstrating that there were no technical problems in the RNAi experiment that would lead to such low expression. Finally, we confirmed that lin-41(RNAi) produces the expected precocious lin-29::gfp expression in L3 staged larvae that developed continuously (Fig 4C). Taken together, contrary to the role of lin-29 during continuous development, our data indicate that lin-41 regulates col-19p::gfp expression in dauer larvae and that lin-29 does not play a significant role in this regulation.

daf-16 acts at least partially independently of lin-29 to regulate col-19p::gfp during dauer As described above, daf-16 promotes lin-41 expression during dauer, and misexpression of lin-41 suppresses the precocious col-19p::gfp expression observed in daf-16(0) dauer larvae. Since lin-29 was not required for misexpression of col-19p::gfp in lin-41(-) dauer larvae, we hypothesized that lin-29 would also be dispensable for col-19p::gfp expression in daf-16(-) dauer larvae. We found that neither of two lin-29 null alleles affected the penetrance of col-19p::gfp expression in daf-16(0) mutant dauers, demonstrating that the misexpression of col-19p::gfp in daf-16(0) dauer larvae does not depend on lin-29 (Fig 5A). However, when we compared levels of expression between the strains, we found a small but statistically significant decrease in expression in dauer larvae that lack lin-29, indicating that the presence of lin-29 bolsters col-19p::gfp expression slightly (Fig 5B). We next asked whether loss of daf-16 affects expression of lin-29. Examining our mRNA-seq data, no significant difference in lin-29 mRNA levels were observed in daf-16(0) vs. control dauer larvae (S6 Fig). However, during continuous development, regulation of lin-29a by lin-41 occurs translationally and may not be evident from mRNA levels [32,48]. We next examined lin-29::gfp expression in daf-16(0) dauer larvae. Unlike the dim lin-29::gfp expression we observed in lin-41(RNAi) dauer larvae, lin-29::gfp expression in the hypodermis was completely undetectable in daf-16(0) dauer larvae. Using confocal microscopy, 0/24 daf-16(0) dauer larvae displayed detectable lin-29::gfp. Taken together, these experiments demonstrate that daf-16 regulates col-19p::gfp expression largely independently of lin-29. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. daf-16 acts at least partially independently of lin-29 to block adult cell fate during dauer. (A) daf-16(0); lin-29(0) dauers expressed col-19p::gfp at a similar penetrance to daf-16(0) dauers. n546 is a nonsense mutation and xe37 is a deletion of all but 27 amino acids. Images were taken with a 63x objective. Numbers indicate the number of dauers expressing hypodermal col-19p::gfp over the total number of dauers. (B) Levels of col-19p::gfp are slightly reduced in daf-16(0); lin-29(xe37) dauer larvae compared to daf-16(0) dauers. Fluorescence levels were determined as described in S7 Fig. ****p<0.0001 (Two-tailed Mann-Whitney Test). n = 34–50; see S8 Table for complete underlying numerical data. https://doi.org/10.1371/journal.pgen.1009881.g005

[END]

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009881

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/