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

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. Overall design: mRNA-seq profiles in VT2317 daf-16(mgDf50); daf-7(e1372) and control CB1372 daf-7(e1372) dauer larvae.

许多组织特异性干细胞（tissue-specific stem cell）在长期非分裂状态（即静息状态，quiescence）下，仍可维持产生多种细胞类型的能力。FOXO转录因子（FOXO transcription factor）可促进干细胞静息状态的维持与自我更新，但FOXO蛋白在静息状态下促进细胞多能性的具体分子机制仍有待阐明。秀丽隐杆线虫（C. elegans）中仅存的FOXO同源基因为daf-16，其可介导线虫在不良环境信号刺激下，进入一种兼具静息与抗逆特性的幼虫阶段——dauer幼虫（dauer larva）。在dauer阶段，干细胞与祖细胞可维持或重新建立多能性，以便在dauer阶段结束后恢复正常发育进程。本研究发现，在dauer阶段，daf-16/FOXO可阻止表皮干细胞（seam cells）过早分化为具备成体特征的细胞。具体而言，缺失daf-16的dauer幼虫会异常表达通常仅在成体中富集的胶原蛋白。本研究以col-19p::gfp作为成体细胞命运标记物，发现在dauer阶段，所有主要的daf-16亚型（isoform）均可抑制col-19p::gfp的表达。与之相反，未进入dauer发育阶段的daf-16(0)幼虫并不会异常表达col-19p::gfp。成体细胞命运及col-19p::gfp的表达时序，受异时性基因调控网络（heterochronic gene network）调控，该网络包含lin-41与lin-29两个关键基因。lin-41编码一种RNA结合蛋白（RNA-binding protein），其与哺乳动物中的LIN41/TRIM71为同源基因；而lin-29则编码一个保守的锌指转录因子（zinc finger transcription factor）。在非dauer发育过程中，lin-41通过抑制lin-29的翻译来对抗成体细胞命运：lin-29可直接激活col-19的转录并促进成体细胞命运的建立。本研究发现，在dauer阶段，lin-41可抑制col-19p::gfp的表达，但令人意外的是，该过程并不依赖lin-29。此外，daf-16可促进dauer幼虫体内lin-41的表达。在daf-16表达降低的dauer幼虫中观察到的col-19p::gfp异常表达表型，依赖于lin-41的下调，但并不需要lin-29的参与。综上，本研究揭示了daf-16/FOXO作为异时性基因的全新功能：在静息干细胞模型中，其通过促进lin-41/TRIM71的表达，以维持细胞的多能性命运。整体实验设计：对VT2317 daf-16(mgDf50); daf-7(e1372)突变体线虫与对照CB1372 daf-7(e1372)突变体线虫的dauer幼虫进行mRNA测序（mRNA-seq）分析，获取其转录组图谱。