Negative feedback regulation of growth factor signaling in adult spermatogonial stem cells

摘要 生态位衍生生长因子(GFS)对哺乳动物的建立和维持至关重要 成人睾丸的精原干细胞(SSCs)。然而，在培养中，最佳条件是 自我更新的干细胞的纯种群的扩张仍然难以捉摸。我们最近发现， 小鼠SSCs拥有出人意料的强大机制，通过以下方式反向调节GF信号 诱导包括Spry成员在内的负反馈调节因子(NFR)的表达 和DUSP基因家族。SSCs中特定NFR的取消导致ERK信号的增加， 干细胞相关基因的体外表达减少和干细胞活性的丧失 移植，提示过度的GF依赖信号对SSC是有害的 功能，并可能有利于分化。微扰后发生的细胞变化 的NFR是未知的。然而，我们的数据暗示，SSCs被编程为限制ERK 在一个狭窄的生理范围内发出信号。这项提案通过以下方式解决这些机制 哪些NFR限制SSC中的GF信令，防止计划外分化，并启用 SSC克隆的长期繁殖。利用遗传小鼠模型，SSC培养， 移植分析，以及新开发的ERK活性实时生物传感器，我们 回答以下问题：(1)SSC自我续订需要哪些NFR？(2)如何 NFR的消融是否会导致干细胞活性的丧失？(3)NFR如何控制这种动态 细胞内信号级联到GF受体下游？以及(4)在 ERK途径NFR发挥作用了吗？除了提供合理的基础来改善 SSC培养系统，这些研究还将发现新的成年SSC标记并阐明 利基和SSC之间的相互作用平衡自我更新和 体内分化。

Abstract Niche-derived growth factors (GFs) are essential for establishing and maintaining mammalian spermatogonial stem cells (SSCs) in the adult testis. In culture, however, optimal conditions for expansion of pure populations of self-renewing SSCs remain elusive. We recently found that mouse SSCs possess unexpectedly robust mechanisms to counter-regulate GF signaling by inducing expression of negative feedback regulators (NFRs), including members of the Spry and Dusp gene families. Abrogation of specific NFRs in SSCs led to increase ERK signaling, decreased expression of stem cell-associated genes in vitro and loss of stem cell activity upon transplantation, suggesting that excessive GF-dependent signaling is detrimental to SSC function and may favor differentiation. The cellular alterations that occur following perturbation of NFRs are unknown. However, our data imply that SSCs are programmed to limit ERK signaling within a narrow physiological range. This proposal addresses the mechanisms by which NFRs restrict GF signaling in SSCs, prevent unscheduled differentiation, and enable long-term propagation of SSC clones. Using genetic mouse models, SSC culture, transplantation analysis, and a newly-developed real-time biosensor for ERK activity, we address the following questions: (1) Which NFRs are required for SSC self-renewal? (2) How does ablation of NFRs result in loss of stem cell activity? (3) How do NFRs control the dynamic intracellular signaling cascades downstream of GF receptors? And (4) At which nodes in the ERK pathway do NFRs exert their actions? In addition to providing a rational basis to improve SSC culture systems, these studies will also identify novel adult SSC markers and shed light on mechanisms by which cross-talk between the niche and SSCs balances self-renewal and differentiation in vivo.