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

摘要 生态位衍生生长因子（gf）是维持哺乳动物生长发育的重要因子。 精原干细胞（SSCs）在成年睾丸。然而，在培养中， 自我更新的精原干细胞纯群体的扩增仍然是难以捉摸的。我们最近发现， 小鼠SSCs具有出乎意料的强大机制来反调节GF信号传导， 诱导负反馈调节因子（NFR）的表达，包括Spry Dusp基因家族SSC中特定NFR的去除导致ERK信号传导增加， 体外干细胞相关基因表达降低， 这表明过度的GF依赖性信号传导对SSC是有害的。 功能和可能有利于分化。扰动后发生的细胞变化 NFRs是未知的。然而，我们的数据表明，SSC被编程为限制ERK， 在狭窄的生理范围内发出信号。该提案通过以下方式处理机制问题： 哪些NFR限制SSC中的GF信号传导，防止非预定分化， SSC克隆的长期繁殖。使用遗传小鼠模型，SSC培养， 移植分析，和一个新开发的实时生物传感器的ERK活性，我们 解决以下问题：（1）SSC自我更新需要哪些NFR？(2)如何 消融NFR是否会导致干细胞活性丧失？(3)NFR如何控制动态 细胞内信号级联下游的GF受体？（4）在哪些节点上， ERK通路NFR发挥作用吗？除了提供一个合理的基础，以改善 SSC培养系统，这些研究还将确定新的成人SSC标记物，并阐明 生态位和SSC之间的串扰平衡自我更新和 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.