| Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
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Author:
Date:
2017-02-05
[Abstract] All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During ...
[摘要] 构成成熟视网膜的所有七种视网膜细胞类型都是由普通的多能视网膜祖细胞池（RPC）产生的（Wallace，2011）。已经产生足够数量的特定细胞类型的RPC知道的一种方式是通过负反馈信号，其由分化细胞发射并且必须达到阈值水平以阻止该细胞类型的额外分化。评估负反馈信号是否由分化细胞发出的关键测定是异源沉淀测定，其中早期RPC被解离并用BrdU标记，然后与20倍过量的解离的分化细胞混合。然后将组合的细胞再次聚集并在细胞膜上培养7-10天。在这段时间内，RPC将会分化，BrdU + RPC的命运可以使用细胞类型特异性标记进行评估。开发这种沉淀测定的研究人员最初表明，当两种细胞类型混合在一起时，新生儿RPC与胚胎RPC相比，在加速进度条件下产生杆（Watanabe和Raff，1990; Watanabe等，1997）。我们已经使用这种测定来证明我们发现作为视网膜神经节细胞（RGC）分化的负调节物的声刺猬（Shh）促进RPC增殖（Jensen和Wallace，1997; Ringuette等，2014）。最近我们修改了异质性沉淀测定法，以评估视网膜无长突细胞的反馈信号的作用，将转化生长因子β2（Tgfβ2）鉴定为负反馈信号，并将Pten作为Tgfβ2应答的调节剂（Ma et al。，2007 ; ...
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| A Method to Analyze Local and Systemic Effects of Environmental Stimuli on Root Development in Plants
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Author:
Date:
2016-09-05
[Abstract] Root development in vascular plants is innately tied to the environment. However, relatively little attention has been paid toward understanding the spatial scales at which the root perceives and responds to external stimuli. While some environmental signals elicit global responses that affect root system architecture, others may have more localized effects. We have observed that various developmental processes can be induced or suppressed along the circumference of the main root depending on local contact with available water in a process termed hydropatterning (Bao et al., 2014). Our studies of hydropatterning indicate that the root can detect and respond to certain external stimuli at the resolution of the diameter of a single organ. In order to characterize developmental ...
[摘要] 维管植物中的根发育与环境固有关系。然而，相对较少的关注已经被用于理解根部感知和响应外部刺激的空间尺度。虽然一些环境信号引起影响根系统结构的全局响应，但其他环境信号可能具有更局部化的效应。我们已经观察到，根据在称为水图案化的过程中与可用水的局部接触，可以沿着主根的圆周诱导或抑制各种发育过程（Bao等人，2014）。我们对水印的研究表明根部可以检测和响应某些外部刺激在单个器官的直径的分辨率。为了表征在该空间尺度的发育图案化，我们开发了使用琼脂培养基在体外改变穿过根的周向轴的环境输入的程序。根在"三明治"中的两块琼脂培养基之间生长。局部环境条件可以根据任一侧的介质的组成而变化。局部作用的刺激可以与基于根的发育反应系统作用的刺激相区分。在这里我们描述了整体的方法，并提供了一个例子，它如何可以用于分析外部水势梯度响应中玉米Zea mays（玉米）的侧根图案。我们还讨论了该方法如何可以更广泛地用于其他植物物种和环境处理。
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