Mechanisms of neural specification and patterning during planarian brain regeneration

数十年的出色工作描述了许多模式生物胚胎中的神经发育，这表明神经发育是神经干细胞在空间和时间上的精确协调，以形成具有适当细胞类型比例的功能神经系统。然而，目前尚不清楚这些发育原理如何应用于成年生物体，因为神经元在损伤后被翻转(内稳态)或被替换(再生)。在我们的第一个NSERC资助学期中，我们开始使用淡水扁平动物(扁虫；米氏沼虾)来研究成年神经稳态和再生，因为它的神经稳态水平很高，并且有能力在7天内从头开始再生新的大脑。我们假设，我们可以采取一种候选的方法来寻找影响行星动物神经命运的转录因子。使用分子工具和RNA干扰来测试基因功能，我们发现平面动物使用同源盒转录因子Lhx1/5和Pitx来指定和再生5-羟色胺能神经元。此外，在中央大脑中，bHLH转录因子指定神经细胞类型，并在邻近大脑的一些干细胞中表达。正是这最后一个观察推动了目前的提议：大脑相邻的干细胞如何通过信号通路协调，以产生适当数量和类型的神经元，以及干细胞之间的潜力是否存在差异(即异质性)？在这里，我们将使用有偏见和无偏见的方法来测试这个生物学问题。基于我们之前的工作，我们知道一条保守的信号通路Hedgehog是由大脑中特定数量的神经元转录的。我们建议测试Hedgehog是否被用作从神经元到邻近干细胞的信号，以控制神经元内稳态的速率。此外，我们假设并不是所有大脑邻近的干细胞都对刺猬有反应，这意味着它们的反应能力是不同的。我们建议，这可以通过采取公正的方法进行测试，使用大脑相邻干细胞的单细胞RNA深度测序来确定这些干细胞是否具有1)异质性，2)我们是否可以在浮游生物中发现一种新的神经干细胞群体。从这一点上，我们还将发现新的干细胞调节器参与了浮游生物的脑内稳态和再生。 NSERC的影响：我们是加拿大唯一一个使用行星动物研究大脑再生的实验室，我们所有的神经再生研究都是由NSERC独家资助的。

Decades of elegant work have described neural development in the embryos of many model organisms, which has shown that neural development is the precise coordination of neural stem cells in space and time in order to make a functioning nervous system with appropriate cell-type ratios. However, it is not known how these developmental principles apply to adult organisms as neurons are turned over (homeostasis) or are replaced following injury (regeneration). In our first NSERC funding term, we started to investigate adult neural homeostasis and regeneration using the freshwater planarian (flatworm; Schmidtea mediterranea) due to its high level of neural homeostasis and the ability to regenerate a new brain from scratch within 7 days. We hypothesized that we could take a candidate approach to finding a transcription factors responsible for neural fates in planarians. Using molecular tools and RNA-interference to test gene function, we discovered that planarians specify and regenerate serotonergic neurons using the homeobox transcription factors lhx1/5 and pitx. Furthermore, in the central brain, bHLH transcription factors specify neural cell types and are expressed in some of the stem cells adjacent to the brain. It is this last observation that drives the current proposal: How are brain-adjacent stem cells coordinated by signaling pathways to make the proper number and type of neurons, and do the stem cells differ in potential between each other (i.e. heterogeneity)? Here we will test this biological question using both biased and unbiased approaches. Biased on our previous work, we know that a conserved signaling pathway, Hedgehog, is transcribed by a specific population of neurons in the brain. We propose to test whether Hedgehog is being used as a signal from neurons, to adjacent stem cells, in order to control rates of neuronal homeostasis. Furthermore, we hypothesize that not all brain-adjacent stem cells respond to Hedgehog, which implies that they are heterogeneous in their competence to respond. We propose that this can be tested by taking an unbiased approach using single-cell RNA-deep sequencing of brain-adjacent stem cells to determine whether these stem cells are 1) heterogeneous, and 2) whether we can find a novel populations of neural stem cells in planarians. From this, we will also find novel stem cell regulators involved in brain homeostasis and regeneration in planarians. Impact of NSERC: We are the only lab in Canada to use planarians to study brain regeneration, and all of our neural regeneration research is exclusively funded by NSERC.