Mechanisms of neural specification and patterning during planarian brain regeneration

几十年来，人们对许多模式生物胚胎中的神经发育进行了细致的描述，这些研究表明，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，尚不清楚这些发育原则如何适用于成年生物体，因为神经元在损伤后被翻转（稳态）或替换（再生）。在我们的第一个NSERC资助期内，我们开始使用淡水涡虫（扁虫；Schmidtea mediterranea）研究成虫的神经稳态和再生，因为它具有高水平的神经稳态和在7天内从头开始再生新大脑的能力。我们假设我们可以采取一种候选的方法来寻找一种负责涡虫神经命运的转录因子。利用分子工具和rna干扰测试基因功能，我们发现涡虫通过同源盒转录因子lhx1/5和pitx来指定和再生血清素能神经元。此外，在中枢大脑中，bHLH转录因子指定神经细胞类型，并在一些邻近大脑的干细胞中表达。正是这最后一个观察结果推动了当前的建议：脑邻近干细胞如何通过信号通路协调产生适当数量和类型的神经元，干细胞之间的电位是否不同（即异质性）？在这里，我们将使用有偏和无偏的方法来测试这个生物学问题。根据我们之前的工作，我们知道一个保守的信号通路，Hedgehog，是由大脑中特定的神经元群转录的。我们建议测试Hedgehog基因是否被用作神经元到邻近干细胞的信号，以控制神经元稳态的速率。此外，我们假设并非所有脑邻近干细胞都对Hedgehog基因有反应，这意味着它们的反应能力是异质的。我们建议采用一种无偏倚的方法对脑邻近干细胞进行单细胞rna深度测序，以确定这些干细胞是否具有1)异质性，以及2)我们是否可以在涡虫中发现新的神经干细胞群体。由此，我们还将发现参与涡虫大脑稳态和再生的新型干细胞调节因子。

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.