Gene-ethanol interactions underlying aberrant neuronal cell behaviors

项目摘要 这一补充的目的是使研究的继续，是有限的阻碍， 由于COVID-19大流行，我将负责研究业务，并协助我过渡到独立PI 位置从这一补充所获得的结果将提高我的竞争力的教师职位。我会 从我的导师那里获得关于关键职业技能的进一步培训。特别是，我们的每周会议将包括 讨论相关技能，如面试，谈判以及建立和维护 独立实验室该补充还将有助于建立网络，以确定潜在的合作者和工作 由于COVID-19严重阻碍了这一关键的职业发展活动，因此我们提供了许多机会。我将参加 UT奥斯汀分子生物科学博士后协会每周专业发展研讨会。与 我还将以虚拟形式参加相关会议，最好是在社交限制放松时亲自参加。 这些会议将包括RSA、神经科学学会和国际斑马鱼会议。 我的初步数据表明，乙醇与vangl 2在切向FBMN迁移过程中相互作用。 切向FBMN迁移未能发生在对照和乙醇暴露的vangl 2突变体。虽然控制 野生型（WT）和vangl 2杂合子正常发育，乙醇诱导切向FBMN迁移缺陷 依赖于剂量和时间。引人注目的是，许多暴露于乙醇的WT和vangl 2杂合子 表型复制突变体。值得注意的是，这些缺陷在暴露于乙醇的vangl 2中更常见。 杂合子与WT相比。值得注意的是，FBMN迁移缺陷是由低至0.25%v/v 乙醇同样，我发现乙醇与Wnt/PCP途径的另一个成员pk 1b相互作用， 在FBMN迁移期间。我推测，一个综合的遗传和乙醇介导的平面细胞衰减 极性导致异常的FBMN迁移行为。 在本补充材料的目标1中，我将使用实时的时间推移共聚焦分析来评估 在乙醇暴露的vangl 2、pk 1b、celsr 2、fzd 3a和scrib 1杂合子胚胎中FBMN的行为， isl 1：eGFP;ubi：nlsmApple和gfap：mCherry转基因系。在本附录的目标2中，我将描述 VANG 12-乙醇相互作用是神经元迁移缺陷的基础。我会用共聚焦分析来评估 使用hoxb 1a：RFP、egr 2b：GFP和mafba：GFP在乙醇暴露的vang 12杂合子中的后脑模式化 转基因我将使用CRISPR/Cas9介导的Vangl 2：EGFP和Celsrl：RFP转基因产生Vangl 2：EGFP和Celsrl：RFP转基因。 同源性定向修复以检查平面细胞极性获取并使用actb 2：Mmu. Ar 113 b-GFP 转基因以检查vangl 2杂合子中的平面细胞极性功能， 与Wnt/PCP途径相互作用。在本补充的目标3中，我将映射和功能特性 通过正在进行的ENU鉴定的具有FBMN迁移缺陷的乙醇敏感性斑马鱼突变体， 和基于转座子的正向遗传筛选。

Project Summary This purpose of this supplement is to enable the continuation of research that was hindered by limited research operations due to the COVID-19 pandemic and to assist in my transition to an independent PI position. The results obtained from this supplement will enhance my competitiveness for faculty positions. I will gain further training from my mentor on crucial career skills. In particular, our weekly meetings will include discussion of relevant skills such as interviewing, negotiating as well as establishing and maintaining an independent lab. The supplement will also aid in networking to identify potential collaborators and job opportunities, as this critical career advancement activity was severely hindered by COVID-19. I will attend the UT Austin molecular biosciences postdoctoral association weekly professional development seminars. With funds I will also attend relevant meetings, in virtual formats and ideally, as social restrictions ease, in person. These meetings will include RSA, the Society for Neuroscience and the International Zebrafish Meeting. My preliminary data demonstrates ethanol interacts with vangl2 during tangential FBMN migration. Tangential FBMN migration fails to occur in both control and ethanol-exposed vangl2 mutants. While control wildtype (WT) and vangl2 heterozygotes develop normally, ethanol induces tangential FBMN migration defects that are dose and time dependent. Strikingly, many ethanol-exposed WTs and vangl2 heterozygotes phenocopy the mutant. Notably, these defects are observed more often in ethanol-exposed vangl2 heterozygotes compared to WTs. Remarkably, FBMN migration defects are induced by as low as 0.25% v/v ethanol. Similarly, I have found that ethanol interacts with another member of the Wnt/PCP pathway, pk1b, during FBMN migration. I hypothesize that a combined genetic and ethanol-mediated attenuation of planar cell polarity results in aberrant FBMN migration behaviors. In Aim 1 of this supplement, I will use live time-lapsed confocal analyses to assess the migratory behaviors of FBMNs in ethanol-exposed vangl2, pk1b, celsr2, fzd3a and scrib1 heterozygous embryos using the isl1:eGFP;ubi:nlsmApple and gfap:mCherry transgenic lines. In Aim 2 of this supplement, I will characterize vangl2-ethanol interactions underlying neuronal migration defects. I will use confocal analyses to assess hindbrain patterning in ethanol-exposed vangl2 heterozygotes using hoxb1a:RFP, egr2b:GFP, and mafba:GFP transgenics. I will generate the Vangl2:EGFP and Celsr1:RFP transgenes using CRISPR/Cas9 mediated homology directed repair to examine planar cell polarity acquisition and use the actb2:Mmu.Arl13b-GFP transgenes to examine planar cell polarity functionality in vangl2 heterozygotes to determine how ethanol interacts with the Wnt/PCP pathway. In Aim 3 of this supplement, I will map and functionally characterize ethanol-sensitive zebrafish mutants with FBMN migration defects that are identified through an ongoing ENU- and transposon-based forward genetic screen.