EH004: Lmx1a encodes a rostral set of mesodiencephalic dopaminergic neurons marked by the Wnt/b-catenin signaling activator Rspo2

Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor Lmx1a is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in Lmx1a-dr/dr mice. Therefore, microarray analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of Lmx1a. Subsequent expression analysis confirmed that Nurr1 is regulated by Lmx1a, and additional downstream targets were identified, like Pou4f1, Pbx1, Pitx2, C130021l20Rik, Calb2 and Rspo2. In line with a specific, rostral-lateral loss of expression of most of these genes during development, Nurr1 and C130021l20Rik were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the Wnt/b-catenin signaling activator Rspo2 in this domain. Expression analysis in Rspo2-/- embryos revealed affected mdDA neurons, partially phenocopying the Lmx1a mutant. Together, in this study we reveal that Lmx1a is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the activation of the Wnt activator Rspo2. Microarray expression study comparing 4 samples of homozygous LMX1A dr/dr mice, midbrain E12.5 with a pooled sample of their wt/wt littermates. Two samples were analyzed in opposite dye orientation.针对中脑间脑多巴胺能（mesodiencephalic dopaminergic, mdDA）神经元的分子编程研究近年取得诸多进展，现已鉴定出多种参与mdDA神经元特化过程的转录因子。LIM同源结构域转录因子Lmx1a对鸡mdDA神经元发育，以及胚胎干细胞（ES细胞）向多巴胺能表型的高效分化均至关重要。本研究旨在更深入解析Lmx1a-dr/dr小鼠的细微表型，因此通过基因芯片分析阐明Lmx1a缺失后神经元缺陷背后的确切分子编程机制。后续表达验证实验证实，Nurr1的表达受Lmx1a调控，并鉴定出Pou4f1、Pbx1、Pitx2、C130021l20Rik、Calb2及Rspo2等额外下游靶基因。鉴于这些基因中的大多数在发育过程中呈现特异性的吻侧-外侧表达缺失特征，Nurr1与C130021l20Rik在成熟mdDA系统的黑质致密部（SNc）中表达受到影响。值得注意的是，该区域的缺陷表现为Wnt/β-连环蛋白信号通路激活因子Rspo2的完全缺失。对Rspo2-/-胚胎的表达分析显示，其mdDA神经元存在异常，表型部分模拟Lmx1a突变体。综上，本研究揭示Lmx1a对mdDA神经元区域的吻侧-外侧亚群至关重要，其可能通过激活Wnt通路激活因子Rspo2，调控mdDA前体细胞的增殖与分化，进而发挥关键功能。本基因芯片表达研究对4例纯合子LMX1A dr/dr小鼠的胚胎期12.5中脑样本，与1例混合野生型（wt/wt）同窝幼崽样本进行了比较分析；其中2份样本采用反向染料标记方向进行检测。