METHODS: To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings.
RESULTS: Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression.
CONCLUSIONS: This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.
方法:为了确定V0v基因调控网络的候选成员,我们FAC分选野生型和evx1;evx2双突变斑马鱼V0v脊髓中间神经元,并使用微阵列和单细胞RNA-seq对它们进行表达分析。我们还使用原位杂交来比较evx1中候选基因子集的表达;evx2双突变体和野生型兄弟姐妹。
结果:我们的数据揭示了斑马鱼V0v脊髓中间神经元在48小时时的两种不同分子亚型,到了这个发展阶段,evx1;evx2双突变细胞转移到抑制性脊髓中间神经元,或者运动神经元.我们的结果还确定了25个转录调节基因,这些基因需要Evx1/2才能在V0v中间神经元中表达,加上另外11个转录调节基因,这些基因在V0v中间神经元中被Evx1/2抑制。后面的两个基因是hmx2和hmx3a。有趣的是,我们发现Hmx2/3a,抑制skor1a和nefma的dI2中间神经元表达,在V0v中间神经元中表达需要Evx1/2的两个基因。这表明Evx1/2可能通过抑制Hmx2/3a表达来调节V0v中间神经元中skor1a和nefma的表达。
结论:这项研究确定了斑马鱼V0v脊髓中间神经元的两个分子不同的亚群,以及多个转录调节因子,它们是作用于Evx1/2下游的强大候选因子,以指定这些细胞的基本功能特征。我们的数据进一步表明,在没有Evx1和Evx2的情况下,V0v脊髓中间神经元最初将其神经递质表型从兴奋性变为抑制性,然后,稍后,开始表达不同类型的抑制性脊髓中间神经元的标记,或者运动神经元.一起来看,我们的研究结果显著增加了我们对V0v和脊柱发育的认识,并使我们更接近确定确定这一关键细胞类型的完整基因调控网络的基本目标.