Monteiro Filipe A, Miranda Rafael M, Samina Marta C, Dias Ana F, Raposo Alexandre A S F, Oliveira Patrícia, Reguenga Carlos, Castro Diogo S, Lima Deolinda
Unidade de Biologia Experimental, Departamento de Biomedicina, Faculdade de Medicina da Universidade do Porto, Porto, Portugal.
Pain Research Group, Instituto de Biologia Molecular e Celular, Porto, Portugal.
Front Cell Dev Biol. 2021 Apr 29;9:642697. doi: 10.3389/fcell.2021.642697. eCollection 2021.
The spinal cord dorsal horn is a major station for integration and relay of somatosensory information and comprises both excitatory and inhibitory neuronal populations. The homeobox gene Tlx3 acts as a selector gene to control the development of late-born excitatory (dILB) neurons by specifying glutamatergic transmitter fate in dorsal spinal cord. However, since Tlx3 direct transcriptional targets remain largely unknown, it remains to be uncovered how Tlx3 functions to promote excitatory cell fate. Here we combined a genomics approach based on chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and expression profiling, with validation experiments in null embryos, to characterize the transcriptional program of Tlx3 in mouse embryonic dorsal spinal cord. We found most dILB neuron specific genes previously identified to be directly activated by Tlx3. Surprisingly, we found Tlx3 also directly represses many genes associated with the alternative inhibitory dILA neuronal fate. In both cases, direct targets include transcription factors and terminal differentiation genes, showing that Tlx3 directly controls cell identity at distinct levels. Our findings provide a molecular frame for the master regulatory role of Tlx3 in developing glutamatergic dILB neurons. In addition, they suggest a novel function for Tlx3 as direct repressor of GABAergic dILA identity, pointing to how generation of the two alternative cell fates being tightly coupled.
脊髓背角是躯体感觉信息整合与中继的主要站点，由兴奋性和抑制性神经元群体组成。同源盒基因Tlx3作为一个选择基因，通过在脊髓背侧指定谷氨酸能递质命运来控制晚期生成的兴奋性（dILB）神经元的发育。然而，由于Tlx3直接的转录靶点仍大多未知，Tlx3如何发挥作用促进兴奋性细胞命运仍有待揭示。在这里，我们将基于染色质免疫沉淀随后进行下一代测序（ChIP-seq）和表达谱分析的基因组学方法，与在基因敲除胚胎中的验证实验相结合，以表征小鼠胚胎背侧脊髓中Tlx3的转录程序。我们发现了大多数先前确定由Tlx3直接激活的dILB神经元特异性基因。令人惊讶的是，我们发现Tlx3还直接抑制许多与替代性抑制性dILA神经元命运相关的基因。在这两种情况下，直接靶点都包括转录因子和终末分化基因，表明Tlx3在不同水平上直接控制细胞身份。我们的发现为Tlx3在发育中的谷氨酸能dILB神经元中的主要调控作用提供了一个分子框架。此外，它们还提示了Tlx3作为GABA能dILA身份直接抑制因子的新功能，指出了两种替代性细胞命运的产生是如何紧密耦合的。
