Abstract
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 Tlx3 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.
Highlights
The dorsal horn of the spinal cord modulates and conveys peripheral somatosensory input related to pain, itch, touch, cold, and warm to higher brain centres
To understand how Tlx3 acts as a selector gene in the specification of the glutamatergic populations of dorsal horn neurons, we began by defining the repertoire of genes directly regulated by Tlx3 by combining genome-wide mapping of Tlx3 binding sites with expression profiling of dorsal spinal cord region
We performed chromatin immunoprecipitation (ChIP)-seq using chromatin extracted from mouse dorsal spinal cord at embryonic day 14.5 (E14.5), a stage at which Tlx3 is highly expressed in newly born postmitotic neurons (Figure 1A)
Summary
The dorsal horn of the spinal cord modulates and conveys peripheral somatosensory input related to pain, itch, touch, cold, and warm to higher brain centres. Most of the neurons that settle in the superficial dorsal horn are generated during the late phase of dorsal neurogenesis [embryonic day (E) 12 to E13.5] from a common pool of progenitors expressing the transcription factors Gsx1/2 and Ascl (Gross et al, 2002; Muller et al, 2002; Mizuguchi et al, 2006; Wildner et al, 2006) From these progenitors, two related subpopulations arise in a salt and pepper manner: GABAergic dILA neurons characterized by the expression of Ptf1a, Lbx, Pax, Lhx1/5, and Gbx homeodomain (HD) transcription factors (Gross et al, 2002; Muller et al, 2002; Glasgow et al, 2005; John et al, 2005; Pillai et al, 2007), and glutamatergic dILB neurons, which co-express Tlx1/3, Lbx, Lmx1b, Prrxl, and Brn3a (Cheng et al, 2004, 2005; Xu et al, 2008; Rebelo et al, 2010). In dILB neurons, Tlx promotes the expression of Prrxl, Lmx1b, and Brn3a/Pou4f1 (Qian et al, 2002; Xu et al, 2008) and antagonizes Lbx1-mediated specification of GABAergic phenotype (Cheng et al, 2005)
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