ABSTRACT
The cell-adhesion molecule NEPH1 is required for maintaining the structural integrity and function of the glomerulus in the kidneys. In the nervous system of Drosophila and C. elegans, it is involved in synaptogenesis and axon branching, which are essential for establishing functional circuits. In the mammalian nervous system, the expression regulation and function of Neph1 has barely been explored. In this study, we provide a spatiotemporal characterization of Neph1 expression in mouse dorsal root ganglia (DRGs) and spinal cord. After the neurogenic phase, Neph1 is broadly expressed in the DRGs and in their putative targets at the dorsal horn of the spinal cord, comprising both GABAergic and glutamatergic neurons. Interestingly, we found that PRRXL1, a homeodomain transcription factor that is required for proper establishment of the DRG-spinal cord circuit, prevents a premature expression of Neph1 in the superficial laminae of the dorsal spinal cord at E14.5, but has no regulatory effect on the DRGs or on either structure at E16.5. By chromatin immunoprecipitation analysis of the dorsal spinal cord, we identified four PRRXL1-bound regions within the Neph1 introns, suggesting that PRRXL1 directly regulates Neph1 transcription. We also showed that Neph1 is required for branching, especially at distal neurites. Together, our work showed that Prrxl1 prevents the early expression of Neph1 in the superficial dorsal horn, suggesting that Neph1 might function as a downstream effector gene for proper assembly of the DRG-spinal nociceptive circuit.
Subject(s)
Ganglia, Spinal , Homeodomain Proteins , Neurites , Spinal Cord Dorsal Horn , Transcription Factors , Animals , Mice , Spinal Cord Dorsal Horn/metabolism , Spinal Cord Dorsal Horn/cytology , Neurites/metabolism , Neurites/physiology , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Ganglia, Spinal/metabolism , Ganglia, Spinal/cytology , Ganglia, Spinal/embryology , Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Membrane Proteins/genetics , Nerve Tissue ProteinsABSTRACT
Prrxl1 encodes for a paired-like homeodomain transcription factor essential for the correct establishment of the dorsal root ganglion - spinal cord nociceptive circuitry during development. Prrxl1-null mice display gross anatomical disruption of this circuitry, which translates to a markedly diminished sensitivity to noxious stimuli. Here, by the use of an immunoprecipitation and mass spectrometry approach, we identify five highly conserved phosphorylation sites (T110, S119, S231, S233 and S251) in PRRXL1 primary structure. Four are phospho-S/T-P sites, which suggest a role for the prolyl isomerase PIN1 in regulating PRRXL1. Accordingly, PRRXL1 physically interacts with PIN1 and displays diminished transcriptional activity in a Pin1-null cell line. Additionally, these S/T-P sites seem to be important for PRRXL1 conformation, and their point mutation to alanine or aspartate down-regulates PRRXL1 transcriptional activity. Altogether, our findings provide evidence for a putative novel role of PIN1 in the development of the nociceptive system and indicate phosphorylation-mediated conformational changes as a mechanism for regulating the PRRXL1 role in the process.
Subject(s)
Ganglia, Spinal/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Spinal Cord/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Conserved Sequence , Embryo, Mammalian , Fibroblasts/cytology , Fibroblasts/metabolism , Ganglia, Spinal/cytology , Ganglia, Spinal/growth & development , Homeodomain Proteins/genetics , Mice , Mice, Knockout , NIMA-Interacting Peptidylprolyl Isomerase/genetics , Nerve Tissue Proteins/genetics , Neurons/pathology , Phosphorylation , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Spinal Cord/cytology , Spinal Cord/growth & development , Transcription Factors/geneticsABSTRACT
The proper establishment of the dorsal root ganglion/spinal cord nociceptive circuitry depends on a group of homeodomain transcription factors that includes Prrxl1, Brn3a and Tlx3. By the use of epistatic analysis, it was suggested that Tlx3 and Brn3a, which highly co-localize with Prrxl1 in these tissues, are required to maintain Prrxl1 expression. Here, we report two Tlx3-dependent transcriptional mechanisms acting on Prrxl1 alternative promoters, referred to as P3 and P1/P2 promoters. We demonstrate that (i) Tlx3 induces the transcriptional activity of the TATA-containing promoter P3 by directly binding to a bipartite DNA motif and (ii) it synergistically interacts with Prrxl1 by indirectly activating the Prrxl1 TATA-less promoters P1/P2 via the action of Brn3a. The Tlx3 N-terminal domain 1-38 was shown to have a major role on the overall Tlx3 transcriptional activity and the C-terminus domain (amino acids 256-291) to mediate the Tlx3 effect on promoters P1/P2. On the other hand, the 76-111 domain was shown to decrease Tlx3 activity on the TATA-promoter P3. In addition to its action on Prrxl1 alternative promoters, Tlx3 proved to have the ability to induce Prrxl1 phosphorylation. The Tlx3 domain responsible for Prrxl1 hyperphosphorylation was mapped and encompasses amino acid residues 76 to 111. Altogether, our results suggest that Tlx3 uses distinct mechanisms to tightly modulate Prrxl1 activity, either by controlling its transcriptional levels or by increasing Prrxl1 phosphorylation state.
Subject(s)
Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Homeodomain Proteins/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Embryo, Mammalian , Gene Expression Regulation, Developmental , HeLa Cells , Humans , Mice , Molecular Sequence Data , Nociception , Phosphorylation , Protein Processing, Post-Translational , Spinal Cord/metabolismABSTRACT
The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5'-UTR variants, named 5'-UTR-A, 5'-UTR-B, and 5'-UTR-C. These 5'-UTR sequences confer distinct mRNA stability and translation efficiency to the Prrxl1 transcript. The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia.