Hepatocyte growth factor-Met signaling is required for Runx1 extinction and peptidergic differentiation in primary nociceptive neurons

Abstract

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.

Figure 1.

A population of CGRP⁺ peptidergic neurons arises from RUNX1⁺ precursors. Fate-mapping analysis of RUNX1 lineage using tamoxifen-inducible Runx1^Cre. Representative examples of CGRP and YFP immunofluorescence in newborn DRG of Runx1^Cre Rosa26^eYFP injected with 4-OHT at E12.5. These lineage experiments clearly demonstrated that a number of CGRP⁺ peptidergic neurons (arrowheads) derive from cells expressing RUNX1 at early developmental stages. Scale bars, 20 μm.

Figure 2.

Met is expressed in a subset of adult DRG neurons. a, Pictures of adult DRG illustrating double labeling of Met together with different markers of specific neuronal populations. As shown, most Met⁺ neurons are TrkA⁺/CGRP⁺ peptidergic neurons. It is also expressed in some TrkC⁺ neurons (arrowheads). In contrast, Met is largely excluded from Ret⁺/RUNX1⁺ population of nociceptive neurons and TrkB⁺ mechanoceptive neurons. Immunofluorescence for CGRP, TrkA, TrkC, Ret, and RUNX1. In situ hybridization for Met and TrkB. b, Proportions of Met⁺ cells expressing different markers (data obtained from three different animals) and size distribution of Met⁺ neurons. c, Pictures illustrating coexpression of Met and RUNX1 at E16 in Runx1^LacZ reporter embryos. Immunoflurescence for LacZ. In situ hybridization for Met. Scale bars: a, 50 μm; b, 30 μm.

Figure 3.

Met controls Runx1 extinction in vivo. a, Increased number of RUNX1 neurons in Nes-Met mutant animals. Pictures of RUNX1 immunofluorescence show a moderate increase of RUNX1⁺ neurons in Nes-Met mice. Quantitative analysis revealed a significant difference in these animals compared with control (*p < 0.05; control, n = 3; Nes-Met, n = 3). b, Persistent expression of RUNX1 in TrkA⁺ neurons in adult Nes-Met mice. TrkA in situ hybridization followed by immunofluorescence for RUNX1 revealed that RUNX1 is completely excluded from peptidergic neurons in control animals (top). In contrast, a number of TrkA⁺ neurons maintains RUNX1 expression in adult Nes-Met mice (bottom) suggesting that Met is necessary for proper Runx1 extinction. Immunofluorescence for RUNX1. In situ hybridization for TrkA. Scale bars, 20 μm.

Figure 4.

RUNX1 represses Met expression in vivo. Met is derepressed in Runx1^−/− DRG neurons. Representative pictures illustrating the increase of Met expression in DRG neurons in the absence of RUNX1. Quantitative analysis demonstrated that Met increase in Runx1^−/− mice was parallel to the expansion of CGRP population. Interestingly, the proportion of CGRP⁺Met⁻ neurons was not affected (**p < 0.001, control, n = 3; Runx1^−/−, n = 3). Immunofluorescence for CGRP. In situ hybridization for Met. Scale bar, 50 μm.

Figure 5.

Conditional Nes-Met mutants do not show defects in the number of neurons or early specification of nociceptive and proprioceptive neurons. a, Quantification of DRG neurons using the pan-neuronal marker SCG10 revealed no differences between Nes-Met and their control littermates (control, n = 3; Nes-Met, n = 3). b, Representative pictures illustrating the distribution of TrkA, TrkC, and PV-expressing neurons in control and Nes-Met DRG. Immunofluorescence for TrkA and TrkC. In situ hybridization for PV. c, Quantitative analysis of the numbers of DRG neurons positive for TrkA, TrkC, and PV showed no significant differences in Nes-Met mutants compared with control mice (control, n = 4; Nes-Met, n = 4). Scale bars, 50 μm.

Figure 6.

Disruption of peptidergic markers in Nes-Met mice. a, Expression of the peptidergic markers CGRP, Trpv1, and Trpa1 in control and Nes-Met DRG. Immunofluorescence for CGRP, IB4, and TrkC. In situ hybridization for Trpv1 and Trpa1. b, Neuronal counts revealed that Met deletion led to a significant reduction in the number of DRG neurons expressing CGRP, Trpv1, and Trpa1 (control, n = 3; Nes-Met, n = 3; **p < 0.01, *p < 0.05). Scale bar, 50 μm.

Figure 7.

Phenotypic switch of nociceptive neurons in Nes-Met mice. a, Double staining of TrkA/Ret and TrkA/SP showed an increase in the number of DRG neurons positive for Ret, SP, and TrkA/Ret (arrowheads) in Nes-Met mice. Conversely, TrkA remained unchanged. Immunofluorescence for TrkA and Ret. In situ hybridization for SP. b, Quantitative analysis of the percentage of Ret and SP-expressing neurons (left) demonstrated a significant increase in Nes-Met DRG. The proportion of double-labeled TrkA⁺/Ret⁺ neurons is also augmented (right) (control, n = 4; Nes-Met, n = 5; p < 0.05). c, Schematic representation of the observed defects of nociceptive maturation in Nes-Met mice. Briefly, a subset of prospective peptidergic CGRP⁺ neurons is lost (dotted line) and switch their fate to become TrkA⁺/Ret⁺ neurons expressing SP. Scale bar, 50 μm.

Figure 8.

Proposed model for late maturation of nociceptive neurons. As described previously, prospective nociceptive neurons are TrkA⁺ at E14.5. At this stage, most of these cells (88%) coexpresses Runx1 (right column), whereas only a minor population remains TrkA⁺Runx1⁻ (left column). Our findings suggest that this latter population give rise to a subset of CGRP⁺ nociceptive neurons independently from Runx1 and Met. According to our hypothesis, Runx1 high expressors would give rise to Ret⁺ nonpeptidergic neurons. Finally, low levels of Runx1 might allow initial Met expression and the apparition of a transitory population of Met⁺Runx1⁺ cells. Due to mutually repressive activity, this population would segregate and become either Met⁺CGRP⁺ or TrkA⁺Ret⁺ neurons. Boxes in the lower part of the scheme represent the final distribution of different subsets of nociceptive neurons in the absence of Met (left) or Runx1 (right).