Prdm12 is a conserved regulator of pain-sensing neurons: a potential target for pain therapy
Prdm12 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA
Desiderio S, Vermeiren S, Van Campenhout C, Kricha S, Malki E, Richts S, Fletcher EV, Vanwelden T, Schmidt BZ, Henningfeld KA, Pieler T, Woods CG, Nagy V, Verfaillie C, Bellefroid EJ.
Cell Rep. 2019 Mar 26;26(13):3522-3536.e5. doi: 10.1016/j.celrep.2019.02.097.
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Desiderio et al. report that, in developing somatosensory neurons, Prdm12 is restricted to the nociceptors and that these are selectively eliminated from Prdm12 mutant mice. In Xenopus and human iPSCs, they show that Prdm12, in conjunction with bHLH proneural proteins, promotes the expression of the neurotrophin receptor TrkA.
In somatosensory ganglia, Prdm12 is specific to the nociceptive lineage
Prdm12 is necessary for the survival of developing nociceptors
Prdm12 initiates and maintains the expression of TrkA in developing nociceptors
Prdm12 acts in conjunction with bHLH proteins Ngn1/2 to promote a nociceptor fate
In humans, many cases of congenital insensitivity to pain (CIP) are caused by mutations of components of the NGF/TrkA signaling pathway, which is required for survival and specification of nociceptors and plays a major role in pain processing. Mutations in PRDM12 have been identified in CIP patients that indicate a putative role for this transcriptional regulator in pain sensing. Here, we show that Prdm12 expression is restricted to developing and adult nociceptors and that its genetic ablation compromises their viability and maturation. Mechanistically, we find that Prdm12 is required for the initiation and maintenance of the expression of TrkA by acting as a modulator of Neurogenin1/2 transcription factor activity, in frogs, mice, and humans. Altogether, our results identify Prdm12 as an evolutionarily conserved key regulator of nociceptor specification and as an actionable target for new pain therapeutics.
Figure 2. Prdm12 Is Expressed in Precursors of Both Ngn1- and Ngn2-Dependent Neurogenic Waves
Transverse sections of DRG of embryos with the indicated genotype, double immunostained at the indicated stage with Prdm12 (purple) and the indicated markers (green).
(A–C) Comparison of the expression of Prdm12 with that of Ngn2- and Ngn1- expressing cells or with boundary cap-derived cells. Yellow arrowheads point to Prdm12+ precursors co-expressing the second analyzed marker (Ngn2 in A, GFP in B, and tdTomato in C).
(D–I) Double immunostaining of Prdm12 and TrkA (D and G), TrkB (E and H), or TrkC (F and I) in DRG of E15.5 WT and Ngn1GFP/GFP knockout embryos. Note the severe loss of Prdm12+ and TrkA+ cells in Ngn1GFP/GFP compared to WT and that the few remaining TrkA+ cells also express Prdm12 (D and G).
(J) Transverse sections of WT and Ngn1GFP/GFP E11.5 embryos hybridized with Ngn1 or Prdm12 antisense probes. Prdm12 expression at that stage appears unaffected by the absence of Ngn1. DRG and spinal cord are delineated with dashed lines. Scale bars, 100 mm.
Figure 6. Prdm12 Is Induced by Retinoic Acid and Cooperates with Ngn1 and Ngn2 to Promote a No- ciceptor Fate in Xenopus
(A) Prdm12, Ntrk1, Ntrk2, and Ntrk3 are expressed in TG (arrowheads) and DRG (arrows) of stage 28 Xenopus tadpoles. Scale bar, 200 mm.
(B) qRT-PCR analysis of Prdm12 expression in stage 16 AC explants derived from embryos injected with mRNA of Pax3-GR (250 pg), Zic1-GR (125 pg), or Ngn2-GR (250 pg). The hatching gland marker Xhe, the pre-placodal marker Six1, and the neuronal-specific gene Tubb2b were also examined as controls of Pax3-, Zic1-, and Ngn2-inducing activity, respectively.
(C) Prdm12 expression in Xenopus neurula-stage embryos injected unilaterally at the 2-cell stage with Pax3-GR (250 pg), Zic1-GR (125 pg), Ngn1 (200 pg), or Ngn2-GR (250 pg). Xhe that is upregulated by Pax3, Tubb2b that is induced by Ngn1/2, and pre-placodal marker Foxi1c that is reduced by Zic1 were examined as controls. Arrow- heads indicate their altered expression in the trigeminal placode on the injected side revealed by X-gal staining. Scale bar, 200 mm.
(D) Treatment of Zic1-GR (125 pg)-overexpressing AC explants with citral (100 mM) blocks Prdm12 and Six1 used as controls.
(E) Treatment of intact embryos at stage 11 with RA (0.01 mM) expands Prdm12.
(F) qRT-PCR analysis of the expression of the indicated genes in stage 31 animal cap explants overexpressing Prdm12 (250 pg), Ngn1 (200 pg), and Ngn2 (200 pg), alone or in combination, as indicated, showing that Prdm12 modulates the inducing activity of Ngn1/2.
(G) qRT-PCR analysis of Ntrk1 and En1 in stage 28 Xen- opus laevis AC explants derived from embryos co-ex- pressing Ngn1 and Prdm12 WT or mutant constructs as indicated, showing that both the PR and the zinc-finger domain of Prdm12 are required for its activity.
In (C) and (E), the total number of injected embryos analyzed and among them those showing the observed phenotype is indicated. In all qRT-PCR experiments, mean ± SEM from at least 2 independent experiments is shown, and the individual values obtained are indicated (red dots).
Figure 7. PRDM12 Promotes a Nociceptor Fate in Hu- man Embryonic Stem Cells Differentiated into Sensory Neurons
(A) Transverse sections at the thoracic level of the spinal cord and DRG (delineated with dashed lines) of a CS12 human embryo stained with PRDM12 and TRKA antibodies. Lower panels are high magnifications of some PRDM12+/TRKA+ cells observed in the DRG (indicated by the yellow arrowhead). Scale bars, 25 mm.
(B) qRT-PCR analysis of the expression of PRDM12, NTRK1, NGN1, and NGN2 in uninduced Prdm12-RMCE iPSCs differ- entiated into sensory neurons and harvested at day 7, 15, or 25 of differentiation. Expression levels were compared to the level in undifferentiated iPSCs, defined as 1 (mean ± SEM).
(C) qRT-PCR analysis of the expression of PRDM12, NTRK1, NTRK2, and NTRK3 in doxycycline-induced Prdm12-RMCE iPSCs differentiated into sensory neurons and harvested until day 7, 15, or 25 of differentiation. Expression levels were compared to the expression level in uninduced control cells, which was defined as 1 (mean ± SEM).
(D) Immunostaining of TRKA and PRDM12 on Prdm12-RMCE iPSCs differentiated into sensory neurons and harvested at day 32. Scale bar, 25 mm. See also Figure S7.
In (B) and (C), individual values for biological replicates are indicated (dots).
Adapted with permission from Cell Press on behalf of Cell Reports: Desederio et al. (2019). Prdm12 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA. Cell Rep. 2019 Mar 26; 26(13):3522-3536.e5. doi: 10.1016/j.celrep.2019.02.097. Copyright (2019).
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