Extrinsic and intrinsic signals converge on the Runx1/CBFβ transcription factor for nonpeptidergic nociceptor maturation
- Howard Hughes Medical Institute, Harvard Medical School, United States
- Howard Hughes Medical Institute, Johns Hopkins School of Medicine, United States
- Burke Medical Research Institute, Weill Medical College of Cornell University, United States
- Seattle Children's Hospital, Seattle Children's Research Institute, United States
Figures
Figure 1 with 1 supplement
The majority of nonpeptidergic nociceptor-specific genes depend on both NGF and Runx1 for expression.
(A–J) Expression of Mrgprd (Control, 12.7% ± 2.4%; Ngf-/-Bax-/-, 0%), Gfra2 (Control, 28.7% ± 2.5%; Ngf-/-Bax-/-, 14.3% ± 1.1%), Ptprt (Control, 19.1% ± 0.3%; Ngf-/-Bax-/-, 10.3% ± 2.1%), Myo1a (Cont…
Figure 1—figure supplement 1
The majority of nonpeptidergic nociceptor-specific genes depend on both NGF and Runx1 for initiation of expression.
(A–P) Expression of Mrgprd, Ptprt, Myo1a and Kif21b in control and Ngf-/-Bax-/- DRGs (A-H), or in control and Runx1 CKO DRGs (I–P) at E16.5, assessed by in situ hybridization analysis. Note that …
Figure 2
Ret is an unusual nonpeptidergic nociceptor-specific gene whose expression is differentially dependent on NGF and Runx1.
(A and B) Greatly diminished Ret expression in Ngf-/-Bax-/- DRGs compared to controls at P0, assessed by in situ hybridization. The NGF-independent Ret+ neurons are mechanoreceptors (Aβ RA-LTMRs). (C…
Figure 3 with 2 supplements
Runx1 functions downstream of NGF to mediate expression of the majority of nonpeptidergic nociceptor-specific genes, whereas it controls Ret expression at least in part by enhancing NGF signaling.
(A–L) In situ hybridization analysis of expression of Mrgprd (A–C), Gfra2 (D–F), Ptprt (G–I) and Ret (J–L) in DRGs of P2 control animals that received BSA injections, Runx1CKO animals that received …
Figure 3—figure supplement 1
Runx1 potentiates TrkA activity without regulating TrkA expression.
(A–D) Double staining of pTrk-SHC and Neurofilament heavy chain (NFH) (A and B) or pTrk-PLCγ and NFH (C and D) in control and Runx1 CKO DRGs at P0 shows greatly diminished pTrk immunoreactivity in …
Figure 3—figure supplement 2
Runx1 controls expression of the majority of nonpeptidergic-specific genes independent of its stimulatory effect on NGF signaling.
(A–D) Real-time PCR analysis of expression of Mrgprd (A), Gfra2 (B), Ptprt (C) and Ret (D) in DRGs of P2 control animals that received BSA injections, Runx1CKO animals that received BSA injections …
Figure 4 with 1 supplement
Runx1 and CBFβ are co-expressed and form a complex in DRG neurons.
(A and B) Double immunostaining of Flag and Runx1 in wildtype and CbfbFlag/+ DRGs at P0 confirms the specificity of the Flag antibody. Note that Flag immunoreactivity in wildtype DRGs is nearly …
Figure 4—figure supplement 1
Generation of the CbfbFlag allele and more detailed characterization of the temporal and spatial patterns of Cbfb expression.
(A) Schematic showing the targeting strategy used for generation of CbfbFlagknockin mice. Following germ-line transmission, the Neo selection cassette was removed by crossing the carrier to a mouse …
Figure 5 with 1 supplement
CBFβ is required for acquisition of molecular and morphological features of nonpeptidergic nociceptors.
(A–J) Expression of Mrgprd (Control, 26.9% ± 2.8%; Cbfb CKO, 0%), Gfra2 (Control, 38.8% ± 2.8%; Cbfb CKO, 11.7% ± 1.9%), Ptprt, (Control, 31.9% ± 3.2%; Cbfb CKO, 7.1% ± 2.8%), Myo1a (Control, 26.9% …
Figure 5—figure supplement 1
Generation of the Cbfbf allele and demonstration of a postnatal requirement for both CBFβ and Runx1 in C-LTMR development.
(A) Schematic showing the targeting strategy for generation of the Cbfbf conditional allele. Following germ-line transmission, the Neo selection cassette was removed by crossing the carrier to a …
Figure 6 with 1 supplement
NGF regulates the Runx1/CBFβ complex through differential control of Cbfb and Runx1 expression.
(A and B) In situ hybridization analysis of Cbfb expression in control and Ngf-/-Bax-/- DRGs at E14.5 shows a significant reduction in the level of transcripts in small diameter neurons that …
Figure 6—figure supplement 1
Cbfb expression is NGF-dependent in vivo.
(A) Real-time PCR analysis of Cbfb expression in control and Ngf-/-Bax-/- DRGs at E14.5 and P0 reveals early onset of NGF dependence for Cbfb expression. An unpaired t test was performed using data …
Figure 7 with 1 supplement
NGF promotes Cbfb expression through the ERK/MAPK signaling pathway.
(A–D) Double staining of Flag (green) and βIII-Tubulin (blue) in DMSO or U0126-treated dissociated DRG neurons from P0 CbfbFlag/+ animals that were cultured without or with NGF. U0126 is a selective …
Figure 7—figure supplement 1
In vitro evidence for the necessity of MAPK signaling for NGF-dependent CBFβ expression.
(A) Immunoblot analysis of expression of Cbfb in DMSO or U0126-treated dissociated DRG neurons from P0 CbfbFlag/ + animals that were cultured in the presence or absence of NGF. Histone 3 serves as …
Figure 8
Islet1 is required for initiation of Runx1, but not Cbfb expression.
(A–D) In situ hybridization analysis of expression of Runx1 (A and B) and Cbfb (C and D) in control and Isl1 CKO DRGs at E12.5 shows that Islet1 deficiency abolishes expression of Runx1 but not Cbfb …
Tables
Table 1
Primers used for real-time PCR analysis
| Cbfb | F-TCGAGAACGAGGAGTTCTTCAGGA | R-AGGCGTTCTGGAAGCGTGTCT |
| Runx1 | F-GCAGGCAACGATGAAAACTACT | R-GCAACTTGTGGCGGATTTGTA |
| Mrgprd | F-TGCTGCTGGAAACACTTCTAGGGA | R-GCTGCTGTCAAGAGTGGAGTTCAT |
| Gfra2 | F-TCGTACAGACCACTTGTGCC | R-ATCAAACCCAATCATGCCAG |
| Ptprt | F-ACCTGCTTCAACACATCACCCAGA | R-TTCATCTTCCTTGGCTGTGTCCCA |
| Myo1a | F-ACAGGTGCTTCAACACAGCCAATC | R-GCCCTTAAACAGTTCACTGGCACA |
| Ret | F-TCAACCTTCTGAAGACAGGCCACA | R-ATGTCAGCAAACACTGGCCTCTTG |
| PGP9.5 | F-CAGACCATCGGAAACTCCTG | R-CACTTGGCTCTATCTTCGGG |
| GAPDH | F-ATGCCTGCTTCACCACCTTCTT | R-ATGTGTCCGTCGTGGATCTGA |
Additional files
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Supplementary file 1.
Microarray analysis of genes that are differentially expressed in E16.5 DRGs of control and Runx1 CKO animals
- https://doi.org/10.7554/eLife.10874.019
