Figure 5. 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% ± 3.2%; Cbfb CKO, 5.6% ± 0.6%) and Kif21b (Control, 20.2% ± 0.1%; Cbfb CKO, 2.4% ± 0.5%) in control and Cbfb CKO DRGs at P0 by in situ hybridization analysis. The gene expression deficits in Cbfb CKO animals phenocopy those observed in Runx1 CKO animals except for Kif21b expression. The discrepancy likely reflects Kif21b expression in proprioceptors where it presumably depends on Runx3 and CBFβ for expression. Shown are the means ± SEMs for the percentage of neurons expressing indicated genes based on counts from a total of at least 9 sections from three independent animals per genotype. DRG neurons were identified and counted based on combined NeuN immunostaining, which was not shown. See also Figure 5—figure supplement 1D, E. (K–N) GFP immunostaining of P0 hairy skin to visualize sensory innervation of the epidermis in control and Runx1 CKO animals (K and L) or control and Cbfb CKO animals (M and N) that also carry the Tau^mGFP allele. The Tau^mGFP allele was introduced to label all Cre-expressing neurons including all sensory neurons. Note that there is a dramatic reduction in fiber density specifically in the epidermis in both Runx1 CKO and Cbfb CKO animals relative to their littermate controls. The yellow dotted line denotes the epidermal-dermal junction which was drawn based on TOPRO3 counterstain (blue). (O and P) Quantification of sensory innervation of the epidermis in control and Runx1 CKO animals (O) or control and Cbfb CKO animals (P) reveals a remarkably similar reduction in the innervation density in both mutants at P0. The innervation density is defined as the fraction of area occupied by GFP⁺ fibers in the epidermis. An unpaired t test was performed on data from three independent animals per genotype. ***p ≤ 0.001. (Q and R) Runx1 immunostaining of control and Cbfb CKO DRGs at P0 shows almost complete loss of Runx1 proteins in the absence of CBFβ. Shown are representative images from at least three independent experiments. (S) Immunoblot analysis of expression of Runx1 and Cbfb in control and Cbfb CKO DRGs at P0 shows dramatic loss of Runx1 proteins as a result of CBFβ depletion. βIII-Tubulin serves as a loading control. Shown are results from three independent experiments. (T and U) In situ hybridization analysis of Runx1 expression in control and Cbfb CKO DRGs at P0 shows comparable levels of Runx1 transcripts in control and mutant animals. (V) Real-time PCR analysis of Runx1 expression in control and Cbfb CKO DRGs at P0 shows increased Runx1 mRNA expression in Cbfb CKO DRGs compared to control, which likely reflects an increased ratio of nociceptors to proprioceptors (data not shown). An unpaired t test was performed on data from four independent pairs of control and mutant animals, **p ≤ 0.01. Cbfb^f/f mice were used as control animals for analysis of Cbfb CKO mutants. Scale bar, 50 μm.

DOI:http://dx.doi.org/10.7554/eLife.10874.011

Figure 5—figure supplement 1. Generation of the Cbfb^f 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 mouse expressing FlpE recombinase in the germ line. A Bstz171 restriction site was introduced immediately downstream of the 3’ loxP site to facilitate southern screening of ES cells. LoxP and FRT sites are shown as open and filled triangles respectively. (B and C) Efficient gene ablation in the DRG shown by in situ hybridization analysis of Cbfb expression in control and Cbfb CKO DRGs at P0. (D and E) Real-time PCR analysis of expression of Gfra2, Mrgprd, Ptprt and Myo1a in control and Cbfb CKO DRGs (D) or control and Runx1 CKO DRGs (E) at P0 further demonstrates the similarity in nonpeptidergic-specific gene expression deficits between Cbfb CKO and Runx1 CKO mutants. Unpaired t tests were performed on data from four independent pairs of control and Cbfb CKO animals, or three independent pairs of control and Runx1 CKO animals. **p ≤ 0.01, ***p ≤ 0.001. (F-I) Expression of TH and GFP in DRGs of P21 Runx1CreER/ + ; TaumGFP/ + and Runx1CreER/f; TaumGFP/ + animals (F and G)(Control, 23.2% ± 1.6%; Runx1 mutant, 3.8% ± 2.7%) or Runx1CreER/ + ;Cbfbf/ + ; TaumGFP/ + and Runx1CreER/ + ; Cbfbf/f; TaumGFP/ + animals (H and I) (Control, 21.4% ± 1.9%; Cbfb mutant, 8.9% ± 2.0%) that received an intraperitoneal injection of tamoxifen at P2. The TaumGFP allele was introduced to indicate the neurons with active Cre expression. Note that there is a substantial reduction in the number of GFP/TH double positive neurons due to a selective loss of TH expression in the GFP + population in both Runx1 and Cbfb mutant DRGs. Shown are means ± SEM for the percentage of GFP + neurons that express TH based on counts from a total of at least 9 sections from three independent animals per genotype. (J–M) Double staining of CGRP and GFP in back hairy skin of P21 Runx1CreER/ + ; TaumGFP/ + and Runx1CreER/f; TaumGFP/ + animals (J and K) or Runx1CreER/ + ;Cbfbf/ + ; TaumGFP/ + and Runx1CreER/ + ; Cbfbf/f; TaumGFP/ + animals (L and M) that received IP injections of tamoxifen at P2. Note that in both mutant animals, there is a marked decrease in the number of GFP + longitudinal lanceolate endings characteristic of C-LTMRs, which is accompanied by an increased frequency of GFP + endings that assume a peptidergic neuron-like morphology. Shown are representative images from more than 3 independent experiments. Scale bar, 50 μm.

DOI:http://dx.doi.org/10.7554/eLife.10874.012