The zinc finger transcription factor Klf7 is required for TrkA gene expression and development of nociceptive sensory neurons

Abstract

TrkA, the high affinity receptor for nerve growth factor (NGF), is essential for the development of nociceptive sensory and sympathetic neurons. The zinc finger transcription factor Klf7 interacts with an important cis element of the TrkA minimal enhancer and is coexpressed with TrkA in these neurons. We show that Klf7 binds to the endogenous TrkA minimal enhancer and can activate transcription from the TrkA minimal enhancer in a sequence-dependent manner. In Klf7(-/-) newborn mice, we find a significant reduction in sensory neurons due to increased apoptosis. The neuronal loss is restricted to nociceptive neurons that normally depend on TrkA for neurotrophic support, while other populations of somatosensory neurons appear normal. The reduction of TrkA expression in sensory neurons is a direct effect of Klf7 gene ablation, rather than a secondary effect of cell death. As a result, Klf7(-/-) mice have deficient response to noxious stimuli. Finally, removal of one TrkA allele exacerbates the loss of TrkA(+) neurons in Klf7(-/-) mice. Thus, Klf7 specifically regulates TrkA gene expression and is required for the development of a subset of nociceptive sensory neurons.

Figure 1.

Klf7 specifically activates transcription from the TrkA minimal enhancer by binding its cognate site IK2. (A) ChIP analysis. Lane 1 is from empty vector (CS2 + MT) transfected cells without immunoprecipitation. Lane 2 is from CS2 + MT transfected cells after immunoprecipitation with an anti-Myc antibody. Lanes 3 and 4 are from myc-mKlf7 transfected cells following immunoprecipitation with (lane 3) and without (lane 4) an anti-Myc antibody. Lanes 5 and 6 are from myc-mKlf7C transfected cells following immunoprecipitation with (lane 5) and without (lane 6) an anti-Myc antibody. After ChIP, PCR was carried out using primers specific for the TrkA minimal enhancer to detect the precipitated DNA fragments. The arrow indicates the 173-bp PCR product. (B) Reporter constructs used in the luciferase assays. pGL3P is the empty vector pGL3-Promoter containing the firefly luciferase coding region under the control of a SV40 minimal promoter (Promega). TrkA#1 and TrkA#2 contain the wild-type TrkA minimal enhancer upstream of the SV40 minimal promoter in opposite orientations. TrkA-IK#1 and TrkA-IK#2 contain the TrkA minimal enhancer with a mutated IK2 core sequence, in opposite orientations. IK2(x3) contains three copies of the wild-type IK2 site in tandem. (C) Klf7 activates transcription from reporter constructs containing either the TrkA minimal enhancer or just its cognate site IK2 in PC12 cells. (D) Klf7 does not activate transcription from reporter constructs containing the TrkA minimal enhancer with a mutated IK2 site in PC12 cells. In both C and D, the empty vector CS2 + MT was used as a negative control. (^★★) p < 0.01, t-test.

Figure 2.

Reduced expression of TrkA in Klf7^-/- DRG. (A-F) Protein expression of TrkA in E12.5 DRG (A,B) and E13.5 DRG (C-F) of wild-type (A,C,E) and mutant (B,D,F) embryos assayed by anti-TrkA immunohistochemistry. (G,H) In situ hybridization of wild-type (G) and mutant (H) P0 DRG using a TrkA antisense RNA probe. (I) Real-time RT-PCR analysis of TrkA, TrkB, and TrkC at E11.5, E12.5, and E13.5. The RNA levels of TrkA, TrkB, and TrkC were normalized against the level of β-actin. The numbers represent the ratios of the relative RNA level of the mutant embryos to that of the wild-type embryos. N = 3 for each embryonic stage and genotype. Bars: A-D,G,H, 100 μm; E,F, 200 μm.

Figure 3.

Increased apoptosis in Klf7^-/- DRG at E13.5. (A,B) DRG sections of E12.5 wild-type (A) and mutant (B) embryos stained with the TUNEL method for the detection of cells undergoing apoptosis. (C,D) DRG sections of E13.5 wild-type (C) and mutant (D) embryos stained with the TUNEL method for the detection of cells undergoing apoptosis. (E,F) DRG sections of E13.5 wild-type (E) and mutant (F) embryos double-labeled with TUNEL (green) and anti-TrkA antibodies (red). (G) Quantification of TUNEL(+) cells showing comparable numbers of apoptotic cells at E12.5 between the wild-type and mutant DRG but a marked increase of apoptotic cells in mutant DRG at E13.5. Shown are the average numbers of TUNEL(+) cells per section from the wild-type (N = 3) and mutant (N = 3) embryos. Twenty sections of lumbar DRG of each embryo were used for counting TUNEL(+) cells. E12.5, p = 0.192; E13.5, p = 2.2 × 10^-9, t-test. Bars: A-D, 50 μm; E,F, 35 μm.

Figure 4.

A significant loss of NGF-dependent primary DRG neurons in culture. (A) Primary DRG neurons were isolated from E13.5 wild-type or Klf7^-/- embryos and cultured in the presence of 10 ng/mL NGF, BDNF, or NT3. The percentages of neuronal survival after 48 and 72 h in culture are shown. N = 8 for each genotype. (^★★) p < 0.01, t-test. (B) Diagram showing the DNA constructs of the full-length Klf7 and dominant-negative Klf7 (DN-Klf7). The number of amino acids is shown. AD represents the transactivation domain, and DBD represents the DNA-binding domain of Klf7. (C) DN-Klf7 inhibits the survival of NGF-dependent wild-type DRG neurons in a dose-dependent manner. DRG neurons from E13.5 wild-type embryos were cultured in the presence of 10 ng/mL NGF, BDNF, or NT3 and infected with recombinant adenoviruses expressing an inactive form of raf (Xraf), Klf7, or DN-Klf7, respectively. The multiplicity of infection (MOI) was 50 for Xraf, Klf7, and DN-Klf7^★, and 10 for DN-Klf7. The percentage of neuronal survival 72 h after the infection is shown. (^★★) p < 0.01, t-test.

Figure 5.

Loss of TrkA-specific neurons in Klf7^-/- DRG. (A,B) Representative images of L4 DRG of wild-type (A) and Klf7^-/- (B) P0 pups. (C,D) Parvalbumin (PV) immunostaining of L4 DRG of wild-type (C) and Klf7^-/- (D) P0 pups. (E,F) CGRP immunostaining of L4 DRG of wild-type (E) and Klf7^-/- (F) P0 pups. (G) Neuron counts of L4 DRG. N = 5 for each genotype. (^★) p < 0.05, t-test. (H) Quantification of CGRP(+) or PV(+) neurons in L4 DRG showing a significant reduction of CGRP(+) neurons in Klf7^-/- DRG but similar numbers of PV(+) neurons in wild-type and Klf7^-/- DRG. Serial sections of spinal columns of wild-type (N = 3) and Klf7^-/- (N = 3) P0 pups were stained with anti-CGRP or anti-PV antibodies, and the numbers of immunoreactive neurons were counted. CGRP, p = 0.015; PV, p = 0.776, t-test. Bar: A-F, 100 μm.

Figure 6.

Neuronal loss in TrkA^+/- or Bax^-/- background. (A,C,E,G,I,K,M,O) Nissl staining of P0 TG sections of indicated genotypes. (B,D,F,H,J,L,N,P) Anti-TrkA immunostaining of P0 TG sections of indicated genotypes. (Q) Quantification of total neurons and TrkA(+) neurons in P0 TG of indicated genotypes. N = 4 for each genotype. (^★) p < 0.05, t-test. Keys: (K) Klf7; (A) TrkA; (B) Bax. Bars: A,C,E,G,I,K,M,O, 200 μm; B,D,F,H,J,L,N,P, 50 μm.

Figure 7.

Reduced nociceptive innervation of spinal cord in Klf7^-/- P0 pups. DiI tracing of the spinal cord of the wild-type (A,B) and Klf7^-/- (C,D) P0 pups. Sections shown are at the same level from the middle of the spinal cord segment. N = 8 for each genotype. Laminae I-V are labeled numerically, and Ia denotes the Ia afferent from TrkC(+) neurons.

Figure 8.

Reduced sensitivity to noxious stimuli in adult Klf7^-/- mice. (A-D) The nociceptive innervation of the dorsal spinal cord is greatly reduced in adult Klf7^-/- mice. A and B show anti-CGRP immunostaining of laminae I and IIo, while C and D show IB4 staining of lamina IIi. Both types of innervation are significantly reduced. (E) Test of thermal sensitivity. The response latency of Klf7^-/- mice is significantly longer than that of wild-type littermates; (^★★★) p < 0.001, t-test. (F) Test of chemical nociception. Injection of a 5% formalin solution into the hind paw evokes two phases of paw licking: Phase I reflects direct activation of primary nociceptors; phase II reflects peripheral inflammation and sensitization of dorsal horn neurons. Both phases of licking are significantly reduced in Klf7^-/- mice; (^★★) p < 0.01; (^★★★) p < 0.001, t-test. (G) Test of sensitivity to innocuous mechanical stimulation using calibrated von Frey filaments. The threshold weight (in grams) necessary to produce a response is similar for Klf7^-/- and wild-type mice (p = 0.979, t-test). (H) Proprioception as determined by a grid-walking test is not significantly different between wild-type and Klf7^-/- mice (p = 0.081, t-test). N = 6 for each genotype in all tests.