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. 2002 Jul 1;21(13):3454-63.
doi: 10.1093/emboj/cdf370.

The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons

Ditsa Levanon  1 , David Bettoun, Catherine Harris-Cerruti, Eilon Woolf, Varda Negreanu, Raya Eilam, Yael Bernstein, Dalia Goldenberg, Cuiying Xiao, Manfred Fliegauf, Eitan Kremer, Florian Otto, Ori Brenner, Aharon Lev-Tov, Yoram Groner
Affiliations

The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons

Ditsa Levanon et al. EMBO J. .

Abstract

The RUNX transcription factors are important regulators of linage-specific gene expression in major developmental pathways. Recently, we demonstrated that Runx3 is highly expressed in developing cranial and dorsal root ganglia (DRGs). Here we report that within the DRGs, Runx3 is specifically expressed in a subset of neurons, the tyrosine kinase receptor C (TrkC) proprioceptive neurons. We show that Runx3-deficient mice develop severe limb ataxia due to disruption of monosynaptic connectivity between intra spinal afferents and motoneurons. We demonstrate that the underlying cause of the defect is a loss of DRG proprioceptive neurons, reflected by a decreased number of TrkC-, parvalbumin- and beta-galactosidase-positive cells. Thus, Runx3 is a neurogenic TrkC neuron-specific transcription factor. In its absence, TrkC neurons in the DRG do not survive long enough to extend their axons toward _target cells, resulting in lack of connectivity and ataxia. The data provide new genetic insights into the neurogenesis of DRGs and may help elucidate the molecular mechanisms underlying somatosensory-related ataxia in humans.

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Figures

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Fig. 1. Generation and phenotype of Runx3 mutant mice. (A) A schematic diagram of the Runx3 genomic locus and Runx3-mutant allele. The SacI site used for insertion of the cassette is marked in blue. (B) Southern blot analysis of EcoRI–HindIII digest of genomic DNA isolated from tail biopsies. The 3′ external probe used here is marked in red. Wild type (+/+) shows the expected 10.5 kb band. Heterozygotes (+/–) and homozygotes (–/–) show a 4.5 kb band, which results from a new EcoRI site in the _targeting vector. PCR analysis, using primers from Neo and exon 2, was employed for confirmation and rapid genotyping (not shown). (C) Western blot analysis (top) and RT–PCR (bottom) of proteins and RNA extracted from thymus and DRG. RT–PCR: I, Runx3; II, actin. Primers used for Runx3 are 5′-GCGGAGTAGTTCTCATCATTG-3′ (exon 1) and 5′-ATGCGTATT CCCGTAGACCCG-3′ (exon 2). (D) Immunostaining for Runx3 in WT DRG (upper panel) and KO (lower panel) mice. (E) Runx3 KO mice were smaller (lower pup) and had a clearly recognizable phenotype, characterized by severe ataxia, extensor rigidity and posture abnormalities. The photograph depicts two 3-week-old littermates (upper) and one 2-month-old KO mouse.
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Fig. 2. Expression of Runx3 in DRG proprioceptive neurons at E10.5 (A) and at E16.5 (B). (C) Co-expression of Runx3 (red) and TrkC (green) at E12.5. (D) Co-expression of PV (red) and Runx3 (green) at P0. (E) Expression of Runx3 (green) and TrkB (red) in different neuronal populations (E16.5). (F) Expression of Runx3 (green) and Runx1 (red) in different neuronal populations (E15.5). (G and H) Typical example of Runx1 (G) and trkA (H) expression in a similar neuronal population (sections of E14.5 ganglia).
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Fig. 3. Electrophysiological measurements. (A) Computer-averaged records (mean of 20 sweeps each) of ventral root potentials produced in the L5 segment of control and KO spinal cord by 0.1 Hz repetitive stimulation of L5 dorsal root afferents at an intensity of twice the synaptic threshold (2T). The arrow denotes a small short-latency VRP component of the Runx3 KO VRP. (B) Computer-averaged records (mean of 20 sweeps each) of L5 VRPs and PSPs produced in L5 motoneurons (IC) and ventral roots (VRP) of control (left) and KO (right) mice, by 0.1 Hz stimulation of L5 dorsal root afferents at 1.1 or 2T (lower and upper trace in each pair, respectively). The arrow denotes the low-amplitude short-latency PSP component produced in the KO motoneurons. Calibration: 5 mV, 2 ms. (C) PSPs (eight superimposed sweeps) produced in L5 motoneurons of a KO mouse by 0.1 Hz stimulation of the L5 dorsal root afferents at 2T. Calibration: 5 mV, 2 ms. Note the scattered motoneuron firing. (D) Measurements of the latency (Lat.), first detectable peak (1st peak, arrow) and time to the first peak (T-1st peak) of a 20-sweep computer-averaged control PSP. Dashed lines mark the stimulus artifact, the beginning of the PSP and the occurrence of its peak. (E) Interrelationship between the latency, peak and time to peak of the first detectable PSP component in control and KO motoneurons. Afferent stimulation intensity (1.3–1.5T) was adjusted to obtain the maximal possible subthreshold PSP. Measurements were taken from computer-averaged records (20–40 sweeps each). One-way ANOVA followed by the modified Tukey’s method for multiple comparisons α = 0.05) was used to compare the means of each of the measured parameters of control EPSPs, Runx3 KO EPSPs and the longer latency Runx3 KO PSPs. These analyses revealed that the amplitude of Runx3 KO EPSPs was much smaller than that of control EPSPs and Runx3 KO PSPs (p < 0.001); that the time to peak of Runx3 KO PSPs was significantly longer than control and Runx3 KO EPSPs (p < 0.05); and that the latency of Runx3 KO PSPs was significantly longer compared with control and Runx3 KO EPSPs (p < 0.001).
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Fig. 4. Analysis of spinal cord proprioceptive afferent projections. (A and D) Schematic diagram of the spinal cord at L4 (A) and C6 (D). I–IX, cytoarchitectonic laminae. (B and C) Anterograde DiI tracing in control (B) and KO (C) mice. Transverse sections of P0 spinal cord at the L4 DRG level. (E and F) Immunostaining of PV in spinal cords of control (E) and KO (F) mice. Transverse sections at C6 DRG level at P0. (G–J) Dorsal column at high cervical level of P30 WT (G) and Runx3 KO (H). Transverse 1 µm epon sections, stained with Toluidine Blue. gr, gracile fascicle; cu, cuneate fascicle; py, pyramidal tract. (I and J) Higher magnification of the squares in (G) and (H), showing a reduced number of large-diameter afferents in the dorsal column of KO mice (J) as compared with WT (I) (the dotted line indicates the midline).
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Fig. 5. (A) Size distribution of dorsal root fibers. Cross-sectional diameter of fibers at P0 and P30–P53. (I) Distribution of large fibers in L5 dorsal root at P0. (II) Distribution of total myelinated fibers in C7–C8 dorsal roots at P30–P53. Analysis disclosed differences between control and KO mice (Kolmogorov–Smirnov p < 0.002 at both ages). (B) Muscle spindles in limb and head muscles. Transverse thin sections of P30 soleus muscle (I and II) and P42 superficial masseter muscle (III and IV) from WT (I and III) and KO mice (II and IV) embedded in epon and stained with Toluidine Blue. Arrows indicate muscle spindles.
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Fig. 6. Developmental pattern of proprioceptive markers in WT and Runx3 KO mice. (A) Expression of TrkC in DRG (I–VI) and trigeminal ganglion (VII–X) of WT and KO mice, between E10.5 and E13.5 (E10.5, thoracic DRG; E11.5 and E13.5, cervical DRG). (B) Expression of PV at P0 in L5 DRG of WT (I) and KO (II) mice. Exposure of KO mice was increased 2.5 times. (C) Expression of Er81 in DRG of WT (I and III) and KO (II and IV) at E13.5 and P10 (E13.5, cervical; P10-L5). (D) Expression of TrkA and TrkB in WT (I and III) and KO (II and IV) at E15.5, cervical ganglion.
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Fig. 7. Quantification of DRG neurons and analysis of β-gal activity in heterozygous and homozygous Runx3-mutant mice. (A) Neuronal count (I) and volume evaluation (II) of L5 DRG of P0 control and KO mice. Total number of neurons, number of large cells (mean diameter ≥ 20 µm) and DRG volume (µm3) were measured. Results are the mean of eight control (four heterozygous and four WT) and seven KO mice ± SEM. (B) Lateral view of dissected brain and spinal cord with attached cranial and DRG. Whole-mount E13.5 heterozygous (I) and homozygous (II) embryos stained for β-gal activity as described previously (Levanon et al., 2001a). (III and IV) E18.5 cervical ganglia of heterozygote (III) and KO embryo (IV). (V and VI) Ventral view showing the trigeminal ganglion bilaterally in heterozygote (V) and KO embryo (VI).
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