Shigeru Ozaki

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3−/−) mice displayed severe motor discoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3−/− DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3−/− DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

Spontaneous motoneuronal activity mediated by glycine and GABA in the spinal cord of rat fetuses in vitro.

1. Spontaneous motoneuronal activity was monitored from the lumbar ventral roots in an isolated spinal cord preparation from rat fetuses at embryonic days (E) 13.5‐18.5. 2. Spontaneous bursts that were synchronized in both left and right ventral roots were observed periodically (mean interval, 1.5‐2.6 min) from E14.5 to 17.5. This activity was abolished in Ca(2+)‐free saline or by application of tetrodotoxin (1 microM), indicating that it was synaptically mediated. 3. The glutamate receptor blocker kynurenate (4 mM) failed to block spontaneous bursts at E14.5‐15.5, though it completely abolished them at E17.5. The glycine receptor antagonist strychnine (10 microM) completely blocked spontaneous bursts at E14.5‐15.5. Bicuculline, a GABAA receptor antagonist, reduced the amplitude of the spontaneous bursts. 4. At E15.5, a brief application of glycine (250 microM to 2 mM) evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. Such glycine‐induced responses were not observed under Ca(2+)‐free conditions, suggesting that they were synaptically evoked. These synaptic responses were not blocked by kynurenate (4 mM), but they were abolished by strychnine (10 microM). 5. It is concluded that glycine and GABA generate the earliest spontaneous motor activity of the fetus and function transiently as excitatory transmitters in the embryonic spinal cord.

Development of locomotor activity induced by NMDA receptor activation in the lumbar spinal cord of the rat fetus studied in vitro.

The development of neuronal circuits generating locomotor activity was characterized in an isolated lumbar spinal cord preparation from of fetal and neonatal rats. Locomotor activity induced by bath application of the NMDA receptor agonists, NMA and NMDA, was monitored from both sides of the corresponding lumbar ventral roots. Activation of NMDA receptors first evoked rhythmic motor activity at E15.5. NMA-induced rhythmic motor activity was not observed under synaptic blockade by TTX or cadmium ions, suggesting that this activity was evoked by synaptic drive from the interneuronal circuits in the spinal cord. At E15.5-E16.5, the rhythmic motor activity on both sides was synchronized. Phase relationship of the rhythmic motor activity between both sides was variable at E17.5-E19.5. The rhythmic motor activity was alternating on both sides at E20.5. Mid-sagittal splitting of the spinal cord did not affect the rhythm generation at all stages examined, suggesting the existence of independent rhythm-generating circuits on each side. The rhythmic motor activity in the presence of strychnine was synchronized on both sides at all stages examined. These results indicate that the changes in rhythm pattern are mediated by development of glycinergic inhibitory pathways, while the basic rhythm can be generated without the glycinergic inhibitory pathways.

Dynamic regulation of the expression of neurotrophin receptors by Runx3

Sensory neurons in the dorsal root ganglion (DRG) specifically project axons to central and peripheral targets according to their sensory modality. However, the molecular mechanisms that govern sensory neuron differentiation and the axonal projections remain unclear. The Runt-related transcription factors, Runx1 and Runx3, are expressed in DRG neuronal subpopulations, suggesting that they might regulate the cell specification and the trajectories of specific axons. Here, we show that parvalbumin-positive DRG neurons fail to differentiate from the onset in Runx3-/- mice. By contrast, TrkC-positive DRG neurons differentiate normally at embryonic day (E) 11.5, but disappear by E13.5 in Runx3-/- mice. Subsequently, TrkC-positive DRG neurons reappear but in smaller numbers than in the wild type. In Runx3-/- mice, central axons of the TrkC-positive DRG neurons project to the dorsal spinal cord but not to the ventral and intermediate spinal cord, whereas the peripheral axons project to skin but not to muscle. These results suggest that Runx3 controls the acquisition of distinct proprioceptive DRG neuron identities, and that TrkC-positive DRG neurons consist of two subpopulations: Runx3-dependent early-appearing proprioceptive neurons that project to the ventral and intermediate spinal cord and muscle; and Runx3-independent late-appearing cutaneous neurons that project to the dorsal spinal cord and skin. Moreover, we show that the number of TrkA-positive DRG neurons is reduced in Runx3-/- mice, as compared with the wild type. These results suggest that Runx3 positively regulates the expression of TrkC and TrkA in DRG neurons.

Runx3 is essential for the target-specific axon pathfinding of trkc-expressing dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons project their axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality (Neuron 30 (2001), 707; Cell 101 (2000), 485; Neuron 31 (2001), 59; J. Comp. Neurol. 380 (1997), 215; Sensory Neurons, Oxford Univ. Press, New York, 1992, p. 131). Expression of runt-related Runx/AML genes (Mech. Dev. 109 (2001), 413) on subtypes of DRG neurons suggests their involvement in lamina-specific afferent differentiation and maturation. Here we show that Runx3-/- mice display severe limb ataxia and abnormal posture and that most of them die shortly after birth. They show that proprioceptive afferent axons fail to reach the ventral horn and have a smaller dorsal funiculus in their spinal cords. Despite the strong resemblance of this phenotype to that of knockout mice deficient in neurotrophin-3 (NT-3) (Cell 77 (1994), 503; Nature 369 (1994), 658) and its receptor, trkC, (Nature 368 (1994), 249), which show proprioceptive afferent loss through selective neuronal cell death, Runx3-/- mice maintain normal number of TrkC/trkC positive DRG neurons throughout development. Our results suggest that Runx3 controls the target-specific axon pathfinding of trkC-expressing DRG neurons in the spinal cord.

Runx1 promotes neuronal differentiation in dorsal root ganglion

Transcription factor Runx1 controls the cell type specification of peptidergic and nonpeptidergic nociceptive dorsal root ganglion (DRG) neurons by repressing TrkA and calcitonin gene-related peptide (CGRP) expression and activating Ret expression during late embryonic and early postnatal periods (Chen et al., 2006b; Kramer et al., 2006; Yoshikawa et al., 2007). Because Runx1 is expressed in DRG from early developmental stages, we examined the roles of Runx1 in the proliferation and the neuronal differentiation of DRG cells. We used transgenic Runx1-deficient (Runx1(-/-)::Tg) mice which are rescued from early embryonic lethality by selective expression of Runx1 in hematopoietic cells under the control of GATA-1 promoter. We found that TrkA-expressing (TrkA(+)) DRG neurons were decreased at embryonic day (E) 12.5 in contrast to the previous study showing that TrkA(+) DRG neurons were increased at E17.5 in Runx1(-/-)::Tg mice (Yoshikawa et al., 2007). The number of DRG neurons which express neuronal markers Hu, NeuN and Islet1 was also reduced in Runx1(-/-)::Tg mice at E12.5, suggesting that the neuronal differentiation was suppressed in these mice. The cell cycle analysis using BrdU/IDU revealed that the number of DRG cells in S-phase and G2/M-phase was increased in Runx1(-/-)::Tg mice at E12.5, while the length of S-phase was not changed between Runx1(+/+)::Tg and Runx1(-/-)::Tg mice, suggesting that Runx1 negatively controls the proliferation of DRG progenitor cell subpopulation in early embryonic period. Hes1 is a negative regulator of neuronal differentiation (Ishibashi et al., 1995; Tomita et al., 1996), and we found that the number of Hes1(+) DRG cells was increased in Runx1(-/-)::Tg mice at E12.5. In summary, the present study suggests a novel function that Runx1 activates the neuronal differentiation of DRG cell subpopulation through the repression of Hes1 expression in early embryonic period.

The effects of sciatic nerve axotomy on spinal motoneurons in neonatal Bax-deficient mice

During development, the survival of spinal motoneurons depends on the integrity of the connection to their peripheral targets. Peripheral nerve axotomy induces apoptosis in neonatal neurons supplying axons to the nerve. Bax is known to promote apoptosis among developing neurons. To examine the effect of axotomy on spinal motoneurons in Bax-deficient (Bax-/-) and wild-type neonatal mice (Bax+/+), the sciatic nerve was axotomized on postnatal day (P) 0, and motoneurons in the fourth lumbar (L4) segment were visualized at P7 by acetylcholinesterase (AChE) histochemical staining. Presumably due to the reduction in naturally occurring cell death resulting from the deficiency of Bax, there were about 50% more AChE-positive cells in Bax-/- than in Bax+/+. Motoneurons in the dorsolateral motor pool of L4 project through the sciatic nerve. In Bax+/+, axotomy of the sciatic nerve induced significant cell loss in the pool. Most motoneurons survived such axotomy in Bax-/-, although they appeared atrophic and their AChE expression was decreased. Motoneurons may receive vital support retrogradely from their targets, and loss of such support may lead to hypofunction of spinal motoneurons, as indicated by the reduced production of AChE by axotomized motoneurons and their small size in Bax-/-.

Species differences in the distribution and coexistence ratio of serotonin and substance P in the monkey, cat, rat and chick spinal cord

Serotoninergic raphe-spinal motor neuron projections exhibit wide species differences in both innervation pattern and coexistence of serotonin and substance P. The coexistence ratios vary widely ranging from more than 80% (rat) to less than 1% (chick). Serotonin and substance P positive fibers are also unevenly distributed in the ventral horn of different species: dense clusters of serotonin and substance P positive fibers were preferentially located in the motor neuron pools of extensor muscles of the hip joint (chick) as well as antigravity muscles of the forelimb (cat and rat).

Runx3 is required for the specification of TrkC-expressing mechanoreceptive trigeminal ganglion neurons

Sensory neurons project axons to specific peripheral and central targets according to their sensory modality. Runx3 is crucially involved in proprioceptive dorsal root ganglion neuron development. Runx3 is also expressed in trigeminal ganglion (TG) neurons. The role of Runx3 in the TG, however, is largely unknown because the TG does not contain proprioceptive neurons. In Runx3-deficient (Runx3(-/-)) mice, TrkB-expressing TG neurons were increased, whereas TrkC-expressing TG neurons were decreased during TG neuron development. In Runx3(-/-) neonatal mice, TrkC-expressing TG neurons did not project to the Merkel cells in the outer root sheath (ORS) of whisker vibrissae peripherally and the spinal trigeminal nucleus pars interpolaris (Sp5I) centrally. These findings suggest that Runx3 is required for the specification of TrkC-expressing TG neurons, conveying mechanoreceptive signals from the Merkel cells in the ORS of the whisker vibrissae to the Sp5I.

Coexpression of Runx1 and Runx3 in mechanoreceptive dorsal root ganglion neurons

Runt‐related transcription factors (Runx) regulate the development of various cells. It has been reported that Runx1 and Runx3 are expressed in distinct subpopulations of primary sensory neurons in the dorsal root ganglion (DRG), and play important roles in the differentiation of nociceptive and proprioceptive neurons, respectively. In the present study, we examined the developmental changes of the expression of Runx1 and Runx3 in the mouse DRG during embryonic and postnatal stages. We found that the expression of Runx3 preceded that of Runx1, but dramatically decreased before birth, whereas the Runx1 expression was maintained during postnatal periods. These results suggest that roles of Runx1 and Runx3 may change dynamically in the differentiation and maturation of DRG neurons. In addition, several DRG neurons expressed both Runx1 and Runx3 throughout embryonic and postnatal stages and many Runx3‐expressing DRG neurons coexpressed Runx1 at postnatal day 28. Double and triple labeling studies suggest that some of the Runx1/Runx3‐double expressing neurons coexpressed TrkB, c‐ret, and TrkC, which have been shown in the mechanoreceptive DRG neurons. These results suggest that Runx1/Runx3‐double expressing neurons may represent mechanoreceptive properties in the DRG.© 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 469–479, 2013