Earlier in situ hybridization studies indicated strong expression of Runx1 mRNA in spinal motor neurons, DRG, cranial ganglia and specialized sensory epithelial structures such as olfactory and gustatory mucosa, and follicles of the vibrissae 20. Subsequently, the generation of specific antibodies against Runx1 and Runx3 and the utilization of Runx1^β-gal or Runx3^β-gal mice revealed the expression of Runx1 and Runx3 in the nervous system in more detail 162122. Runx1 is synthesized in both the central and peripheral nervous systems of mouse embryos. In the central nervous system, Runx1 is synthesized in selective populations of somatic motor neurons in the spinal cord and in cholinergic branchial and visceral motor neurons in the hindbrain, such as dorsal vagal nucleus and nucleus ambiguus 2122. In the peripheral nervous system, Runx1 is localized to DRG and selective cranial ganglia, including trigeminal (V) and vestibulocochlear (VIII) ganglia and the glossopharyngeal-vagal (IX-X) ganglia complex 2122. In contrast to Runx1, Runx3 is confined to the peripheral nervous system, specifically to DRG and cranial ganglia 1621. Although Runx1 and Runx3 are almost exclusively found in postmitotic neurons in the central nervous system and peripheral ganglia 162122, a rare exception is the expression of Runx1 in proliferating progenitors of the olfactory epithelium 23. These observations suggest Runx1 and Runx3 have extensive functions in the mammalian nervous system.

DRG neurons convey peripheral somatosensory stimuli to the spinal cord. There are three major subpopulations of DRG neurons – nociceptive, mechanoreceptive, and proprioceptive – which differ in their cell size, dependency on neurotrophins, and distinct axonal terminal fields in the spinal cord and peripheral tissues. Runx1 and Runx3 are synthesized initially in TrkA⁺ nociceptive and TrkC⁺ proprioceptive neurons, respectively (Figure 1) 172425. This complementary expression pattern suggests specific roles for Runx1 and Runx3 in subtypes of DRG neurons. Indeed, the phenotype of Runx3 knockout mice is similar to that of NT3 and TrkC knockout mice 161726272829. Namely, Ia/Ib type DRG neurons fail to form a stretch reflex circuit with motor neurons in the spinal cord, resulting in severe motor discoordination 1617. What is the molecular basis of the phenotype? Several elegant studies have been performed to answer this question.

First, the role of Runx3 in the neurotrophin receptor phenotype was shown by Arber and her colleagues 25, who thoroughly compared neurotrophin receptor synthesis in mouse strains in which Runx3 had been disrupted or expressed ectopically. In DRG neurogenesis, dynamic changes are observed during the synthesis of neurotrophin receptors (TrkB, TrkC) 25. At early developmental stages, most DRG neurons synthesize TrkC protein first before the onset of TrkB synthesis. Thus, some TrkC⁺ DRG neurons co-synthesize TrkB (Figure 1a). Subsequently, the ratio of TrkB/TrkC-hybrid neurons declines to produce DRG neurons that synthesize either TrkC or TrkB (Figure 1a). During this segregation, Runx3 is observed in most TrkC⁺ neurons but not in TrkB⁺ neurons 25. One of the functions of Runx3 is to repress TrkB when DRG neurons acquire TrkC⁺ identity (Figure 1a) 25.

Second, the axonal outgrowth and/or axonal guidance of propiroceptive DRG neurons are also regulated by Runx3. Two different interpretations were proposed for the phenotype of the Runx3-/- DRG. One group proposed that Runx3 controls the appropriate axon targeting of trkC-expressing proprioceptive DRG neurons to motor neurons 16. However, another group observed massive cell death of TrkC⁺ neurons in Runx3-/- DRG in apparent contradiction to the previous proposition 17. A recent study with Runx3 and Bax-double knockout mouse revealed clearly that the axonal projection of propioceptive DRG neurons to motor neurons is still lost in the Runx3 mutant even in the absence of apoptosis 30. The study further clarified that the initial model 'Runx3 → TrkC and Runx1 → TrkA' might not apply to later developmental stages 30. They observed that Runx3 co-localizes not only with TrkC, but also TrkA and TrkB at postnatal day 0 (P0) 30. Of note, Runx1⁺ and Runx3⁺ neurons were clearly segregated at embryonic day 16.5 (E16.5) but almost all Runx3⁺ neurons co-synthesize Runx1 at E18.5 and P0 30. It is possible that Runx3 has some functions not only in proprioceptive neurons, but also in nociceptive neurons 30. Overall, the evidence obtained from Runx3 and Bax compound mutants support a role for Runx3 in the control of axonal projection, although the molecular mechanisms remain unknown 30. Prior studies showed that DRG explants from Runx3-knockout mouse embryos extended short neurites in the presence of NT3, a ligand for TrkC, but not in the presence of NGF, a ligand for TrkA 16. This suggests that Runx3 may regulate the axonal outgrowth of specific DRG neurons independently of the target tissue. On the other hand, Chen et al. 24 revealed, using a tour de force method, that Runx3 activity determines the dorso-ventral position of axonal termination of DRG neurons in the spinal cord. DRG neurons with high Runx3 activity extended their axons far into the ventral spinal cord like proprioceptive neurons, whereas those neurons with low Runx3 activity extended their axons into the dorsal spinal cord. Ectopic expression of Runx3 is sufficient to drive axons from the dorsal to the ventral spinal cord, indicating that Runx3 per se has instructive roles in central axon targeting in DRG neurons.

Thus, Runx3 controls the neurotrophin receptor phenotype as well as the axonal projection of proprioceptive DRG neurons. The two functions may not be mutually exclusive but closely related to each other. For example, NGF/TrkA signalling and NT3/TrkC signalling are required for proper axonal projection 3132.

In contrast to Runx3, the study of Runx1 function in DRG development was delayed owing to the early embryonic lethality of the targeting mouse 222333. Thus, Runx1 knockout mice die due to a lack of definitive hematopoiesis by E12.5, which is before the onset of major events in the development of TrkA⁺ DRG neurons. However, recent studies have investigated the roles of Runx1 in DRG neurons using different experimental models.

First of all, Runx1 controls the lineage diversification of nociceptive neurons 253334. During late embryonic and early postnatal periods, trkA-expressing neurons differentiate into two subpopulations of nociceptive neurons; trkA-retaining peptidergic neurons, and non-peptidergic neurons that repress trkA and instead activate Ret, a receptor for glial-derived neurotrophic factor (GDNF; Figure 1b). During the late embryonic stages, most trkA-expressing DRG neurons coexpress Runx1 (Figure 1b). Postnatally, Runx1 disappears in trkA-retaining peptidergic neurons but continues to exist in Ret-inducing non-peptidergic neurons (Figure 1b). Using the Runx1-conditional knockout mouse, it was shown that Runx1 is dispensable for the de novo induction of TrkA 34. This was confirmed by Shiga and his colleagues 33, who used a different gene-targeting method that relied on the rescuing of Runx1 expression in hematopoietic cells. However, Marmigere et al. 35 showed that virally expressed Runx1 induced de novo synthesis of TrkA in the DRG and spinal cord of chick embryos. One possible explanation is that the minimal enhancer of trkA, which Runx1 regulates 35, may not be required for the de novo induction of trkA expression 36. On the other hand, Runx1 is essential for the late repression of trkA and induction of Ret when TrkA⁺ and Ret⁺ neurons segregate (Figure 1b) 34. In addition to trkA, Runx1 also represses the neuropeptide, calcitonin-gene-related peptide (CGRP; Figure 1b) 253334. More surprisingly, nearly all the known marker genes for nociception are under the control of Runx1. In the conditional Runx1 mutant DRG, expression of a number of nociceptor-specific G protein coupled receptors, ATP channels, and TRPV channels is attenuated (Figure 1b) 34.

Similar to Runx3, Runx1 also regulates the axonal outgrowth and guidance of nociceptive neurons. Marmigere et al. 35 revealed that the transfection of Runx1 into boundary cap-derived neural crest stem cells increased neurite length and branching. In Runx1-knockout mice, the axonal projection to laminae IIi of the dorsal spinal cord was perturbed 3334. In the wild type, peptidergic nociceptive axons project to layer I/IIo in the superficial dorsal horn, whereas non-peptidergic nociceptive axons project to deeper layer IIi. In Runx1-knockout mouse, non-peptidergic axonal projection displays dorsal shift to layer I/IIo 34.

Thus, Runx1 controls a battery of genes that are associated with the generation of non-peptidergic nociceptive neurons. The findings that both Runx3 and Runx1 play critical roles in distinct sensory neurons suggest that Runx factors are involved in the evolution of sophisticated sensory systems in higher vertebrates.

The upstream signals and transcriptional regulation of RUNX genes have been studied in non-neuronal tissues 37. However, only limited studies have addressed this issue in the nervous system. Both Runx3 and Runx1 genes contain Brn-3a binding sites in their 5'-upstream regions, suggesting that Runx3 and Runx1 are candidate downstream targets of Brn-3a, a well characterized transcription factor in sensory neurons 3839. Microarray studies have shown decreased levels of Runx1 and Runx3 transcripts in the sensory neurons of Brn-3a-knokout mice 4041. Kramer et al. 25 investigated the putative upstream signal of Runx1/Runx3 in DRG neurons. Plausible candidates are TrkC/TrkA signalling and the basic helix-loop-helix transcription factors Ngn2/Ngn1; however, a genetic study has excluded these possibilities 25. Ginty and colleagues 42 investigated the roles of NGF and the Ret receptor in DRG neurons. In Ngf-Bax compound knockout DRG, TrkA neurons are hypotrophic although de novo Runx1 expression is unaffected 42. However, Runx1 expression is not maintained to the neonate stage and the expression of all putative Runx1 target genes is altered 42. Thus, NGF signalling is essential for sustained expression of Runx1. In Ret conditional knockout DRG, Runx1 expression is normal but a part of Runx1 target genes are affected, suggesting the GFR/Ret dependent transcriptional regulation by Runx1 in DRG neurons 42. Although this study placed Runx in a pivotal position in developmental signalling cascades, the upstream signalling event(s) still remains elusive.

On the other hand, how does Runx1/Runx3 regulate downstream transcriptional cascades? In DRG neurons, TrkC is a critical signalling receptor involved not only in the control of cell survival, but also in axon path-finding and fate determination of proprioceptive DRG neurons 324344. Therefore, it is natural to infer that trkC is a transcriptional target of Runx3 17. However, unbiased computational analysis suggested that a cis-regulatory element exists in the gene locus of TrkB, rather than in the gene locus of TrkC 45. This was unexpected because trkB is expressed in neurons of an alternative sensory fate, TrkB⁺TrkC^- neurons 43. The strategy "to repress alternative traits" appears to be a common feature in neuronal lineage commitment 46. At the molecular level, trkB possesses a conserved cluster of Runx binding sites that function as a silencer of the trkB promoter in cultured DRG neurons 45. In Runx3 knockout DRG, derepression of trkB seems to be a crucial event, influencing lineage commitment 2545, and, eventually, resulting in drastic behavioural consequences 1617.

Runx protein works both as an activator and repressor, depending on the molecular context 47. The finding that Runx3 represses trkB raises a question as to the identities of its partner molecules in the transcriptional repressor complex. The function of Runx1 as a transcriptional repressor has been widely studied 4849. A plausible candidate in the context of DRG is the Groucho corepressor. In motoneuron fate specification, Groucho-mediated repression is a common mechanism for homeodomain proteins containing the EH1 domain 46. Runx proteins have the evolutionarily conserved VWRPY carboxy-terminal motif, which is considered to be critical for Groucho binding/function 5051. Yarmus et al. 52 generated mice in which Runx3 lacks these amino acids. Surprisingly, VWRPY knockout mice displayed the normal development in DRG neurons, though they showed the phenocopy to Runx3 knockout mice in dendritic cells 52. The results suggest that Runx3 represses trkB through a Groucho independent mechanism. Recently, Ma and his colleagues 53 investigated the significance of the VWRPY motif of Runx1 in DRG neurons. Runx1 cDNA, which lacks the VWRPY coding sequence, was knocked into the native Runx1 locus in delta446 mice 54. In the delta446 mice, derepression of Mrg class G-protein-coupled receptor genes was observed, suggesting that Mrg genes are repressed by a Groucho-dependent mechanism (Figure 1c) 53. Interestingly, two putative target genes that are repressed by Runx1, trkA and CGRP, were unaffected in the delta446 mice 53. These results suggest that Runx1 represses target genes through either a Groucho-dependent or an independent mechanism in DRG neurons.

Chen et al. 34 indicated that Runx1 controls nearly all known marker genes critical for nociceptive functions. Such global control by Runx over the transcription landscape is also observed in other physiological functions, such as hematopoietic stem cell formation (Runx1) and osteoblast maturation (Runx2). How this unique transcription factor has such a huge influence on many different transcriptional cascades remains a challenging question.

Stifani and his colleagues 2223 have worked on the neurological phenotypes of the Runx1 knockout mouse other than those arising from defects in DRG neurons. They analysed the cranial sensory neurons as well as cholinergic branchial and visceral motor neurons of hindbrain at an early embryonic stage 22. The expression of Runx1 was restricted to post-mitotic neurons, and disruption of Runx1 resulted in massive neuronal apoptosis 22. In contrast to this finding, Runx1 is expressed in the proliferating neuronal progenitors/precursors of olfactory receptor neurons (ORNs) 23. Runx1 drives the cell cycle in ORN progenitors through transcription repression of the cyclin dependent kinase inhibitor p21 23. Unlike DRG, they did not observe any changes in the lineage markers in the neurons examined (cranial, hindbrain and olfactory), indicating that Runx1 has distinct functions in different types of neurons 2223.

The study of the neurological function of Runx3 other than in DRG is very limited. Levanon et al. 17 reported that TrkC⁺ neurons in the trigeminal ganglion survive in contrast to DRG neurons in Runx3-/- mouse. Most Runx3 knockout mice of the C57/B6 strain die within one day after birth 916. The main cause of death may be starvation, as little milk is found in the stomachs of these mice 9. As this is probably related to the pups being unable to swallow milk, it is interesting to note that Runx3 is strongly expressed in cranial ganglia, including the glossopharyngeal ganglion 1617. It is possible that Runx3 is essential for the functional glossopharyngeal system (swallowing), suggesting the critical roles in developmental cranial neurons.