Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC

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Functional Genomics of Runx3 and DRG Neurogenesis

Yoram Groner, Yael Bernstein, David Bettoun, Catherine Harris-Cerruti, Ofer Fainaru, Dalia Goldenberg, Ditsa Levanon, Joseph Lotem, Varda Negreanu, Amir Pozner, Eilon Wolf, Cuiying Xiao, Merav Yarmus
Department of Molecular Genetics the Weizmann Institute Rehovot ISRAEL
Telephone: 972-8-9343-972
Fax: 972-8-9344-108

The mammalian runt-related transcription factors (Runx) belong to a small gene family of three genes (Runx1, Runx2 and Runx3). They all contain a highly conserved DNA binding domain designated "runt domain," which is also found in the Drosophila gene Runt. Runx1 and Runx2 are associated with human diseases and act as master regulators of gene expression in hematopoiesis and osteogenesis. We have cloned, sequenced, and elucidated the genomic structure of the human RUNX1 and RUNX3 genes. RUNX1 resides on human chromosome 21 and RUNX3 on chromosome 1. Chromosomal translocations involving RUNX1 are prevalent in human leukemias. Disruption of RUNX1 obliterates definitive hematopoiesis and impairs formation of vascular capillaries. The mammalian RUNX genes arose early in evolution and maintained extensive structural similarities between them. Sequence analysis suggested that RUNX3 is the most ancient of the three mammalian genes, consistent with its role in neurogenesis of the monosynaptic reflex arc, the simplest neuronal response circuit, found in the most primitive animals, the Cnidarians.

The three RUNX proteins bind to the same DNA motif; thus, their pleiotropic functions are likely to result from a regulated spatial/temporal expression pattern. Interestingly, Runx1 and Runx3 genes contain RUNX binding sites in their promoter region, raising the possibility of cross regulation (both positive and negative) between them. We used immunohistochemistry and -galactosidase (LacZ) activity of targeted Runx3 and Runx1 loci to determine the expression pattern of Runx1 and Runx3 during mouse embryogenesis. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, Runx3 expression was confined to different compartments. The data provided new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross regulation between them plays a role in embryogenesis.

Of the three RUNX genes, RUNX3 is the least studied. In adults, Runx3 is highly expressed in the hematopoietic system, but its biological function is largely unknown. We generated knockout (KO) mice with disrupted Runx3 alleles by inserting a LacZ-neo cassette into exon 2. Heterozygous Runx3-mutant mice appeared phenotypically normal, whereas homozygous mutant mice showed posture abnormalities and severe limb ataxia. To understand the biological significance of what seemed to be a neuronal defect, we first examined in great detail the expression pattern of Runx3 in the nervous system. We found that Runx3 is highly expressed during mouse development in a subset of sensory neurons within the dorsal root ganglia (DRG), which we subsequently identified as TrkC, group Ia proprioceptive neurons. These neurons form monosynaptic connections with both muscle spindles and motor neurons to generate the monosynaptic stretch reflex circuit. Interestingly expression of the other family member Runx1 was also detected in the DRGs, but was confined to the small diameter TrkA nociceptive neurons. To further evaluate the physiological defect of the Runx3 mutant, electrophysiological studies were performed. Measurements revealed complete disruption of monosynaptic connectivity between intraspinal afferents and MNs. Tracing experiments, using anterograde DiI labeling, demonstrated an absence of afferent projections in the spinal cord of the KO mice, and further analysis revealed marked reduction in the number of Ia neurons in the KO DRGs. Taken together, the data demonstrate that Runx3 is a neurogenic TrkC neurons 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 DRGs neurogenesis and may help elucidate the molecular mechanisms underlying somatosensory-related ataxia in humans.

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