A striking feature of vestibular hair cells is the polarized arrangement of their stereocilia as the basis for their directional sensitivity. In mammals, each of the vestibular end organs is characterized by a distinct distribution of these polarized cells. We utilized the technique of post-fixation transganglionic neuronal tracing with fluorescent lipid soluble dyes in embryonic and postnatal mice to investigate whether these polarity characteristics correlate with the pattern of connections between the endorgans and their central targets; the vestibular nuclei and cerebellum. We found that the cerebellar and brainstem projections develop independently from each other and have a non-overlapping distribution of neurons and afferents from E11.5 on. In addition, we show that the vestibular fibers projecting to the cerebellum originate preferentially from the lateral half of the utricular macula and the medial half of the saccular macula. In contrast, the brainstem vestibular afferents originate primarily from the medial half of the utricular macula and the lateral half of the saccular macula. This indicates that the line of hair cell polarity reversal within the striola region segregates almost mutually exclusive central projections. A possible interpretation of this feature is that this macular organization provides an inhibitory side-loop through the cerebellum to produce synergistic tuning effects in the vestibular nuclei. The canal cristae project to the brainstem vestibular nuclei and cerebellum, but the projection to the vestibulocerebellum originates preferentially from the superior half of each of the cristae. The reason for this pattern is not clear, but it may compensate for unequal activation of crista hair cells or may be an evolutionary atavism reflecting a different polarity organization in ancestral vertebrate ears.

Numerous experiments in mutant and transgenic mice have implicated Hox transcription factors in development of the skeletal system, postulating a role for these proteins in cell proliferation of precursor cells and regulation of cell differentiation. Our own data from Hoxc8 and Hoxd4 transgenic mice suggest that Hoxc8 is involved in cell proliferation during cartilage development. In order to directly assess its role in cell proliferation of a specific skeletal cell type, the cartilage-producing chondrocyte, we performed morpholino-mediated knockdown experiments in normal primary chondrocytes. Through analysis of PCNA expression and staining for phosphorylated Histone 3, two cell cycle markers, we show that interference with Hoxc8 expression in chondrocytes reduces cell proliferation, but in the absence of apoptosis. Instead, cells with a knockdown in Hoxc8 expression appear to be delayed in their progression through the cell cycle. Our results provide evidence for prolonged duration of and delayed exit from M-phase, thus implicating a role for Hoxc8 in controlling cell cycle progression at this critical check point.

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