SUMMARYDevelopment of a functional neuronal network during embryogenesis begins with pioneer axons creating a scaffold along which later-outgrowing axons extend. The molecular mechanism used by these follower axons to navigate along pre-existing axons remains poorly understood. We isolated loss-of-function alleles of fmi-1, which caused strong axon navigation defects of pioneer and follower axons in the ventral nerve cord (VNC) of C. elegans. Notably follower axons, which exclusively depend on pioneer axons for correct navigation, frequently separated from the pioneer. fmi-1 is the sole C. elegans ortholog of Drosophila flamingo and vertebrate Celsr genes, and this phenotype defines a new role for this important molecule in follower axon navigation. FMI-1 has a unique and strikingly conserved structure with cadherin and C-terminal G-protein coupled receptor domains and could mediate cell-cell adhesion and signaling functions. We found that follower axon navigation depended on the extracellular but not on the intracellular domain, suggesting that FMI-1 mediates primarily adhesion between pioneer and follower axons. By contrast, pioneer axon navigation required the intracellular domain, suggesting that FMI-1 acts as receptor transducing a signal in this case. Our findings indicate that FMI-1 is a cell-type dependent axon guidance factor with different domain requirements for its different functions in pioneers and followers.

Cytokeratins in taste buds were immunocytochemically evaluated with monoclonal antibodies. In each of six different epithelial sites in the rat oral cavity, intragemmal cells of taste buds were immunoreactive for keratin polypeptides 8, 18, and 19, as well as for keratin 7, which has not been previously reported in taste buds. Keratin-18-like immunoreactivity was present in fewer than half of the intragemmal cells, whereas all intragemmal cells were immunopositive for keratins 7, 8, and 19. Apart from some salivary duct cells, no other cells in the tongue were immunoreactive for any of these four keratins. Morphological and immunocytochemical profiles indicate that taste buds are islets of simple epithelium embedded in an expanse of stratified squamous epithelium. These simple epithelial cells and their keratins are nerve-dependent, since denervation eliminated all four keratins and replaced elongated taste cells of the vallate papilla with stratified squamous epithelium. We conclude that antibodies against keratins 7, 8, or 19 are useful markers for intragemmal cells in studies of taste bud development, degeneration, regeneration, turnover and tissue culture.

In a genetic screen for regulators of synaptic morphology, we identified the single C. elegans flamingo-like cadherin fmi-1. fmi-1 mutants exhibit defective axon pathfinding, reduced synapse number, aberrant synapse size and morphology, as well as an abnormal accumulation of synaptic vesicles at non-synaptic regions. Although FMI-1 is primarily expressed in the nervous system, it is not expressed in the Ventral D-type (VD) GABAergic motorneurons, which are defective in fmi-1 mutants. The axon and synaptic defects of VD neurons could be rescued when fmi-1 was expressed exclusively in non-VD, neighboring neurons, suggesting a cell non-autonomous action of FMI-1. FMI-1 protein that lacked its intracellular domain still retained its ability to rescue the vesicle accumulation defects of GABAergic motorneurons, indicating that the extracellular domain (ECD) was sufficient for this function of FMI-1 in GABAergic NMJ development. Mutations in cdh-4, a Fat-like cadherin, cause similar defects in GABAergic motorneurons. cdh-4 is expressed by the VD neurons, and appears to function in the same genetic pathway as fmi-1 to regulate GABAergic neuron development. Thus, fmi-1 and cdh-4 cadherins might act together to regulate synapse development and axon pathfinding.

All or nearly all intragemmal (elongated) cells of rat taste buds were immunopositive for keratins 7, 8, and 19. In contrast, keratin 18 was detected in 19 +/- 5 cells per taste bud (mean +/- sd), or about 25% of the intragemmal cells. During taste bud development keratins 7, 8, and 19 were evident initially in polygonal cells and later in elongated taste cells. Keratin 8 appeared in vallate taste cells at P0 (postnatal day 0), followed by keratins 7 and 19 at P1, and keratin 18 at P2-P3. Keratin 18 was always limited to elongated cells. The assemblage of elongated taste cells comprising a taste bud began with a single elongated cell, rather than with the synchronous elongation of a cluster of cells. Developmental errors were observed at P2-P3, e.g., some vallate taste cells had a misoriented axis. In order to study the pace of keratin differentiation during cell turnover we injected bromodeoxyuridine (BrdU) into adult rats to monitor taste cell age. Keratin-19-positive intragemmal cells differentiated within 1 day. In contrast, keratin 18 was first detected in cells aged 3 days. Hence, both in taste cell development and replacement, keratin 18 was restricted to the older cells; it was the last taste cell keratin to become expressed during differentiation.

Three monoclonal antibodies, 4.62, LP2K and 170.2.14, were used to evaluate keratin 19-like immunoreactivity in gustatory epithelia. Keratin 19-like immunoreactivity was restricted to the intragemmal cells for all types of mammalian taste buds examined. These taste buds included fungiform, foliate and vallate taste buds in rat, gerbil and rabbit, and nasopalatine, epiglottal and palatine taste buds in rat. There was no keratin 19-like immunoreactivity in basal cells or in perigemmal cells lateral to the immunoreactive taste receptor cells. Denervation of the rat vallate papilla eliminated all taste buds, as well as all immunoreactive taste cells. That the immunoreactive material in the taste cells was keratin 19 was supported by the comparable staining of rat taste buds with each of three monoclonal antibodies specific for keratin 19. Furthermore, as predicted, these antibodies selectively stained luminal cells of rat bile ducts, bladder, salivary ducts, trachea, ureter and uterus. It was concluded that monoclonal antibodies against keratin 19 can usefully distinguish intragemmal taste receptor cells from keratinocytes, and from the perigemmal and basal cells of gustatory epithelia. Anti-keratin 19 antibodies may serve to identify differentiated taste cells in gustatory epithelia undergoing taste bud development, renewal, degeneration or regeneration.