In the present study, we investigated expression and function of several molecules in the development of the mouse optic chiasm. One of the molecules is CD44, a cell adhesion molecule which is expressed in an inverted V-shaped configuration caudal to the optic chiasm. Demonstration of functions of CD44 molecule per se in the axon routing has been lacking although a previous study reported that removal of CD44 expressing cells leads to retinal projection stalled at the pre-midline area (Sretavan et al., 1995). We used two CD44 functional blocking antibodies to specifically determine CD44's roles in the critical period of retinal axon pathfinding in the diencephalon in a brain slice preparation of the mouse retinofugal pathway. We found that perturbation of CD44 functions resulted in a reduction of the midline crossing of the first axons to the contralateral diencephalon and a reduction of the ipsilaterally projecting axons at a later developmental stage. These findings suggested that CD44 was necessary for crossing and sorting of the axons at the midline of the chiasm.; A large body of evidence has shown that hyaluronate, a ligand for CD44, is involved in the CD44 mediated cell-cell and cell-matrix adhesion. We asked whether HA was present in the retinal pathway and how it was related to the functions of CD44. By using a specific probe for hyaluronate, we observed that expression of hyaluronate was overlapped with CD44 and colocalization of hyaluronate CD44 was found in the central area of the V-shaped array of early diencephalic neurons. The morphological data suggested a close association of hyaluronate with CD44 mediated activity in the formation of the optic chiasm. To support this notion, we carried out an experiment by application of hyaluronate or hyaluronidase into the brain slice preparation of the optic pathway. We found that treatment with hyaluronate or enzymatic removal of hyaluronate resulted in a severe failure of the first axon crossing at the midline. At a later developmental stage, disturbing HA functions led to a significant decrease in the amount of the ipsilaterally projecting axons. These findings were basically comparable with the data obtained in the experiments of blocking CD44 functions, indicating that CD44 involved hyaluronate in regulation of routing and sorting of axons during the formation of the optic chiasm. Given the fact that CD44 mediates the metabolism of hyaluronate, we speculated that it may be hyaluronate rather than CD44 interacting with the axons to regulate axon growth and guidance. It was also possible that CD44 or hyaluronate may also have independent roles in the process of the guidance regulation at the chiasm since blocking CD44 function at E14 reduced axon crossing at the midline but hyaluronate treatment did not have effects on this activity. (Abstract shortened by UMI.)
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