Transgenic mice were generated in which sequences that flank the rat tyrosine hydroxylase (TH) gene were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Mice bearing 4.8 kilobases (kb) of 5' flanking DNA exhibited correct tissue-specific expression in the CNS and periphery. Expression was more robust in the CNS than in the periphery, although CAT activity was clearly detected in sympathetic ganglia (superior cervical ganglia) and the adrenal, the two peripheral tissues that contain TH-positive cells. Within the brain, CAT expression was seen in all the expected areas containing TH-positive cell bodies, with little or no expression in other regions. In the olfactory bulb, which contains the majority of the CNS TH cells, developmental expression of CAT was quantifiable and was found to parallel the postnatal rise in endogenous TH, with both TH and CAT reaching adult levels by postnatal day 21. Since TH activity in the olfactory bulb requires afferent input, the dependence of CAT activity on transsynaptic input was also assayed in transgenic mice. Like the endogenous TH activity, CAT levels were also reduced by deafferentation, in parallel with loss in endogenous dopamine levels. While previous experiments demonstrated that shorter 5' flanking regions (2.5 kb and 3.5 kb of 5' upstream sequences of the human and mouse TH gene, respectively) failed to direct accurate tissue-specific expression, our data demonstrate that 4.8 kb of 5' flanking sequence of the rat TH gene contains sufficient regulatory information to mediate appropriate tissue-specific expression in all CNS and PNS tissues, as well as to mediate developmental and transsynaptic expression in the olfactory bulb.

The tetraspan cell surface glycoprotein, CD9, has been implicated in cellular signaling during growth and differentiation in the hematopoietic and nervous systems. Because CD9 expression is induced early in development in sensory and sympathetic neuroblasts, we investigated the role of CD9 in neurite outgrowth. We plated dissociated cells from neonatal sympathetic ganglia on immobilized anti-CD9 antibodies or antibodies against other cell surface molecules. We show here that B2C11, an anti-CD9 antibody that has been shown previously to activate Schwann cells in vitro, promotes robust neurite outgrowth from sympathetic neurons that is greater than that on other antibody surfaces and is comparable to neurite outgrowth on a collagen substratum. In addition, B2C11 causes dramatic morphological changes in neurons and glia from dissociated ganglia, including a flattening of these cells.Because CD9 interacts with integrins in many cell types including Schwann cells, and specifically with the ␣3␤1 integrin in some cells, we tested whether the effect of B2C11 on neurite outgrowth is mediated by this integrin. An anti-␣3␤1 antibody, Ralph 3-1, attenuates the extent of neurite outgrowth on B2C11 and collagen, but not on laminin. Because the ␣3␤1 integrin has been shown to mediate neurite outgrowth on different substrates, these results provide a functional significance for the CD9-␣3␤1 interaction; downstream signaling may be activated by this cis interaction on the cell surface in response to external cues that promote neurite outgrowth.

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