THE ROLE OF INTRACELLULAR ALKALINIZATION IN THE ESTABLISHMENT OF ANTERIOR NEURAL FATE IN XENOPUS

Our previous work demonstrated that Xenopus ectoderm cells undergo an alkalinization in response to planar inductive signals during neural i nduction in explants. We have examined the role of intracellular alkal inization in the establishment of anterior neural fate. first, RT-PCR was used to examine neural-specific gene expression in planar explants in which the alkalinization is prevented by treatment with '-dihydrod iisothiocyanatostilbene-2,2'-disulfonate (H2DIDS). In explants culture d in the presence of H2DIDS, expression of NCAM and the anterior neura l gene otx2 is greatly reduced or absent. Second, neural-specific gene expression was examined in isolates of uninduced animal cap ectoderm cultured in the presence of either methylamine or ammonium chloride. N CAM, otx2, and the anterior neural inducer noggin were expressed in al kalinized ectoderm, while the more posterior neural markers krox-20 an d Hox B3 were undetectable. Expression of NCAM, otx2, and noggin was o bserved at stage 11 in both alkalinized ectoderm and the newly induced neural plate, suggesting that intracellular alkalinization could cont ribute to propagation of noggin signaling through the dorsal ectoderm. Alkalinization of uninduced ectoderm at stage 10.5 led to an upregula tion of otx2 within 15 min. Activation of NCAM expression in alkaliniz ed dissociated cells was identical to that observed in intact animal c aps, indicating that alkalinization-mediated changes in gene expressio n do not require cell-cell contact. Finally, the effects of intracellu lar alkalinization on protein tyrosine phosphorylation were investigat ed using 2D gel electrophoresis and immunoblots probed with an antipho sphotyrosine antibody. Several phosphorylated proteins detected in ind uced and alkalinized ectoderm were greatly reduced or absent in unindu ced ectoderm, indicating that alkalinization elicits alterations in ty rosine phosphorylation similar to some of those observed during neural induction in vivo. Our results indicate that intracellular alkaliniza tion plays a critical role in the activation of anterior neural-specif ic gene expression and that alkalinization may act by regulating the a ctivity of a tyrosine kinase or phosphatase. (C) 1998 Academic Press.