The Role of Intracellular Alkalinization in the Establishment of Anterior Neural Fate inXenopus

• Published in: Developmental biology 1998-01, Vol.193 (1), p.10-20
• Main Authors: Uzman, J.Akif, Patil, Sonali, Uzgare, Aarti R, Sater, Amy K
• Format: Article
• Language: English
• Subjects: intracellular pH; neural induction; otx2; Xenopus
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1006/dbio.1997.8782

Our previous work demonstrated thatXenopusectoderm cells undergo an alkalinization in response to planar inductive signals during neural induction in explants. We have examined the role of intracellular alkalinization 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 4,4′-dihydrodiisothiocyanatostilbene-2,2′-disulfonate (H2DIDS). In explants cultured in the presence of H2DIDS, expression ofNCAMand the anterior neural geneotx2is 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.NCAM, otx2,and the anterior neural inducernogginwere expressed in alkalinized ectoderm, while the more posterior neural markerskrox-20andHox B9were undetectable. Expression ofNCAM, otx2,andnogginwas observed at stage 11 in both alkalinized ectoderm and the newly induced neural plate, suggesting that intracellular alkalinization could contribute to propagation of noggin signaling through the dorsal ectoderm. Alkalinization of uninduced ectoderm at stage 10.5 led to an upregulation ofotx2within 15 min. Activation ofNCAMexpression in alkalinized dissociated cells was identical to that observed in intact animal caps, indicating that alkalinization-mediated changes in gene expression do not require cell–cell contact. Finally, the effects of intracellular alkalinization on protein tyrosine phosphorylation were investigated using 2D gel electrophoresis and immunoblots probed with an anti-phosphotyrosine antibody. Several phosphorylated proteins detected in induced and alkalinized ectoderm were greatly reduced or absent in uninduced ectoderm, indicating that alkalinization elicits alterations in tyrosine phosphorylation similar to some of those observed during neural inductionin vivo.Our results indicate that intracellular alkalinization plays a critical role in the activation of anterior neural-specific gene expression and that alkalinization may act by regulating the activity of a tyrosine kinase or phosphatase.