Uzman JA et al. (1998), The role of intracellular alkalinization in the...

XB-ART-15439

Dev Biol 1998 Jan 01;1931:10-20. doi: 10.1006/dbio.1997.8782.

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The role of intracellular alkalinization in the establishment of anterior neural fate in Xenopus.

Uzman JA , Patil S , Uzgare AR , Sater AK .

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Our previous work demonstrated that Xenopus ectoderm 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 of NCAM and the anterior neural 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. NCAM, otx2, and the anterior neural inducer noggin were expressed in alkalinized ectoderm, while the more posterior neural markers krox-20 and Hox B9 were undetectable. Expression of NCAM, otx2, and noggin was 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 of otx2 within 15 min. Activation of NCAM expression 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 antiphosphotyrosine antibody. Several phosphorylated protein 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 induction in 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.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a ctrl egr2 en2 gsc ncam1 nog not otx2 tbx2

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	FIG. 1. Effects of H2DIDS on expression of neural-specific genes in planar explants of the dorsal marginal zone. Planar explants of the dorsal marginal zone were isolated at the onset of gastrulation (st. 10), stripped of external epithelium, and cultured in the presence (lane 2) or absence (lane 1) of 200 mM H2DIDS, an inhibitor of Na/-dependent Cl0/HCO03 exchange that prevents intracellular alkalinization during neural induction. Explants were collected for RNA isolation and RT-PCR when intact control embryos reached st. 20. Treatment with H2DIDS from gastrulation onward (st. 10â 20) inhibits expression of NCAM and the anterior neural gene otx2; expression of the posterior neural-specific gene Hox B9 is unaffected (lanes 1 and 2). In whole embryos at this stage, otx2 is expressed in the presumptive anterior neural (forebrain and midbrain) and cement gland regions (Blitz and Cho, 1995), while Hox B9 is expressed throughout the spinal cord (Sharpe et al., 1987). Treatment of planar explants with H2DIDS after gastrulation (st. 13â 20) does not inhibit expression of otx2 or NCAM (lane 3). Planar explants treated with both H2DIDS and methylamine during gastrulation (st. 10â13) express otx2, but not NCAM, indicating that artificial alkalinization by methylamine partially rescues the effects of H2DIDS (lane 4) (n = 4 experiments).
	FIG. 2. Expression of otx2, not1, and gsc in planar explants cultured in the presence or absence of H2DIDS. DMZ explants were cultured either alone (A, D, and G) or in H2DIDS (B, E, and H); a third set was cultured in H2DIDS / methylamine until mid-10.5, and then in H2DIDS alone (C, F, and I). Explants were fixed when controls reached the end of gastrulation and hybridized with antisense probes directed against otx2 (A, B, and C), not1 (D, E, and F), or gsc (G, H, and I). Explants are oriented with the ectoderm toward the top. Treatment with H2DIDS blocks expression of otx2 in the ectoderm without affecting mesodermal otx2 expression (B); not1 is moderately reduced, but still present (E). Addition of methylamine to H2DIDS-treated explants restores ectodermal otx2 expression, but virtually abolishes mesodermal expression of otx2 (C) or not1 (F). Expression of gsc (arrowheads) is similar in all three explants (G, H, and I). Each in situ hybridization was performed on 14â22 explants obtained from four to seven independent experiments.
	FIG. 3. Expression of anterior neural genes in alkalinized unin- duced ectoderm. Animal cap ectoderm was isolated at st. 8 and transferred at st. 10 to HS containing either 15 mM methylamine or 10mMNH4Cl. After either 3 h in methylamine or 4 h in NH4Cl, animal caps were returned to MBS and cultured until st. 24, when they were collected for RNA isolation and RT-PCR. Genes assayed by RT-PCR are expressed in the following domains: otx2, anterior neural ectoderm (primarily) (Blitz and Cho, 1995); noggin, anterior neural ectoderm (Vodicka and Gerhart, 1995); NCAM, throughout the central nervous system; en-2, midbrainâhindbrain boundary (Hemmati-Brivanlou and Harland, 1989); krox-20, hindbrain (rhombomeres 3 and 5) (Bradley et al., 1993); and Hox B9, spinal cord (Sharpe et al., 1987). Muscle actin was assayed as a control for mesodermal contamination and EF-1a was used to monitor RNA recovery. Lanes: (1) 0RT, sample prepared without reverse transcriptase; (2) WE, whole embryo st. 24; (3) Ctrl, untreated animal caps; (4) Me, methylamine-treated animal caps; and (5) NH4 , NH4Cl-treated animal caps. Artificial alkalinization leads to expression of anterior neural-specific genes, but it does not activate expression of posterior neural-specific genes (n Â 6 experiments).
	FIG. 4. Artificial alkalinization elicits anterior neural-specific gene expression by the midgastrula stage. Animal cap ectoderm was isolated at st. 8 and alkalinized at st. 10 as described earlier. Ectodermal isolates were collected for RT-PCR analysis at st. 11 (midgastrula). Artificial alkalinization activates expression of NCAM and noggin and up-regulates expression of otx2 (lanes 5 and 6). NCAM, noggin, and otx2 are detectable in the neural plate at st. 11 (lane 7). Lanes: (1) 0RT, no reverse transcriptase; (2) WE 24, whole embryo st. 24; (3) WE 11, whole embryo st. 11; (4) Ctrl, untreated animal caps; (5) Me, methylamine-treated animal caps; (6) NH4, NH4Cl-treated animal caps; and (7) NP11, neural plates isolated at st. 11 (n Â 6 experiments).
	FIG. 5. Up-regulation of otx2 expression is a rapid response to alkalinization. Time course of otx2 expression in response to alka-linization. (A) Animal caps were isolated at st. 8 and transferred at st. 10 to HS in the presence or absence of 10mMNH4Cl. Samples of treated (even-numbered lanes) and untreated (odd-numbered lanes) animal caps were collected at intervals after the onset of the alkaLinizing treatment. Expression of otx2 and EF-1a was assessed bY RT-PCR. An increase in otx2 expression in response to alkaliniza- tion is not detected until 1 h after exposure to NH4Cl (lanes 5 and 6) (results shown are from a single experiment; n Â 3 experiments). (B) An identical experiment except that alkalinization was initiated at early st. 10.5. An alkalinization-inducible increase in otx2 expression occurs within 15 min after exposure to NH4Cl (lanes 1 and 2). Low levels of otx2 are detected in untreated animal caps in both experiments (results shown are from a single experiment; n Â 4 experiments).
	FIG. 6. Cell contact is not required for the up-regulation of NCAM expression in response to alkalinization. Animal caps were dissociated at st. 10, and cells were incubated in the presence or absence of NH4Cl or methylamine as described earlier. At the end of the treatment period, they were reaggregated and collected for RNA isolation (see Methods); at this time, intact control embryos were at st. 11.5. Intact animal caps were treated in parallel. Expression of NCAM is up-regulated in cells that were dissociated during the alkalinization period (lanes 6 and 7), as it is in alkalinized intact animal caps (lanes 3 and 4) (n Â 4 experiments).
	FIG. 7. Artificial alkalinization elicits changes in protein tyrosine phosphorylation similar to those observed during neural induction in vivo. Comparison of protein tyrosine phosphorylation in induced, uninduced, and artificially alkalinized ectoderm. Protein samples were prepared from the following tissues: (A) animal caps isolated and lysed at st. 10/ (AC-10/), (B) uninduced ectodermâanimal caps isolated at st. 10/ and aged in culture until st. 11 (Aged AC-11), (C) alkalinized ectodermâanimal caps isolated at st. 10/ and treated with 15 mM methylamine until st. 11 (Me AC-11) and (D) induced ectodermâneural plates isolated at st. 11 (NP-11). For each sample, 100 mg protein was run on a 2D gel; the gels were blotted, and the blots were probed with the anti-phosphotyrosine antibody PY20. Black arrows indicate two of the proteins that are more heavily phosphorylated in induced or alkalinized ectoderm than in uninduced ectoderm at the same stage. White arrows identify a protein spot that does not change in intensity among these samples; it serves as a control for differences in loading. Samples were run in duplicate for each of five experiments.