An increase in intracellular pH during neural induction in Xenopus

Development ◽  
1994 ◽  
Vol 120 (2) ◽  
pp. 433-442 ◽  
Author(s):  
A.K. Sater ◽  
J.M. Alderton ◽  
R.A. Steinhardt
Keyword(s):  
Intracellular Ph ◽  
Marginal Zone ◽  
Phorbol Esters ◽  
Regulation Of Gene Expression ◽  
Homeobox Gene ◽  
Neural Induction ◽  
Ph Indicator ◽  
Pharmacological Agents ◽  
Dorsal Ectoderm ◽  
Intracellular Alkalinization

In this paper, we show that an intracellular alkalinization of the dorsal ectoderm cells is among the earliest responses to neural induction in Xenopus. Planar explants of the dorsal marginal zone were prepared from embryos that had been microinjected during cleavage stages with the fluorescent pH indicator bis-carboxyethyl-carboxyfluorescein-dextran (BCECF-dextran), and intracellular pH (pHi) was monitored continuously by emission ratio microfluorimetry. During stage 10.5, the dorsal ectoderm cells undergo a sustained intracellular alkalinization of approximately 0.1 pH units in response to neural induction; in the absence of the inductive signal, the pH of the dorsal ectoderm cells decreases slightly. Ectoderm cells within planar explants of the ventral marginal zone show little change in pH during a similar period. This increase in intracellular pH is inhibited by 4, 4′-dihydrodiisothiocyanatostilbene-2, 2′-disulfonate (H2DIDS) or a low Na+/high Cl- medium, treatments that presumably affect anion transport. Under these conditions, expression of the anterior neural-specific homeobox gene engrailed is not detected, while the notochord-specific epitope recognized by the Tor-70 antibody is expressed in the presence of H2DIDS. This characteristic alkalinization is not evoked by pharmacological agents that reportedly alter ectodermal developmental pathways in Xenopus embryos, such as NH4Cl, phorbol esters, or cAMP-dependent protein kinase agonists. Our results suggest that an ionic regulatory event may participate in the regulation of gene expression in response to neural induction.

Intracellular pH in human fibroblasts: effect of mitogens, A23187, and phospholipase activation

1985 ◽  
Vol 249 (1) ◽  
pp. C140-C148 ◽  
Author(s):  
L. L. Muldoon ◽  
R. J. Dinerstein ◽  
M. L. Villereal
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Human Fibroblasts ◽  
Phospholipase Activity ◽  
Ph Indicator ◽  
Calcium Indicator ◽  
Peptide Growth Factors ◽  
Acid Loading ◽  
Intracellular Alkalinization ◽  
Stimulation Of

Serum-deprived human fibroblasts (HSWP cells) were loaded with either the fluorescent pH indicator 6-carboxy-4',5'-dimethylfluorescein or the calcium indicator quin 2, and the fluorescence of the intracellular probes was continuously monitored with a microspectrofluorometer. Addition of a cocktail of peptide growth factors causes intracellular alkalinization, which is blocked by amiloride or by replacement of extracellular Na+ with choline, confirming that mitogenic stimulation activates a Na+-H+ exchanger in HSWP cells. The exchanger is also activated by A23187, acid loading the cells, and stimulation of phospholipase activity with melittin. Growth factors and melittin activation of the exchanger have been demonstrated to be dependent on the mobilization of Ca2+ from intracellular stores. The intracellular alkalization and increase in Ca2+ activity due to both melittin and growth factors is inhibited by phospholipase inhibitors, indicating that phospholipase activity is necessary for the activation of the Na+-H+ exchanger and the mobilization of intracellular Ca2+.

scholarly journals Ion exchange mechanisms on the erythrocyte membrane of the aquatic salamander, Amphiuma tridactylum

1987 ◽  
Vol 133 (1) ◽  
pp. 329-338
Author(s):  
B. L. Tufts ◽  
M. Nikinmaa ◽  
J. F. Steffensen ◽  
D. J. Randall
Keyword(s):  
Steady State ◽  
Ion Exchange ◽  
Water Content ◽  
Intracellular Ph ◽  
Erythrocyte Membrane ◽  
Incubation Medium ◽  
Cell Water ◽  
Pharmacological Agents ◽  
Intracellular Alkalinization

The effects of different pharmacological agents and incubation media on the intracellular pH and water content of Amphiuma erythrocytes were investigated in vitro. Adrenaline had no significant effect on the intracellular pH or cell water content. DIDS caused an intracellular alkalinization that could be abolished by amiloride, ouabain or removal of sodium from the incubation medium. In addition, amiloride and DIDS both caused a decrease in cell water content. The data indicate that sodium/proton and chloride/bicarbonate exchangers are present on the membrane of Amphiuma erythrocytes and these exchangers are active under steady-state conditions.

scholarly journals Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator

1998 ◽  
Vol 74 (3) ◽  
pp. 1591-1599 ◽  
Author(s):  
Malea Kneen ◽  
Javier Farinas ◽  
Yuxin Li ◽  
A.S. Verkman
Keyword(s):  
Green Fluorescent Protein ◽  
Intracellular Ph ◽  
Fluorescent Protein ◽  
Ph Indicator ◽  
Green Fluorescent

Mechanism of anteroposterior axis specification in vertebrates. Lessons from the amphibians

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 285-302 ◽  
Author(s):  
J.M. Slack ◽  
D. Tannahill
Keyword(s):  
Molecular Markers ◽  
Expression Patterns ◽  
Signal Transduction Pathway ◽  
Homeobox Gene ◽  
Neural Induction ◽  
High Sensitivity ◽  
Neural Plate ◽  
Mesoderm Induction ◽  
Inducing Factors ◽  
Almost All

Interest in the problem of anteroposterior specification has quickened because of our near understanding of the mechanism in Drosophila and because of the homology of Antennapedia-like homeobox gene expression patterns in Drosophila and vertebrates. But vertebrates differ from Drosophila because of morphogenetic movements and interactions between tissue layers, both intimately associated with anteroposterior specification. The purpose of this article is to review classical findings and to enquire how far these have been confirmed, refuted or extended by modern work. The “pre-molecular” work suggests that there are several steps to the process: (i) Formation of anteroposterior pattern in mesoderm during gastrulation with posterior dominance. (ii) Regional specific induction of ectoderm to form neural plate. (iii) Reciprocal interactions from neural plate to mesoderm. (iv) Interactions within neural plate with posterior dominance. Unfortunately, almost all the observable markers are in the CNS rather than in the mesoderm where the initial specification is thought to occur. This has meant that the specification of the mesoderm has been assayed indirectly by transplantation methods such as the Einsteckung. New molecular markers now supplement morphological ones but they are still mainly in the CNS and not the mesoderm. A particular interest attaches to the genes of the Antp-like HOX clusters since these may not only be markers but actual coding factors for anteroposterior levels. We have a new understanding of mesoderm induction based on the discovery of activins and fibroblast growth factors (FGFs) as candidate inducing factors. These factors have later consequences for anteroposterior pattern with activin tending to induce anterior, and FGF posterior structures. Recent work on neural induction has implicated cAMP and protein kinase C (PKC) as elements of the signal transduction pathway and has provided new evidence for the importance of tangential neural induction. The regional specificity of neural induction has been reinvestigated using molecular markers and provides conclusions rather similar to the classical work. Defects in the axial pattern may be produced by retinoic acid but it remains unclear whether its effects are truly coordinate ones or are concentrated in certain regions of high sensitivity. In general the molecular studies have supported and reinforced the “pre-molecular ones”. Important questions still remain: (i) How much pattern is there in the mesoderm (how many states?) (ii) How is this pattern generated by the invaginating organizer? (iii) Is there one-to-one transmission of codings to the neural plate? (iv) What is the nature of the interactions within the neural plate? (v) Are the HOX cluster genes really the anteroposterior codings?

Intracellular pH measurements in gastric mucosa.

1979 ◽  
Vol 237 (1) ◽  
pp. E82
Author(s):  
S J Hersey
Keyword(s):  
Gastric Mucosa ◽  
Acid Secretion ◽  
Intracellular Ph ◽  
Ph Indicator ◽  
Active Secretion ◽  
Ph Measurements ◽  
Tubular Cells ◽  
A Value ◽  
Indicator Dye ◽  
Secretion Rates

Intracellular pH was measured in bullfrog gastric mucosa using a pH-indicator dye, bromthymol blue (BTB), with a spectrophotometric technique. Studies showed that BTB is taken up by the gastric mucosa and bound to intracellular components. The binding of BTB was shown to cause a shift in the pKa of the dye from the solution value of 6.95 to a value of 8.0. During the nonsecreting state, intracellular pH was estimated to be 7.4 (metiamide inhibition) or 7.1 (SCN inhibition). During active secretion of acid, intracellular pH increased with increasing secretion rates, reaching values in excess of pH 8. Using preparations from which the surface epithelial cells had been removed, it was shown that at least a portion of the alkaline response to stimulation occurs in the oxyntic or tubular cells. The results are interpreted in view of existing models for the chemical reaction involved in gastric acid secretion.

A role for the vegetally expressed Xenopus gene Mix.1 in endoderm formation and in the restriction of mesoderm to the marginal zone

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2371-2380 ◽  
Author(s):  
P. Lemaire ◽  
S. Darras ◽  
D. Caillol ◽  
L. Kodjabachian
Keyword(s):  
Marginal Zone ◽  
Ectopic Expression ◽  
Homeobox Gene ◽  
Early Response ◽  
Endoderm Differentiation ◽  
Head Formation ◽  
Mutual Repression ◽  
Regulatory Loop ◽  
Strong Activator

We have studied the role of the activin immediate-early response gene Mix.1 in mesoderm and endoderm formation. In early gastrulae, Mix.1 is expressed throughout the vegetal hemisphere, including marginal-zone cells expressing the trunk mesodermal marker Xbra. During gastrulation, the expression domains of Xbra and Mix.1 become progressively exclusive as a result of the establishment of a negative regulatory loop between these two genes. This mutual repression is important for the specification of the embryonic body plan as ectopic expression of Mix.1 in the Xbra domain suppresses mesoderm differentiation. The same effect was obtained by overexpressing VP16Mix.1, a fusion protein comprising the strong activator domain of viral VP16 and the homeodomain of Mix.1, suggesting that Mix.1 acts as a transcriptional activator. Mix.1 also has a role in endoderm formation. It cooperates with the dorsal vegetal homeobox gene Siamois to activate the endodermal markers edd, Xlhbox8 and cerberus in animal caps. Conversely, vegetal overexpression of enRMix.1, an antimorphic Mix.1 mutant, leads to a loss of endoderm differentiation. Finally, by targeting enRMix.1 expression to the anterior endoderm, we could test the role of this tissue during embryogenesis and show that it is required for head formation.

Xenopus axis formation: induction of goosecoid by injected Xwnt-8 and activin mRNAs

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 499-507 ◽  
Author(s):  
H. Steinbeisser ◽  
E.M. De Robertis ◽  
M. Ku ◽  
D.S. Kessler ◽  
D.A. Melton
Keyword(s):  
Uv Irradiation ◽  
High Frequency ◽  
Marginal Zone ◽  
Homeobox Gene ◽  
Dorsal Side ◽  
Axis Formation ◽  
Gene Products ◽  
Turn On ◽  
Xenopus Embryos ◽  
Spemann's Organizer

In this study, we compare the effects of three mRNAs-goosecoid, activin and Xwnt-8- that are able to induce partial or complete secondary axes when injected into Xenopus embryos. Xwnt-8 injection produces complete secondary axes including head structures whereas activin and goosecoid injection produce partial secondary axes at high frequency that lack head structures anterior to the auditory vesicle and often lack notochord. Xwnt-8 can activate goosecoid only in the deep marginal zone, i.e., in the region in which this organizer-specific homeobox gene is normally expressed on the dorsal side. Activin B mRNA, however, can turn on goosecoid in all regions of the embryo. We also tested the capacity of these gene products to restore axis formation in embryos in which the cortical rotation was blocked by UV irradiation. Whereas Xwnt-8 gives complete rescue of anterior structures, both goosecoid and activin give partial rescue. Rescued axes including hindbrain structures up to level of the auditory vesicle can be obtained at high frequency even in the absence of notochord structures. The possible functions of Wnt-like and activin-like signals and of the goosecoid homeobox gene, and their order of action in the formation of Spemann's organizer are discussed.

A novel Xenopus mix-like gene milk involved in the control of the endomesodermal fates

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2577-2585 ◽  
Author(s):  
V. Ecochard ◽  
C. Cayrol ◽  
S. Rey ◽  
F. Foulquier ◽  
D. Caillol ◽  
...  
Keyword(s):  
Marginal Zone ◽  
Ectopic Expression ◽  
Homeobox Gene ◽  
Early Response ◽  
Response Gene ◽  
Mesodermal Cell ◽  
Early Response Gene ◽  
Mesoderm Formation ◽  
Definition Of ◽  
Early Gastrula

Here we describe a novel Xenopus homeobox gene, milk, related by sequence homology and expression pattern to the vegetally expressed Mix.1. As is the case with Mix.1, milk is an immediate early response gene to the mesoderm inducer activin. milk is expressed at the early gastrula stage in the vegetal cells, fated to form endoderm, and in the marginal zone fated to form mesoderm. During gastrulation, expression of milk becomes progressively reduced in the involuting mesodermal cells but is retained in the endoderm, suggesting that it may play a key role in the definition of the endo-mesodermal boundary in the embryo. Overexpression of milk in the marginal zone blocks mesodermal cell involution, represses the expression of several mesodermal genes such as Xbra, goosecoid, Xvent-1 or Xpo and increases the expression of the endodermal gene, endodermin. In the dorsal marginal zone, overexpression of milk leads to a severe late phenotype including the absence of axial structures. Ectopic expression of milk in the animal hemisphere or in ectodermal explants induces a strong expression of endodermin. Taken together, we propose that milk plays a role in the correct patterning of the embryo by repressing mesoderm formation and promoting endoderm identity.

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