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Intracellular acidification of gastrulaectoderm is important for posterior axial development in Xenopus.
Gutknecht DR
,
Koster CH
,
Tertoolen LG
,
de Laat SW
,
Durston AJ
.
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There is evidence suggesting that pHi elevation can induce differentiation to cement gland, an extremely anterior structure, during the early development of Xenopus laevis (Picard, J. J. (1975) J. Embryol. exp. Morphol. 33, 957-967; Sive, H. L., Hattori, K. and Weintraub, H. (1989) Cell 58, 171-180). We wanted to investigate whether axial development or neural induction are mediated in Xenopus via regulation of pHi. Our interest was stimulated further because certain signal transduction pathways, which are thought to mediate anterior neural induction (Otte, A. P., Van Run, P., Heideveld, M., Van Driel, R. and Durston, A. J. (1989) Cell 58, 641-648; Durston and Otte (1991), Cell-Cell Interactions in Early Development, pp. 109-127), are also known to modify the activity of proton extruders (Mitsuka and Berk (1991) Am. J. Physiol. 260, C562-C569; Wakabayashi, S., Sardet, C., Fafournoux, P., Counillon, L., Meloche, S., Pages, G. and Pouysségur, J. (1993) Rev. Physiol. Biochem. Pharmacol. Vol. 119, pp. 157-186). We therefore measured pHi in explants of gastrulaectoderm and neurectoderm and identified ion exchangers that regulate pHi in these tissues. The measurements showed that pHi decreases in explants of both neurectoderm and uninduced ectoderm during the time course of gastrulation, this pHi decrease thus fails to correlate with neural induction. One important regulator of this cytoplasmic acidification is the Na+/H+ exchanger. The pHi set point, at which the acid extrusion activity of this alkalizing exchanger is shut off, shifts to more acidic values during the time course of gastrulation, thus permitting cytoplasmic acidification. We found also that preventing cytoplasmic acidification and thereby elevating pHi in late gastrula cells led to the specific suppression of posterior development. Neural induction and anterior development were unaffected by treatments leading either to an elevation of or a decrease in pHi. These findings indicate that the cellular processes mediating anterior development and neural induction are pHi tolerant, while the signals mediating posterior development require a sustained pHi decrease for their action, suggesting that downregulation of pHi is necessary for posterior axial development.
Fig. 1. The fluorescence contributions
of different cellular fractions, as
demonstrated by one representative
experiment using stage 13 gastrula cells
(fluorescence intensities expressed in
arbitrary units). The fluorescence
contributions of the different cellular
fractions, expressed as percentages, and
averaged over three independent
experiments, are also indicated above
the specific columns. The absolute
values could not be compared directly
between different measurements, due to
batch-dependent differences in dye
loading. (A,B) After fluorophore
loading the cells were incubated for 45
minutes before they were lysed and
fractionated. (C,D) 90 minute
incubation after dye loading. Please
note the differences in fluorophore
localisation between BCECF and Fura-
2, especially the low cytoplasmic
concentration of Fura-2. Error bars =
s.e.m.
Fig. 2. False colour images of pHi in a part of
an explant (30-50 cells). (A) Stage 10 ectoderm
and (B) stage 13 neurectoderm. Colour scale
indicates colour representation of different pH
values; yellow pH 7.9; light green pH 7.8;
green pH 7.7, blue pH 7.3. Note that nuclei
(one pointed out by arrowhead) are slightly
more acidic than the surrounding cytoplasm, as
can be deduced from the darker colour. The
particular pHi pattern shown in this image does
not represent a constant tendency. The
examined explants (n=7) displayed a variety of
different pHi patterns including edge and center
localized pHi minima or maxima. Bar indicates
50 mm.
Fig. 3. (A) False colour image of pHi in stage 10
ectoderm cells calibrated to pH 7.0 using
nigericin. Note that all cells are calibrated and
that differences beween cytoplasmic and nuclear
pH as well as differences between cells are now
absent. Colour scale gives colour representation
of different pH values; blue is now pH 7.0.
(B) Image of intracellular BCECF distribution in
the cells shown in A, taken at the isobestic point
of BCECF fluorescence (438 nm), at the end of
the recording. Note that the nucleus (arrowhead)
exhibits strong labelling, as can be deduced from
the strong (bright) fluorescence emission. Bar
indicates 50 mm.
Fig. 4. (A) During gastrulation, intracellular pH (pHi) decreases
continuously in neurectoderm (hatched bars) and in uninduced
ectoderm (white bars) cultured in HCO3- free MFM at pH 7.6.
Importantly, we failed to observe differences in pHi between
uninduced ectoderm cultured between stages 10 and 11 and
uninduced ectoderm excised at stage 11, indicating, that the observed
acidification is not a reaction to explant excision. (B) In ectoderm
and neurectoderm, the absolute pHi is adjusted according to the pH
of the medium (pHo). At stage 10 and stage13 the pHi of cells
incubated in FM at pHo 8.3 (hatched bars) is higher than in cells
incubated in FM at pHo 6.9 (white bars) or in MFM pHo 7.6
(compare with A). The extent of the pHi decrease observed during
gastrulation is also influenced by the nature of the medium. It was
less extensive in phosphate-buffered medium than in Hepes-buffered
MFM. It is unclear what actually causes this difference. Error bar =
s.e.m. in all figures; n: number of experiments.
Fig. 5. pHi recordings in ectoderm and neurectoderm. (A) Acid load
experiment. Anterior neurectoderm (stage 11) in HCO3- free MFM
pH 7.6. Arrows indicate change of medium: P, prepulse by adding 10
mM NH4Cl; AL, acid loading the cells; 7.6, calibration buffer pH 7.6
with nigericin; 8.0, calibration buffer pH 8.0 with nigericin. (B) Acid
load experiment in neural plate cells (stage 13) in medium as in A,
containing the Na+/H+ inhibitor dimethyl-amiloride (+DMA). The
velocity and the extent of the acid load recovery is reduced (compare
with A). (B insert) Ectoderm (stage 10) acid load experiment in Na+
free medium (Na+ replaced with 58 mM cholinechloride) in MFM
pH 7.6 with 2.4 mM HCO3-. (C) Ectoderm (stage 10) cultured in
MFM pH 7.6 with 2.4 mM HCO3- and 0.5 mM FCCP.
Developmental stage of control embryos is indicated under pHi
recording. Ratios were converted to pH units by calibration
procedure. Bars: 10 minutes in A and B; 30 minutes in C. During the
pHi recordings of the explants, pHi decreased continuously (Fig.
4A,B). The results from the fractionation studies rule out the
possibility that this decrease occurs because of BCECF uptake into
acidic organelles (see text and Fig 1A,C). We also tested whether the
observed acidification could be blocked by incubating cells in
medium containing increasing concentrations of bicarbonate. This
increased the buffering capacity of the cells, as can be deduced from
the reduced pHi increase after the addition of identical [NH4Cl]
(compare A and B with B insert). We found that the cytoplasmic
acidification, though predictably reduced, could not be blocked by
this treatment, indicating that the observed pHi decrease reflects a
developmentally regulated cytoplasmic acidification, rather than a
change in the buffering capacity of the cells.
Fig. 6. Acidic shift of the pHi set points. During the acid load
recovery, acid extrusion slows down with increasing pHi and
extrapolates to zero at the set point value of the ion exchangers. A
representative recording is shown from each experimental series. pHi
set point values are given in the text. (A) The set point value of the
Na+/H+ exchanger is shifted to more acidic pHi, in good correlation
with the acidification recorded during gastrulation. The slope
indicates the pHi sensitivity of the ion exchanger, which does not
change significantly during gastrulation. The slope is at stage 10
–0.31±0.05 (n=4), at stage 11 –0.31±0.01 (n=6) and at stage 13
–0.275±0.04 (n=4) (B) The pHi set point of the HCO3-/DMA
insensitive acid extruding mechanism is shifted even more
extensivley to acidic pHi and this acid extruder is less pH sensitive as
depicted by the slopes: stage 10 –0.134±0.01 (n=5), stage11
–0.09±0.01 (n=6) and stage 13 –0.221±0.02 (n=3). The values for
the Na+/H+ exchanger were calculated by subtracting the DMA
insensitive fraction of the acid load recoveries from the inhibitor free
ones, recorded in HCO3- free medium. The experiments were
performed in HCO3- free medium, in order to inhibit activity of the
Na+-dependent HCO3-/Cl- exchanger. We verified that this
exchanger was effectively inhibited because addition of it’s specific
inhibitor SITS (up to 0 mM; Sigma) to HCO3-free medium failed to
result in any additional reduction in recovery rates or in any other
effects, e.g. on pHi.
Fig. 7. Examples of pHi-dependent phenotypes. Morphological structures are identified as shown in F. (A-C) Explants cultured in MFM at
different pHs. (A) Head development only: development of tail and trunk structures is suppressed (explant cultured at pHo 8.8). (B) Head and
trunk development: tail development is suppressed (explant cultured at pHo 7.6). (C) Full axial development, (head, trunk and tail) differentiate
completely (explant cultured at pHo 6.8). (D-F) Explants cultured in MFM of varying pH, containing FCCP. (D) Head structures only: tail and
trunk development is suppressed (pHo 8.8, with FCCP). (E) Head and trunk development: tail development is suppressed (pHo 7.6, with
FCCP). (F) Complete axial development (pHo 6.8 with FCCP). e, eye; cg, cement gland; solid asterisk, trunk; open asterisk, tail. Scale bar:
500 mm.
Fig. 8. Dorsal axial structures always develop in dorsal meso/ecto
explants cultured at different pHo in the presence of FCCP. Explants
were fixed when controls reached stage 42. (A) Head morphology of
an explant cultured at pHo 8.8, in which trunk and tail formation are
suppressed. n, neural tissue; e, eye; cg, cement gland; no, notochord
(n=10). (B) Dorsal structures of an explant cultured at pHo 6.8,
which failed to develop a proper axis, while dorsal tissues such as
neural tissue, notochord and muscle (m) differentiate (n=9). Scale
bar: 100 mm.
Fig. 9. Examples of pHi effects on Hox
gene expression patterns at stage 16 in
dorsal meso/ecto explants. All explants are
oriented as indicated in A: a, anterior; p,
posterior; d, dorsal; v, ventral. (A-D)
Hoxb-9 expression (purple staining) (A)
Explants cultured at pH 7.6 (n=5) (also
true for pH 6.8; n=8) express Hoxb-9 along
the presumptive spinal cord, thus in the
posterior region. (B,C) In explants cultured
at pHo 8.8 (n=6) (also true for pH 7.6 +
FCCP; n=10), Hoxb-9 expression is
compressed to an increasingly smaller
zone, or, as shown in D, is completely
absent. (E-H) Hoxb-3 expression (purple
staining) (E) Hoxb-3 is expressed in a band
in the postotic region of the hindbrain in
explants cultured at pHo 7.6 (n=7) or 6.8
(n=6). (F-H) In explants cultured at pHo
8.8 (n=9) or 7.6 + FCCP (n=7) Hoxb-3
expression remains strong but is shifted
towards the posterior end of the neurula.
Scale bar: 500 mm.
