scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Patterns of free calcium in zebrafish embryos.

15 Jun 1998-Journal of Cell Science (COMPANY OF BIOLOGISTS LTD)-Vol. 111, Iss: 12, pp 1613-1622
TL;DR: The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca 2+ in cellular differentiation and pattern formation.
Abstract: Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 microm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.

Summary (2 min read)

Use of aequorin

  • The authors injected either th usual, near natural, recombinant, R-aequorin (at 0.57 mM) or a se synthetic recombinant h-aequorin (at 0.24 mM); these were kindly provided by Dr Osamu Shimomura.
  • With an egg volume of about 200 nl the final aequorin concentrations in embryo were 5.7 µM for R-aequorin and 2.4 µM for h-aequorin.
  • When focusing on pattern a the animal-vegetal axis, the needle was pushed through the e lateral region to avoid vegetal wound signals.
  • Large wounds obscured the Ca2+ patterns and decreased embryonic survival.
  • To map the Ca2+ patterns during embryonic development, the authors hav measured a total of 25 embryos using the imaging photon dete and 17 embryos using the photomultiplier tube.

Calibration of the imaging system

  • The IPD system was calibrated using BAPTA buffers in which fre Ca2+ was set at specific concentrations.
  • Fig. 1 shows the result of this calibration.
  • The luminescence of both R- and h-aequorin rises with the 2.1 power of free Ca2+ over the mid range needed to interpret spik heights from developing embryos.
  • This is the same power recen reported by Shimomura and Inouye (1996).
  • To clamp the Ca2+ level in the embryo, the authors injected high concentrations of a Ca2+ buffer, in which the free Ca2+ concentration was set at pCa 7 (or 100 nM).

F).

  • At 10-11 hours of development, distinct Ca2+ patterns can be recognized along the antero-posterior axis of the embryo (F 4F,G).
  • Most apparent is the high Ca2+ zone which appears in the presumptive fore and midbrain regions in contrast to presumptive hindbrain region.
  • The overall luminescence remains 1.7 times (± 0.2, n=4) higher in the forebrain as compared to the hindbrain.
  • From the burnout experiments the authors learned that aequorin concentrati are low in the yolk region.

The cleavage period

  • The authors results sup these earlier observations.
  • At the onset of first cleavage the authors observed three distinct Ca2+ peaks.
  • Moreover, the data of Webb et al. (1997) do show furrowin and deepening waves with velocities of 0.5 and 0.5 to 0 µm/second, respectively.

The blastula and gastrula period

  • The first sign of Ca2+ signaling during the blastula period is an increase in spike frequency at 3.5 hours after fertilization.
  • The level of free Ca2+ reaches a maximum during early gastrulation (6.5 hours), when epiboly resumes and th embryonic shield starts to extend towards the animal pole.
  • At 75% epiboly (8 hours), luminescence is high in the blastoderm margin, with peak levels in the dorsal blastoder margin.
  • While this anterio spread does suggest a corresponding, ultraslow Ca2+ wave, it may only indicate extension of a thicker embryonic region.
  • So does evidence which indicates that neural induction amphibians involves an increase in cytosolic Ca2+ within the re f ese ce 1621Calcium patterns in zebrafish embryos n e ent art cts rt nts t a a.

The segmentation period

  • At 10-11 hours of development, distinct Ca2+ patterns can be recognized along the antero-posterior axis of the embryo.
  • The boundary betw high and low Ca2+ may be involved in formation of the midbrain/hindbrain boundary and high Ca2+ itself may be required for rostral differentiation.
  • The authors can imagine at least two roles Ca2+ in brain regionalization: (1) Ca2+ may activate transcription, as it does in other neural systems (Ghosh Greenberg, 1995; Ginty, 1997; Hardingham et al., 1997).
  • Defe in the embryonic heart may act back on further hea development, as suggested by studies in the zebrafish muta sih, web, str, pip, and hip.

Did you find this useful? Give us your feedback

Figures (6)

Content maybe subject to copyright    Report

University of Groningen
Patterns of free calcium in zebrafish embryos
Creton, Robbert; Speksnijder, Johanna E.; Jaffe, Lionel F.
Published in:
Journal of Cell Science
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1998
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Creton, R., Speksnijder, J. E., & Jaffe, L. F. (1998). Patterns of free calcium in zebrafish embryos.
Journal
of Cell Science
,
111
, 1613-1622. http://jcs.biologists.org/content/111/12/1613
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.
More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-
amendment.
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 10-08-2022

INTRODUCTION
The intracellular concentration of free Ca
2+
is generally low,
in the order of 0.1 µM, but can rapidly increase to reach values
in the 1 µM or even 10 µM range. When elevated, Ca
2+
activates a wide variety of intracellular events such as
contraction, secretion, and gene expression (Ghosh and
Greenberg, 1995; Ginty, 1997). With this broad range of
effects, Ca
2+
should be an important regulator in embryonic
development. In early development, this role of Ca
2+
is well
established. Thus so-called fast (10 to 30 µm/second) Ca
2+
waves are known to restart development during fertilization in
metazoa from sponges to man (Jaffe, 1991, 1993); while slow
(~1 µm/second) Ca
2+
waves both accompany and are needed
to start and to extend early cleavage furrows in the large eggs
of Xenopus (Miller et al., 1993; Snow and Nuccitelli, 1993),
medaka fish (Fluck et al., 1991) and zebrafish (Chang and
Meng, 1995; Webb et al., 1997). Moreover, the segregation of
cytoplasm and of oil droplets to the opposite poles of the
medaka egg before first cleavage is accompanied and
dependent upon the prolonged presence of high Ca
2+
zones at
these poles (Fluck et al., 1992, 1994). Additional roles of Ca
2+
in axis formation and pattern formation are suggested by
studies in fucoid eggs and slime molds. In the exceptionally
symmetrical fucoid zygote, formation of a localized,
subsurface zone of high, free Ca
2+
is needed to establish the
region of future rhizoidal outgrowth and thus the basal pole of
the organism (Speksnijder et al., 1989). In multicellular
aggregates of the cellular slime mold, D. discoideum,
formation of high free Ca
2+
regions presage future regions of
stalk as opposed to spore cell differentiation (Cubitt et al.,
1995; Jaffe, 1997).
Most of our knowledge on roles of Ca
2+
in late vertebrate
development comes from the amphibian embryo, in which
inhibition or activation of the inositol-Ca
2+
signaling pathway
affects specification of the dorso-ventral axis (Ault et al., 1996;
Kume et al., 1997). Moreover, there is very interesting
evidence that neural induction in amphibians involves an
increase in cytosolic Ca
2+
via L-type Ca
2+
channels (Moreau
et al., 1994; Drean et al., 1995; Leclerc et al., 1995, 1997).
However, Ca
2+
patterns have never been imaged during long
periods in vertebrate development. For example, no Ca
2+
measurements have been made during regionalization of the
vertebrate central nervous system, which depends on various
secreted signaling proteins such as WNT-1 and FGF-8 (Bally-
Cuif and Wassef, 1995). Perhaps Ca
2+
plays a role in the
secretion of these signaling proteins or in the transduction of
the extracellular signal to the nucleus.
In order to directly explore the character and roles of free
Ca
2+
during vertebrate development, we have imaged Ca
2+
patterns in embryos of the zebrafish, Danio rerio, using the
luminescent Ca
2+
indicator aequorin. Aequorin-based Ca
2+
1613
Journal of Cell Science 111, 1613-1622 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS7254
Direct knowledge of Ca
2+
patterns in vertebrate
development is largely restricted to early stages, in which
they control fertilization, ooplasmic segregation and
cleavage. To explore new roles of Ca
2+
in vertebrate
development, we injected the Ca
2+
indicator aequorin into
zebrafish eggs and imaged Ca
2+
throughout the first day of
development. During early cleavages, a high Ca
2+
zone is
seen in the cleavage furrows. The high Ca
2+
zone during
first cleavage spreads as a slow wave (0.5 µm/second) and
is preceded by three Ca
2+
pulses within the animal pole
region of the egg. When Ca
2+
concentrations are clamped
at the resting level by BAPTA buffer injection into the
zygote, all signs of development are blocked. In later
development, Ca
2+
patterns are associated with cell
movements during gastrulation, with neural induction,
with brain regionalization, with formation of the somites
and neural keel, with otic placode formation, with muscle
movements and with formation of the heart. Particularly
remarkable is a sharp boundary between high Ca
2+
in the
presumptive forebrain and midbrain versus low Ca
2+
in the
presumptive hindbrain starting at 10 hours of
development. When Ca
2+
changes are damped by injection
of low concentrations of BAPTA, fish form with greatly
reduced eyes and hearts. The present study provides a first
overview of Ca
2+
patterns during prolonged periods of
vertebrate development and points to new roles of Ca
2+
in
cellular differentiation and pattern formation.
Key words: Calcium signaling, Imaging, Aequorin, Cell cycle,
Differentiation, Pattern formation, Central nervous system, Danio
rerio
SUMMARY
Patterns of free calcium in zebrafish embryos
Robbert Créton
1
, Johanna E. Speksnijder
2
and Lionel F. Jaffe
1
1
Marine Biological Laboratory, Woods Hole, MA 02543, USA
2
Department of Genetics, Biological Center, University of Groningen, 9751 NN Haren, The Netherlands
*Author for correspondence (e-mail: rcreton@mbl.edu)
Accepted 3 April; published on WWW 27 May 1998

1614
imaging is non-disturbing to the embryo allowing continuous
imaging during the first days of development. Although it is
possible to express apo-aequorin genetically (Rizzuto et al.,
1994; Brini et al., 1995), we chose to directly microinject
aequorin since this circumvents slow uptake of the aequorin
co-factor (Créton et al., 1997). We observed free Ca
2+
patterns
during normal development over 24 hours and thus throughout
the cleavage, blastula, gastrula and segmentation periods
(Kimmel et al., 1995).
MATERIALS AND METHODS
Zebrafish embryos
A small colony of zebrafish (100 fish in two aquaria) was kept on an
artificial 14 hour day/10 hour night cycle at 28.5°C. Fertilized eggs
were collected in a spawning cage 30 minutes after ‘dawn’. The
embryos were kept at 28.5°C in spring water containing 1 mg/l of
methylene blue during all measurements up to 24 hours. Afterwards,
the embryos were immobilized by adding 1% agarose to this same
medium.
Use of aequorin
Zygotes were injected with 2 nl of aequorin dissolved in a buffer
containing 100 mM KCl, 0.05 mM EDTA and 5 mM MOPS, pH 7.05,
using a high pressure system PLI-100 from the Medical Systems Co.,
Greenvale, NY 11548 (Miller et al., 1994). We injected either the
usual, near natural, recombinant, R-aequorin (at 0.57 mM) or a semi-
synthetic recombinant h-aequorin (at 0.24 mM); these were kindly
provided by Dr Osamu Shimomura. The more sensitive h-aequorin
was used to image the first 6 hours of development, whereas the longer
lasting R-aequorin was used to image Ca
2+
in later development. With
an egg volume of about 200 nl the final aequorin concentrations in the
embryo were 5.7 µM for R-aequorin and 2.4 µM for h-aequorin. The
microinjection needle was pushed through the vegetal regions of the
chorion and the plasma membrane to inject the aequorin into the
center of the uncleaved egg. However, when focusing on pattern along
the animal-vegetal axis, the needle was pushed through the egg’s
lateral region to avoid vegetal wound signals. The use of aequorins
for Ca
2+
imaging has been reviewed by Miller et al. (1994).
In the course of 25 experiments, we injected approximately 250
embryos with aequorin. From a batch of about ten injected embryos,
we typically selected one embryo for Ca
2+
imaging and one embryo
for Ca
2+
measurements with the photomultiplier tube. In selecting the
embryos, we looked for an embryo which had little or no cytoplasm
leaking from the injection wound. The injection wounds were visible
on the imaging photon detector as high Ca
2+
regions. Large wounds
obscured the Ca
2+
patterns and decreased embryonic survival. Small
wounds healed up quickly (within half an hour) and did not affect
embryonic development.
To map the Ca
2+
patterns during embryonic development, we have
measured a total of 25 embryos using the imaging photon detector
and 17 embryos using the photomultiplier tube. To calculate the
average luminescence and the standard error of the mean (s.e.m.) we
used those embryos, which developed normally, were imaged at a
specific embryological stage, had the same orientation (side or top-
view), and had the same type of aequorin injected (h- or R-aequorin).
Ca
2+
measurement and imaging
The total level of luminescence versus time was monitored with a
Hamamatsu model 464 photomultiplier tube or PMT. The PMT does
not provide spatial information, but does provide an overview of Ca
2+
levels throughout development. PMT values were calibrated to match
the output of our imaging system.
Our Ca
2+
imaging system is based on a Zeiss 100TV axiovert
microscope with a 20× fluar objective (NA=0.75). To minimize
instrumental noise and allow the recording of data over many hours
without overloading memory, we record the dim aequorin light with
a so-called Imaging Photon Detector or IPD made by Photek Inc., East
Sussex TN389NS, England (reviewed by Miller et al., 1994). This
instrument stores data in a computer as a list of photon events, each
recorded photon having two horizontal space coordinates and one time
coordinate. The two space coordinates identify an individual pixel in
the imaging field and with the 20× objective, each pixel corresponded
to a specimen area of 10 µm × 10 µm. The digital storage of photons
allows flexible reviewing of the Ca
2+
patterns. Thus images of light
emitted over any desired period can be created pointillist style. In
general, we first graphed the total luminescence emerging from an
embryo against time to detect any peaks in Ca
2+
. Then, subsequent
images of 10 minute photon exposures are analyzed to detect any Ca
2+
localizations. Whenever a Ca
2+
peak or Ca
2+
localization is found, the
data is analyzed in more detail, making graphs of specific regions
within the embryo and forming images using shorter exposure times.
Our imaging system collects data from a wide depth of field along the
z-axis (at least 200 µm with the 20× objective). To obtain three-
dimensional information on Ca
2+
patterning we routinely imaged
developing embryos in various orientations. This wide depth of field
also makes the system more sensitive for differences in tissue
thickness than for instance a confocal imaging system would. To
control for such differences we consistently compared embryonic
regions of equal thickness.
In order to determine Ca
2+
pulse frequencies, we needed to
distinguish genuine pulses from statistical fluctuations. To do this, we
compared the time course of luminescence from developing embryos
to that from BAPTA buffered droplets containing comparable levels
of free Ca
2+
and of aequorin. A Ca
2+
pulse was defined as an increase
in luminescence which was twice the resting level since such
fluctuations were never seen in the droplet dummies.
To control for differential distribution or consumption of aequorin,
we added 0.1 ml of 10% Triton to the water surrounding the embryo
at the end of a typical experiment. The Triton treatment causes a
massive increase in cellular Ca
2+
and ‘burns out’ the aequorin still
present. These burnouts indicated that the aequorin was still evenly
distributed in the embryos and that little decay of the aequorin had
occurred.
Calibration of the imaging system
The IPD system was calibrated using BAPTA buffers in which free
Ca
2+
was set at specific concentrations. The BAPTA buffers contained
either 5.7 µM R-aequorin or 2.4 µM h-aequorin in 100 mM KCl, 10
mM monopotassium Hepes, pH 7.0, 1 mM MgCl
2
, 5 mM
tetrapotassium BAPTA and either 1.0 mM, 1.6 mM, 2.4 mM or 3.2
mM CaCl
2
to set the free Ca
2+
concentration at 50, 100, 200 or 400
nM (Pethig et al., 1989). Immediately after adding aequorin, 0.2 µl of
the luminescent buffer was injected as a spherical droplet under
halocarbon oil. The luminescent spheres were imaged on the IPD,
using the 20× objective as if they were eggs.
Fig. 1 shows the result of this calibration. We measured 6.7 times
more light with h-aequorin at a 2.4 times lower concentration. This
16-fold ratio (6.7 × 2.4) in sensitivities of R and of h aequorins
corresponds well to the figure reported earlier by Shimomura et al.
(1993). The luminescence of both R- and h-aequorin rises with the
2.1 power of free Ca
2+
over the mid range needed to interpret spike
heights from developing embryos. This is the same power recently
reported by Shimomura and Inouye (1996).
Ca
2+
clamping and damping in the embryo
To clamp the Ca
2+
level in the embryo, we injected high
concentrations of a Ca
2+
buffer, in which the free Ca
2+
concentration
was set at pCa 7 (or 100 nM). The Ca
2+
buffer contained 250 mM
tetrapotassium BAPTA, 81 mM CaCl
2
and 10 mM Tris, pH 7.5. With
an injection volume of 1 nl, the final cytosolic concentration of
BAPTA was estimated to be about 2.5 mM (assuming that half of the
R. Créton, J. E. Speksnijder and L. F. Jaffe

1615Calcium patterns in zebrafish embryos
egg volume is non-cytosolic). As a control we injected 1 nl of a Tris-
buffered, pH 7.5, KCl solution, which was isotonic to the BAPTA
buffer. To dampen Ca
2+
changes in the embryo, we injected the same
BAPTA buffer at a five times lower concentration, thus giving a final
cytosolic BAPTA concentration of approximately 0.5 mM.
RESULTS
An overview of Ca
2+
signals during embryonic
development
Fig. 2 shows a representative record of total luminescence
versus time from a whole, normally developing embryo during
the first day and thus up to the end of the segmentation period.
It shows increasing levels of luminescence and hence of free
cytosolic Ca
2+
during three main periods: first, during each of
the first 5 relatively synchronous cleavages in the period
between 0.7 and 2.0 hours. Second, during specification of the
dorso-ventral axis and the beginning of gastrulation from about
4 to 7 hours. Third, from about 10 to 13 hours during early
segmentation. In addition to relatively slow changes in Ca
2+
,
Fig. 2 shows numerous Ca
2+
spikes which appear as vertical
lines on this time scale. All of these vertical lines represent
discrete Ca
2+
signals rather than noise. These brief signals
become larger and more frequent after about 10 hours when
segmentation starts.
The three periods of Ca
2+
elevation and the late Ca
2+
spikes
shown in Fig. 2 were similar in all of the embryos studied (n=9
for 0.5-2 hours, n=7 for 2-3 hours, n=6 for 3-22 hours). Thus
the relative changes in luminescence were essentially the same
in all embryos; however, the absolute levels of luminescence
covered a fourfold range in different embryos. This
considerable range in absolute luminescence was probably due
to variability in microinjection volume, in the amount of
aequorin burned up by Ca
2+
leakage during injection, as well
as variability in Ca
2+
levels from embryo to embryo. To
estimate the total amount of aequorin present in each of the
embryos, we burned out all of the available aequorin after 24
hours of development. These burnouts averaged 72 million
photons for R-aequorin (±36, n=4). By comparing these
burnouts with the total luminescence emitted during
development, we calculated that 71% of the injected aequorin
was still present after 24 hours of development (±9%, n=3).
This corresponds to an in vivo half-life of R-aequorin of 48
hours. The ultra sensitive h-aequorin is expected to decay 16×
faster (Shimomura et al., 1993), and was thus used for Ca
2+
imaging during early development only (0-6 hours).
Ca
2+
patterns during the cleavage period
Ca
2+
patterns during the cleavage period were imaged using
the ultra-sensitive h-aequorin. The earliest imageable
luminescence (at half an hour after fertilization) was uniformly
Fig. 1. Calibration of aequorin luminescence with known
concentrations of free Ca
2+
. The Ca
2+
concentration (Ca) can be
calculated from the luminescence (L) using the equations or the
curves in the graph. The equations are represented by dotted lines
and are only valid for the range in which they overlap with the
calibration curve. The exponential portions of both curves show
luminescence rising with the 2.1 power of free Ca
2+
.
Fig. 2. Representative time
course of luminescence from an
R-aequorin injected zebrafish
embryo during the first 22
hours of development. The
inferred average Ca
2+
level
cycles during the first five
cycles (0.75-2 hours) and is
elevated during mid-
gastrulation (6-8 hours) and
early segmentation (11-16
hours). Note the greatly
increased frequency of Ca
2+
spikes after 10 hours. The
emitted light was measured
with a photomultiplier tube.

1616 R. Créton, J. E. Speksnijder and L. F. Jaffe
Fig. 3. Representative Ca
2+
patterns during early cell division obtained by using the ultra-sensitive h-aequorin. (A-E) Side views in the median
focal plane. (F-J) Top views with focal plane 30% down from the animal pole. High Ca
2+
is seen at the sites of cytokinesis. The images (A-J)
are 30,000 photon exposures (~ 1 minute), in which the level of luminescence was color-coded, red representing high Ca
2+
, blue representing
low Ca
2+
. (K) Graph showing Ca
2+
levels during the early cleavage cycles. Cleavage signals can be observed up until the 10
th
cleavage cycle.
From 3.5-4.5 hours of development, small spikes are seen which clearly exceed the noise levels shown between cycles 8 and 9. (L) Three peaks
were observed during first cleavage initiation. The arrow indicates the time of furrow deepening. (M-Q) Subsequent 50 second exposures show
that the Ca
2+
elevation during furrow deepening spreads as a slow wave (0.5 µm/second) along the cleavage furrow. Bars, 200 µm.

Citations
More filters
Journal ArticleDOI
TL;DR: The universality of calcium as an intracellular messenger depends on its enormous versatility, which is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion.
Abstract: The universality of calcium as an intracellular messenger depends on its enormous versatility. Cells have a calcium signalling toolkit with many components that can be mixed and matched to create a wide range of spatial and temporal signals. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of calcium being highly toxic. Exceeding its normal spatial and temporal boundaries can result in cell death through both necrosis and apoptosis.

5,369 citations

Journal ArticleDOI
TL;DR: Findings fail to support the dichotomy in calcium signaling modes that had previously been proposed for protostomes vs deuterostomes and instead suggest that various features of fertilization-induced calcium signals are widely shared throughout the animal kingdom.

685 citations

Journal ArticleDOI
TL;DR: It is concluded on balance that the evidence points to a need for phosphoinositide/calcium signaling during resumption of meiosis, and it is concluded that calcium signals are essential at all three stages of cell division in early embryos.
Abstract: Fertilization calcium waves are introduced, and the evidence from which we can infer general mechanisms of these waves is presented. The two main classes of hypotheses put forward to explain the ge...

451 citations


Cites background or methods from "Patterns of free calcium in zebrafi..."

  • ...the presumptive ventral region was observed in only four of seven embryos; in two others, no difference was observed, and in the seventh, calcium was higher dorsally (103)....

    [...]

  • ...furrow deepening was very evident (103, 594)....

    [...]

  • ...Using a very sensitive aequorin that might be expected to reveal small changes in resting calcium as well as calcium transients, Jaffe and co-workers (103) found that average calcium levels in the zebrafish embryo rose after fertilization, dipped at midblastula, rose again to peak just before halfway through gastrulation, and finally rose substantially early in the segmentation stage....

    [...]

  • ...The use of aequorin together with imaging photon detectors has proved very fruitful in revealing calcium signals during zebrafish development (103, 172, 596)....

    [...]

  • ...single-cell stage, transforming into a stationary ring of elevated calcium that is coincident with a ring of contracting actin (103, 319, 320)....

    [...]

Journal ArticleDOI
TL;DR: The major findings from recent work in zebrafish have been elucidated, including the signaling pathways controlling induction of the Otic placode, morphogenesis and patterning of the otic vesicle, and elaboration of functional attributes of inner ear as mentioned in this paper.
Abstract: Recent years have seen a renaissance of investigation into the mechanisms of inner ear development. Genetic analysis of zebrafish has contributed significantly to this endeavour, with several dramatic advances reported over the past year or two. Here, we review the major findings from recent work in zebrafish. Several cellular and molecular mechanisms have been elucidated, including the signaling pathways controlling induction of the otic placode, morphogenesis and patterning of the otic vesicle, and elaboration of functional attributes of inner ear.

233 citations


Cites background from "Patterns of free calcium in zebrafi..."

  • ...The developing placode shows high levels of intracellular calcium at this time (Créton et al., 1998)....

    [...]

  • ...The developing placode shows high levels of intracellular calcium at this time (Créton et al., 1998)....

    [...]

Journal ArticleDOI
TL;DR: This review summarizes knowledge on the processes involved in the production and redistribution of maternal factors during zebrafish oogenesis and early development, as well as the understanding of the function of these factors in axis formation, germ layer and germ cell specification, and other early embryonic processes.
Abstract: All processes that occur before the activation of the zygotic genome at the midblastula transition are driven by maternal products, which are produced during oogenesis and stored in the mature oocyte. Upon egg activation and fertilization, these maternal factors initiate developmental cascades that carry out the embryonic developmental program. Even after the initiation of zygotic gene expression, perduring maternal products continue performing essential functions, either together with other maternal factors or through interactions with newly expressed zygotic products. Advances in zebrafish research have placed this organism in a unique position to contribute to a detailed understanding of the role of maternal factors in early vertebrate development. This review summarizes our knowledge on the processes involved in the production and redistribution of maternal factors during zebrafish oogenesis and early development, as well as our understanding of the function of these factors in axis formation, germ layer and germ cell specification, and other early embryonic processes.

230 citations


Cites background from "Patterns of free calcium in zebrafi..."

  • ...For example, their furrows during cytokinesis are associated with slow waves of intracellular calcium (Chang and Meng, 1995; Webb et al., 1997; Créton et al., 1998)....

    [...]

  • ...For example, their furrows during cytokinesis are associated with slow waves of intracellular calcium (Chang and Meng, 1995; Webb et al., 1997; Créton et al., 1998)....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: A series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio is described, providing for flexibility and continued evolution of the staging series as the authors learn more about development in this species.
Abstract: We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.

10,612 citations

Journal ArticleDOI
14 Apr 1995-Science
TL;DR: In this article, the authors have shown that, depending on the route of entry into a neuron, calcium differentially affects processes that are central to the development and plasticity of the nervous system, including activitydependent cell survival, modulation of synaptic strength, and calcium mediated cell death.
Abstract: Neuronal activity can lead to marked increases in the concentration of cytosolic calcium, which then functions as a second messenger that mediates a wide range of cellular responses. Calcium binds to calmodulin and stimulates the activity of a variety of enzymes, including calcium-calmodulin kinases and calcium-sensitive adenylate cyclases. These enzymes transduce the calcium signal and effect short-term biological responses, such as the modification of synaptic proteins and long-lasting neuronal responses that require changes in gene expression. Recent studies of calcium signal-transduction mechanisms have revealed that, depending on the route of entry into a neuron, calcium differentially affects processes that are central to the development and plasticity of the nervous system, including activity-dependent cell survival, modulation of synaptic strength, and calcium-mediated cell death.

1,401 citations

Journal ArticleDOI
16 Jan 1997-Nature
TL;DR: It is shown that gene expression is differentially controlled by nuclear and cytoplasmic calcium signals which enable a single second messenger to generate diverse transcriptional responses.
Abstract: Calcium entry into neuronal cells through voltage or ligand-gated ion channels triggers neuronal activity-dependent gene expression critical for adaptive changes in the nervous system. Cytoplasmic calcium transients are often accompanied by an increase in the concentration of nuclear calcium, but the functional significance of such spatially distinct calcium signals is unknown. Here we show that gene expression is differentially controlled by nuclear and cytoplasmic calcium signals which enable a single second messenger to generate diverse transcriptional responses. We used nuclear microinjection of a non-diffusible calcium chelator to block increases in nuclear, but not cytoplasmic, calcium concentrations following activation of L-type voltage-gated calcium channels. We showed that increases in nuclear calcium concentration control calcium-activated gene expression mediated by the cyclic-AMP-response element (CRE), and demonstrated that the CRE-binding protein CREB can function as a nuclear calcium-responsive transcription factor. A second signalling pathway, activating transcription through the serum-response element (SRE), is triggered by a rise in cytoplasmic calcium and does not require an increase in nuclear calcium.

745 citations


"Patterns of free calcium in zebrafi..." refers background in this paper

  • ...…imagine at least two roles Ca2+ in brain regionalization: (1) Ca2+ may activate transcription, as it does in other neural systems (Ghosh Greenberg, 1995; Ginty, 1997; Hardingham et al., 1997). thus seems possible that the rostral high Ca2+ zone induces the transcription of rostral-specific genes....

    [...]

Journal ArticleDOI
TL;DR: Evidence that vas RNA is a germ-cell-specific marker is presented, allowing a description of the zebrafish PGCs for the first time and providing the basis for further studies on this novel RNA localization pattern and on germ-line development in general.
Abstract: Identification and manipulation of the germ line are important to the study of model organisms. Although zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, we cloned the zebrafish homologue of the Drosophila vasa gene, which, in the fly, encodes a germ-cell-specific protein. Northern blotting revealed that zebrafish vasa homologue (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, we investigated the expression pattern of vas RNA in zebrafish embryos from the 1-cell stage to 10 days of development. Here we present evidence that vas RNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. Furthermore, vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. These results suggest new ways in which one might develop techniques for the genetic manipulation of zebrafish. Furthermore, they provide the basis for further studies on this novel RNA localization pattern and on germ-line development in general.

588 citations


"Patterns of free calcium in zebrafi..." refers background in this paper

  • ...Interestingly, a germ-cell-specific mark (vas RNA) localizes to the cleavage plane in 2- and 4-cell sta zebrafish embryos (Yoon et al., 1997)....

    [...]

Journal ArticleDOI
TL;DR: The goal here is to set out the types of unitary decisions made by heart progenitor cells, from their appearance in the heart field until they form the simple heart tube, which provides a context to evaluate cell fate, lineage and, finally, morphogenetic decisions that configure global heart form and function.
Abstract: Our goal here is to set out the types of unitary decisions made by heart progenitor cells, from their appearance in the heart field until they form the simple heart tube. This provides a context to evaluate cell fate, lineage and, finally, morphogenetic decisions that configure global heart form and function. Some paradigms for cellular differentiation and for pattern generation may be borrowed from invertebrates, but neither Drosophila nor Caenorhabditis elegans suffice to unravel higher order decisions. Genetic analyses in mouse and zebrafish may provide one entrance to these pathways.

494 citations


"Patterns of free calcium in zebrafi..." refers background in this paper

  • ...…nt. r lar in on ins 5; and nd er ic ion e ced wt 27 eart t 22 of these Ca2+ signals shows a closer correlation with separatio of the heart chambers (24-30 hours) or with looping of th heart, which occurs at between 30 and 36 hours of developm (Stainier et al., 1993; Fishman and Chien, 1997)....

    [...]

Frequently Asked Questions (2)
Q1. What are the contributions mentioned in the paper "Patterns of free calcium in zebrafish embryos" ?

The role of free Ca2+ signals in early vertebrate development has been investigated in this paper, where aequorin-based CA2+imaging is used to directly microinject apo-aequorin into the zebrafish embryo. 

Confocal Ca2+ imaging has already resulted in detailed knowledge of neural activity in posthatching zebrafish embryos ( O ’ Malley et al., 1996 ; Fetcho et al., 1997 ) and seems a promising approach to study specific Ca2+ signals in zebrafish development with greater spatiotemporal detail. Moreover, the large number of mutant zebrafish that has become available, will provide a unique opportunity to study how specific genes affect the Ca2+ patterns.