Use of transgenic zebrafish reporter lines to study calcium signalling in development

Rachel Ashworth

is a BBSRC research fellow in

the Department of Physiology,

University College London,

London.

Caroline Brennan

is group leader in the

Department of Cell Signalling

and Development, School of

Biological Sciences, Queen

Mary University of London,

London.

Keywords: transgeniczebrafish, calcium signalling,bioluminescence, fluorescenceimaging, embryogenesis

Caroline Brennan,

School of Biological Sciences,

Queen Mary College,

University of London,

London, E1 4NS, UK

Tel: +44 (0)20 7882 3011

E-mail: [email protected]

Technique review

Use of transgenic zebrafishreporter lines to study calciumsignalling in developmentRachel Ashworth and Caroline BrennanDate received: 5th May 2005

AbstractCalcium signals are associated with many of the events common to animal development.

Understanding the role of these calcium signals requires the ability to visualise and manipulate

calcium levels in the developing embryo. Recent work has led to the development of sensitive

protein-based probes that can be used to generate transgenic animals for the analysis of

calcium signalling in vivo. This paper focuses on the use of genetically encoded calcium probes

to follow calcium signals in zebrafish. It reviews progress and speculates on the potential for

use in the future.

INTRODUCTIONCalcium signalling is critical for many of

the cell processes essential for embryo

development, including regulation of gene

expression, secretion and cell movement.

Calcium signals have been demonstrated

in single embryonic cells, explanted tissue

and whole animals, with proposed

developmental roles ranging from

initiation of fertilisation through

embryonic patterning and organogenesis.

Temporal and spatial properties are central

to the signalling role of calcium.1 Thus,

the study of this messenger during

development requires the ability to

visualise and manipulate calcium dynamics

in living cells or embryos. The transparent

nature of the zebrafish embryo, coupled

with its amenability to genetic

manipulation and the recent development

of genetically encoded calcium reporters,

makes the transgenic zebrafish a powerful

model system for such studies.

GENETICALLY ENCODEDPROBES AS A TOOL FORFOLLOWING CALCIUMDYNAMICS IN VIVOThere are two types of calcium reporters:

synthetic indicators and protein-based

probes, both of which have been used to

good effect to study calcium signals in vivo

during zebrafish embryogenesis.2,3 The

high temporal–spatial resolution of

fluorescent dyes enables analysis of rapid

localised events; however, it is difficult to

target dyes to specific individual cells or

populations of cells in vivo. This problem

can be overcome by the use of genetically

encoded protein-based probes. Two types

of protein-based calcium reporters are

available: those based on fluorescence

resonance energy transfer (FRET) and

those based on bioluminescent proteins

such as aequorin.

Calcium probes based onfluorescent proteinsSeveral different genetically encoded

calcium indicators based on fluorescent

proteins (such as green fluorescent protein

[GFP] and its derivatives) have been

described.4,5 One characteristic of the

currently available fluorescent probes is

their design around the calcium-binding

protein calmodulin (although others have

been described just recently). Binding of

calcium to calmodulin induces a

conformational change, resulting in

FRET to associated GFP molecules. The

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cameleon proteins (eg YC2.1) (Figure 1)

were among the first genetically encoded

calcium indicators to be developed.6,7

Recently, the generation of transgenic

zebrafish lines expressing neuronally

targeted cameleon (YC2.1) has been

described. The HuC:YC2.1 line has been

used to study the activity of neurones in

intact larvae8 and offers the possibility of

recording calcium signals from identified

neurones in the embryo. Previously,

calcium signals have been recorded from

cells within the developing spinal cord of

zebrafish using synthetic indicators (eg

BAPTA-based dyes);9 however, the

indicator was not targeted to specific cell

types. Expression of fluorescent protein in

spinal neurones can be observed in

HuC:YC2.1 embryos at early

developmental stages (Figure 2A) and it is

possible to track fluorescence changes in

neuronal cell bodies over time (Figure

2B). The optimisation of signal to noise

when collecting the fluorescence

emission, and the identification of

changes that reflect calcium signals, are

important considerations when using

protein-based indicators.10 Changes in

fluorescence from YC2.1-generated

signals were identified which: 1) deviated

more than 5 per cent from baseline; 2)

were detected as a decrease for cyan

fluorescent protein and an increase for

yellow fluorescent protein; and 3)

displayed a ratio change of greater than 10

per cent. The percentage of embryonic

cells displaying spontaneous calcium

signals measured using either synthetic

indicator or the protein-based probe at

identified developmental stages were

similar (Figure 2C), suggesting that the

genetically encoded calcium indicator

YC2.1 can detect spontaneous calcium

signals in developing neurones of the

embryo. There are, however, still

limitations to the technique: the

cameleon, with its slow time course for

time to peak (hundreds of milliseconds)

and decay (several seconds), is not suitable

for resolving the rapid temporal dynamics

of intracellular calcium transients. The

other major disadvantage of the cameleon

is the poor signal to noise fluorescence

emission levels when expressed in

organisms (Figure 2B), suggesting that the

protein may be modified in vivo.8

Potentially, the signal to noise levels could

be improved by higher expression, newer

probes that emit stronger signals and

better detection instruments (eg (Charge

Coupled Device) CCD, two-photon

confocal imaging).

Calcium probes based onluminescent proteinsUnlike fluorescence-based probes, those

based on bioluminescent proteins do not

require the input of radiative energy. This

is of considerable advantage when

studying processes over prolonged

periods, such as during development, as

Transgenic zebrafishexpressing cameleonproteins

Detecting calcium-mediated FRET

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Figure 1: Fluorescence resonance energy transfer (FRET) between thetwo fluorescent proteins in the cameleon protein used to detect calciumchanges. The YC2.1 cameleon consists of a cyan fluorescent protein(ECFP), calmodulin (CaM), calmodulin-binding peptide M13 and yellowfluorescent protein (EYFP). Binding of calcium induces a conformationalchange in the Cam which then binds to 26-residue calmodulin-bindingpeptide of myosin light-chain kinase (M13). Adapted from the papers byGriesbeck5 and Miyawaki et al.5,6 On binding, calcium emission at awavelength of 440–480 nm will decrease and emission at a wavelength of510–530 nm will increase.

& HENRY STEWART PUBLICATIONS 1473-9550. BRIEF INGS IN FUNCTIONAL GENOMICS AND PROTEOMICS . VOL 4. NO 2. 186–193. JULY 2005 1 8 7

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Figure 2: Measuring calcium signals in nerve cells in zebrafish embryos using the HuC:YC2.1 stable transgenic line. (A)Fluorescence image and bright field superimposed to show YC2.1-expressing neurones within the spinal cord of a 20 hourspost fertilisation hpf zebrafish embryo. (B) Single optical images were taken every 3.5 seconds over a 20-minute period(100-second period shown here). Average fluorescence intensity within a defined area corresponding to the cell body wasplotted against time for each wavelength. Fluorescence changes were normalised to baseline and * denotes changes influorescence emission with greater than 5 per cent change in the two dyes. CFP, cyan fluorescent protein; YFP, yellowfluorescent protein. (C) Calcium transients in cells within the embryo were measured using either Oregon Green BAPTAdextran (black bars) or YC2.1 (grey bars). Developmental stage was determined by the somite number and converted tostandard developmental time (hpf) at 28.58C. The number of cells displaying calcium transients at each time point wasdisplayed as the total cells (�SEM, as calculated from the binomial distribution). Images were taken using a laser scanningconfocal microscope (LSM 510 meta), using a 458 nm excitation wavelength and emission collected at 482–514 nm and535–567 nm.


Rachel Ashworth and Caroline Brennan



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problems with photobleaching,

phototoxicity and autofluorescence are

avoided. Aequorin is the primary

bioluminescent protein used. Pioneering

work by the groups of A. Miller11 and

L. Jaffe12 have used aequorin to reveal

orchestrated waves of calcium signals

associated with significant patterning

events in zebrafish.2 Although providing a

good signal to noise ratio, however,

aequorin signals are very hard to detect, as

aequorin has a very low quantum yield.

This limits the temporal and spatial

resolution possible. To address this

problem, two groups13–15 have generated

chimeric GFP-aequorins that allow

efficient chemiluminescent resonance

energy transfer (CRET) (Figure 3) from

activated aequorin to GFP to increase the

quantum yield approximately 20-fold.13,14

In mammalian cells in culture, and in

retinal explants, such GFP-aequorin based

probes have been able to detect calcium

signals in targeted subcellular domains.14

Genetically encoded calcium probes

can be introduced into zebrafish embryos

by injection of mRNA or DNA into the

fertilised oocyte. Injection of RNA leads

to widespread transient expression

(Figure 4), whereas injection of DNA

allows for cell type-specific expression

and the generation of stable transgenic

lines (see below). When mRNA

encoding GFP-aequorin is injected into

zebrafish zygotes, fluorescent cells and

bioluminescence can be detected

throughout the embryo (Figure 4A).

Localised calcium signals can be detected

from as early as 50 per cent epiboly (not

shown). This is later than those

previously reported with injection of

reconstituted apoaequorin,11,16

presumably reflecting the time necessary

for protein synthesis and reconstitution

in vivo. At later stages of development,

tissue-specific luminescence can also be

detected (Figure 4B). When embryos are

injected with DNA to generate

transiently expressing cells,

bioluminescence can be detected in

individual cells distributed randomly

throughout the embryo (Figure 4C and

Figure 5). This is a significant advance in

spatial resolution over that possible with

injected aequorin. These cells produce

calcium signals with different on–off

characteristics as the embryo develops

(Figure 5). Despite the enhanced

emission intensity afforded by the

development of new chimeric GFP-

aequorin proteins, however, it is still

difficult to resolve the temporal and

spatial characteristics of inter- and

intracellular calcium signals in vivo. Thus,

here, the detailed kinetics of endogenous

calcium signals could only be resolved

using a photomultiplier tube (Figure 5C

and D). The targeting of the reporter

protein to subcellular domains,14 and the

Enhancing aequorinbioluminescence

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Figure 3: Chemiluminescent resonance energy transfer (CRET)between activated aequorin and green fluorescent protein (GFP). Activeprotein (apoaequorin) is formed from aequorin and its luciferin,coelenterazine, in the presence of molecular oxygen. The binding ofcalcium to apoaequorin results in a conformational change that leads tothe oxidation and excitation of coelenterazine, with a resultant emissionof blue light (ºmax 470 nm) when the coelenterazine returns to its groundstate.16 The fusion of the aequorin to GFP allows CRET between theactivated aequorin and GFP. Modified from Rudolf et al.4





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use of modified coelenterazine f or

coelenterazine fcp — which have

increased luminescent intensities

compared with native coelenterazine (18-

and 135-fold, respectively)17 — may help

to overcome this difficulty. The difficulty

in detecting GFP-aequorin signals may

reflect difficulty in generating active

apoaequorin in vivo (see ref. 26 for

discussion). Although coelenterazine is

membrane permeable, at later stages

zebrafish embryos have a relatively

impermeable outer layer that restricts

drug access. Thus incubation of embryos

in relatively high concentrations of

coelenterazine from very early stages of

development is necessary.

GENERATION OFTRANSGENIC ZEBRAFISHA number of techniques for generating

transgenic zebrafish have been

reported.18–24 All involve the injection of

picogram amounts of DNA into the

newly fertilised oocyte. The highest

frequency for obtaining transgenic

founder fish (50 per cent) was achieved

using the newly developed Tol2

transposon system.23,24 Here, mRNA

encoding the Tol2 transposase is co-

injected with a plasmid DNA containing

the promoter–reporter construct and a

mutant Tol2 sequence that lacks the

transposase activity but retains cis elements

necessary for insertion.23,24 In the

presence of active transposase, protein

generated from the co-injected mRNA

the Tol2 element promotes insertion of

the plasmid DNA into the genome. The

frequency of obtaining founder fish with

this methodology (50 per cent) is

considerably higher than the frequencies

achieved by other transgenic methods:

frequencies of between 5 per cent

(injection of naked plasmid DNA 18,19)

and 30 per cent (other available

transposon systems) have been

reported.20–22 The development of these

new transposon systems makes the

generation of transgenic zebrafish an

attractive proposition for even the smallest

of laboratories.

Figure 4: Spatially distributed green fluorescent protein (GFP)-aequorinbioluminescence in zebrafish embryos. Embryos were injected with 100pg RNA (A, A9; B, B9) or 50 pg DNA (C, C9) encoding GFP-aequorin atthe one-cell stage. Embryos were allowed to develop until the mid-gastrula stage, at which time they were hand-dechorionated andincubated in 50 �M coelenterazine for two hours prior to imaging orovernight. GFP-fluorescence (º 510 nm) was used to localise theexpressing cells (A, B, C) and bioluminescence was detected using anintensified CCD camera (ICCD, Photek Ltd, East Sussex, UK) connectedto the baseport of an inverted microscope which assigns an x,ycoordinate for each detected photon.25 The ICCD camera produces verylow background counts (approximately 5 photons/second in a 256 3 256pixel region). The microscope is housed in a light-tight dark box, with ahalogen lamp mounted outside the dark box, connected to themicroscope by fibre optic guides. The luminescence images shown in A9and B9 represent 120-second integrations. The image shown in C9 is anoverlay of the two 240-second integrations inset in C9. The scalerepresents luminescence in photons/pixel.





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Figure 5: Detecting green fluorescent protein (GFP)-aequorin bioluminescence in zebrafish. Overnight recordings ofbioluminescence from a representative embryo that had been injected with 50 pg of an expression plasmid-encoding GFP-aequorin under the control of the i.e.CMV (immediate early cytomegol-ovirus) promoter (A, C, D) and from an uninjectedcontrol embryo (B). Embryos were injected at the 16-cell stage and allowed to develop until mid-gastrulation, when theywere analysed for expression of GFP-aequorin using fluorescence microscopy. Individual embryos that showed clearexpression in a number of cells were then incubated in 5 �M coelenterazine for one hour and subjected to luminescencerecordings. Luminescence was recorded from the end of gastrulation until the 28-somite stage in a photomultiplier tube inphoton-counting mode. (A) Overnight recording of luminescence from a representative injected embryo expressing GFP-aequorin in a number of cells distributed randomly throughout the embryo (shown in E). (B) Control recording from anuninjected embryo. (C, D) Details of the regions of the overnight trace, as indicated. In A and B, marked intervals representone hour. In C and D, marked intervals represent one minute. Vertical axes show luminescence in photons per second.Note that the scale of the vertical axis alters in C and D. (E). Localisation of the GFP-aequorin-expressing cells followingrecording of the bioluminescence (shown in panels A, C and D). The left-hand panel shows a low-power image showingGFP fluorescence in the tail region and over the yolk extension. The right-hand panel shows a higher magnification of thesame embryos, showing GFP fluorescence within a number of muscle fibres.

& HENRY STEWART PUBLICATIONS 1473-9550. BRIEF INGS IN FUNCTIONAL GENOMICS AND PROTEOMICS . VOL 4. NO 2. 186–193. JULY 2005 19 1




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SUMMARYThe publication of the first transgenic

zebrafish lines expressing neuronally

targeted, genetically encoded calcium

sensors has proved to be an important

step. The recent development of

enhanced bioluminescent reporter

proteins also bodes well for the future.

Furthermore, a large and ever-increasing

number of cell-specific promoters for the

generation of reporter lines in zebrafish

are available, facilitating the use of

genetically encoded probes to study

specific aspects of calcium signalling

during development. The creation of

stable transgenic zebrafish lines containing

calcium reporters is already underway

and, while this technology is still in its

infancy, it promises to be an important

tool for the study of calcium signalling

and cell function during development.

Acknowledgments

R. A. is funded by the BBSRC David Phillips

Fellowship and Wellcome Trust. C. B. is funded

by the BBSRC, the Royal Society and the Central

Research Fund, University of London. The

authors would like to thank Joseph R. Fetcho for

supplying the HuC:YC2.1 line and for comments

on the work. They also thank Karl Swann, at

Cardiff University, for help with the photon

counting studies, and William Hinkes, in the

imaging suite, UCL, for his technical support.

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