Download technicolour transgenics: imaging tools for functional

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TECHNICOLOUR TRANSGENICS:
IMAGING TOOLS FOR FUNCTIONAL
GENOMICS IN THE MOUSE
Anna-Katerina Hadjantonakis*, Mary E. Dickinson‡, Scott E. Fraser‡
and Virginia E. Papaioannou*
Over the past decade, a battery of powerful tools that encompass forward and reverse genetic
approaches have been developed to dissect the molecular and cellular processes that regulate
development and disease. The advent of genetically-encoded fluorescent proteins that are
expressed in wild type and mutant mice, together with advances in imaging technology, make it
possible to study these biological processes in many dimensions. Importantly, these technologies
allow direct visual access to complex events as they happen in their native environment, which
provides greater insights into mammalian biology than ever before.
THERMOLABILE
Unstable at moderate or
increased temperatures.
CHIMAERA
An animal that is generated
from, and comprises, several
genetically distinct populations
of cells that are derived from
more than one individual.
*Department of Genetics
and Development, College of
Physicians and Surgeons of
Columbia University,
New York 10032, USA.
‡
Beckman Institute,
California Institute of
Technology, Pasadena,
California 91125, USA.
Correspondence to A.-K.H.
e-mail:
[email protected]
doi:10.1038/nrg1126
NATURE REVIEWS | GENETICS
The mouse is the premier mammalian model organism1
for studying development and disease. The mouse
genome sequence is now available and the mouse
germline can be manipulated in almost every way imaginable, in both a random2–7 and a directed fashion8–11, to
study the phenotypic consequences of perturbations. As
techniques for manipulating the genome have multiplied,
so have the tools for the study of complex phenotypes.
Recently, there has been a surge of interest in using
fluorescence microscopy for phenotypic analysis (BOX 1).
This is attributable to three converging factors. First,
genetically-encoded fluorescent proteins have been
developed that provide high-contrast multicolour vital
markers for monitoring gene activity and protein function. Second, improvements in fluorescence-based imaging technology have allowed deeper imaging and better
spectral separation in live tissues. Third, in vitro culture
systems have been developed that allow the normal
growth of mammalian embryos and explanted tissues,
which is a requirement for vital-imaging experiments.
These developments allow direct connections to be
made between genetic lesions and their cellular consequences, and so pave the way for high-resolution highthroughput functional genomics. Indeed, these features
are already being exploited by many laboratories in
studies of dynamic biological events in several model
organisms, including Drosophila12,13 and zebrafish14–17
(see links in online links box). Having overcome some of
its original challenges, such as THERMOLABILITY and quantum yield of wtGFP at 37°C, which made it unsuitable for
use in mammalian systems, the mouse is fast catching up
with these organisms. In this review, we outline the use of
fluorescent proteins in mice and, to illustrate their power,
we describe some examples of the use of transgenic
mouse lines in investigating mammalian development
and neurobiology.
Development of GFP-based reporters
For almost two decades, lacZ, which is the bacterial
gene that encodes β-galactosidase, has been the marker
of choice for the generation of reporter-expressing
mouse strains18,19, as it is easy to detect and has a high
cellular resolution. Although advantageous for marking
distinct populations of cells in CHIMAERAS or reporting
the activity of specific promoters, lacZ cannot be used
to mark cells in living tissues, because staining for lacZ
requires tissue fixation.
In 1994, green fluorescent protein (GFP), which is
derived from the bioluminescent jellyfish Aequorea
victoria20, was first introduced into Caenorhabditis
elegans as a genetically stable fluorescent marker21 that
could be detected in living tissues. The potential uses
VOLUME 4 | AUGUST 2003 | 6 1 3
REVIEWS
Box 1 | Imaging requirements
The advent of genetically-encoded fluorescent reporters has a Transgenic mouse model
prompted researchers to find new ways to either get their
Genetically-encoded
fluorescent protein
mice under the microscope or bring the microscope to their
mice. Non-invasive time-lapse imaging of vital markers in
mouse embryos, adults and organs in a physiological
environment requires the availability of a transgenic mouse
model that expresses one or more gene-based vital molecular
markers (a), a microscope equipped with an excitation
source and detection system (b), and a computer equipped
with data acquisition and processing software (c).
Image-processing algorithms can then be applied to
extract quantitative data and generate multidimensional
reconstructions.
b Microscope with in vitro culture and
c Image workstation
imaging capabilities
Data acquisition and processing
Maintain specimen under physiological
conditions and acquire data
Heater box
Gas
inlet
Culture
chamber
Humidifier
EMBRYONIC-STEM-CELLMEDIATED TRANSGENESIS
A method in which DNA is
introduced into embryonic stem
(ES) cells and integrates
randomly, or through gene
targeting, into the genome.
Transgenic ES cells are delivered
to the germline through the
generation of (ES cell↔embryo)
chimaeras.
FATE MAP
A spatial map of the fates of
different embryonic cells at a
particular stage of development.
CHORDONEURAL HINGE
A region at the posterior end of a
vertebrate embryo that gives rise
to both neural and mesodermal
cells.
614
of GFP in mice were immediately obvious but issues of
thermolability, low fluorescence and insolubility of fluorescent proteins hampered their initial application.
These problems were overcome through mutagenesis of
GFP, and the first strains of transgenic mice that
expressed readily detectable GFP variants were reported
approximately five years ago22,23. Fluorescent proteins
were also mutated to give reporters with different spectral properties, so in vivo multicolour labelling experiments are now possible if different colour variants are
expressed in the same individual.
Today, several, but not all, genetically-encoded autofluorescent reporters can be used successfully in mice
(BOX 2), and hundreds of different strains have been
generated that carry GFP or its spectral variants.
Fluorescent proteins not only make it possible to mark
specific cells in living organisms, but these cells can be
followed using time-lapse fluorescence-imaging techniques. So, it is now possible to describe normal events
and understand aberrant cellular behaviour in mutant
lines by watching the cells in question.
The versatility of genetic techniques in the mouse
offers several approaches for introducing fluorescent-protein reporters into the germline. Fluorescent reporters can
also be incorporated in mutagenesis regimes24,25, which
paves the way for higher-throughput and increased-resolution functional genomics in the mouse. Mouse strains
| AUGUST 2003 | VOLUME 4
can be engineered to show fluorescence in a constitutive26,27, conditional28,29 or lineage-restricted fashion. This
affords tremendous creativity in experimental design, as
illustrated in the following examples.
Constitutive reporters
The first strains of transgenic mice expressed fluorescent proteins constitutively. They were generated
either by traditional zygote-injection-based transgenic approaches22 or EMBRYONIC STEM (ES) CELL-MEDIATED
23
TRANSGENESIS , so the fluorescent reporters were randomly integrated into the genome. These experiments
were crucial in showing the developmental neutrality of
GFP-variant reporters. Mice that expressed fluorescent
proteins showed widespread fluorescence and were
viable, fertile and morphologically indistinguishable
from their wild type (non-transgenic) littermates.
Mice that constitutively express fluorescent proteins are a source of genetically-marked cells that can
be explanted, recombined, transplanted and used to
generate chimaeras. Cells from such mice have
recently been used to refine FATE MAPS of gastrula-stage
embryos30,31 and to investigate the presence and developmental potential of cells in the CHORDONEURAL-HINGE
region of the developing tail bud in embryos32.
Also, constitutively expressed fluorescent proteins
can be used to mark cells for the isolation of stem cells,
www.nature.com/reviews/genetics
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TROPHOBLAST
An extraembryonic lineage that
is derived from the
trophectoderm of the blastocyst,
which gives rise to the fetal
portion of the placenta.
which is now an area of active research. Fluorescentlytagged cells can be easily identified when introduced
into progeny and separated using flow cytometry33.
One of the first examples of the use of fluorescent stem
cells was the evaluation of putative TROPHOBLAST stem
cells34. Since then, fluorescent-protein reporters have
proved to be an indispensable reagent in stem-cell
research and have been used to mark several types of
35
PLURIPOTENT cell, including HAEMATOPOIETIC stem cells
and neural stem cells36–38.
Fluorescent mosaics. Genetic MOSAICS, such as mouse chimaeras, provide a powerful way to study development
and disease processes in multicellular organisms39.
Box 2 | Genetically-encoded fluorescent proteins used in mice
NATURE REVIEWS | GENETICS
Normalized emission
Normalized excitation
Here we describe the most
a
b
ECFP
EGFP EYFP DsRed HcRed
ECFP EGFP EYFP DsRed HcRed
popular autofluorescent
1.0
1.0
proteins, their origin, evolution
and prominent features.
0.8
0.8
Green fluorescent protein
0.6
0.6
(GFP) and other fluorescent
proteins that are cloned from
0.4
0.4
COELENTERATES share a similar
structure, which comprises a
0.2
0.2
β-barrel-like structure
enclosing a cyclic tripeptide
0
0
375 400 425 450 475 500 525 550 575 600
450 475 500 525 550 575 600 625 650 675 700
(Ser65-Tyr66-Gly67)
109,110
Wavelength (nm)
Wavelength (nm)
FLUOROPHORE
. Mutagenesis
has been carried out to increase
Colour
Name
Origin
Features
References
the THERMOSTABILITY and
maturation kinetics of these
GFP and mutagenized variants
fluorochromes and to create
Green
wtGFP
Aequorea victoria
Original (wild type) GFP
20,21
different spectral variants
EGFP
Phe64Leu/Ser65Thr
GFP variant with increased
55
(as described in the table).
thermostability and fluorescence
For example, enhanced green
Blue
ECFP
Phe64Leu/Ser65Thr/
Cyan (blue-shifted) GFP
120
fluorescent protein (EGFP),
Tyr66Trp/Asn146Ile/
spectral variant
which is, at present, the most
Met153Thr/Val163Ala
popular variant for use in
Yellow
EYFP
Ser65Gly/Val68Leu/
Yellow/green (red-shifted) GFP
109
mice55, carries mutations that
Gln69Lys/Ser72Ala/
spectral variant
have increased the QUANTUM
Thr203Tyr
YIELD and thermostability of
Yellow
Venus
Phe49Leu/Phe64Leu/
Reaches maturity faster and has
54
GFP. Although mutations in
Ser65Gly/Val68Leu/
increased fluorescence compared
Gln69Lys/Ser72Ala/
with EYFP
the vicinity of the fluorophore
Met153Thr/Val163Ala/
have resulted in variants with
Ser175Gly/Thr203Tyr
distinct spectral properties
Yellow
Citrine
Ser65Gly/Val68Leu/
Improved photostability and sensitivity
121
(see table and figure), there is
Gln69Met/Ser72Ala/
to pH changes compared with EYFP
a continuing search for new
Thr203Tyr
sources of fluorescent proteins,
DsRed1 and mutagenized variants
especially those that extend
Red
DsRed1
Discosoma sp.
Original red fluorescent protein
114
into the red part of the visible
Red
DsRed2
Arg2Ala/Lys5Glu/
Reaches maturity faster
115
spectrum111–113.
Lys9Thr/Ala105Val/
than DsRed1
DsRed1 from the sea
Ile161Thr/Ser187Ala
anemone Discosoma sp. was
Red
DsRed-Express/
Arg2Ala/Lys5Glu/Asn6Asp/
Reaches maturity faster than
122
the first commercially
DsRed-T1
Thr21Ser/His41Thr/Asn42Gln/
DsRed variant
available red-fluorescentVal44Ala/Cys117Ser/
protein reporter114,115.
Thr217Ala
Unfortunately, it has several
Red
mRFP1
33
mutations introduced:
Fast maturing, monomeric red
117
limitations113,116. For example,
13 internal to the β-barrel
fluorescent protein
it functions as an obligate
and 20 external to the β-barrel
tetramer and matures slowly,
HcRed
which limits its use for realFar red
HcRed
Heteractis crispa
Far-red fluorescent protein
119
time gene-expression studies.
ECFP,
enhanced
cyan
fluorescent
protein;
EYFP,
enhanced
yellow
fluorescent
protein;
mRFP1,
monomeric
red
fluorescent
protein
1.
Although strains of mice that
express DsRed1 in neurons
have been generated71, attempts to produce lines of transgenic EMBRYONIC STEM (ES) CELLS and mice with constitutive expression of DsRed1, or its
derivative DsRed2, have failed42. Several laboratories have directed their efforts towards generating an improved red fluorescent reporter116–118.
There is a continuing search for new sources of spectrally-distinct fluorescent proteins111–113,118, and one such reporter that has recently become
commercially available is HcRed119. Emission spectra reproduced with permission from Clontech (BD Biosciences) Catalogue (2002).
VOLUME 4 | AUGUST 2003 | 6 1 5
REVIEWS
a
d
b
e
c
f
PLURIPOTENT
Able to give rise to a wide range
of, but not all, cell lineages
(usually all fetal lineages and a
subset of extraembryonic
lineages).
HAEMATOPOIETIC
Giving rise to the cellular
elements of the blood, such as
the white blood cells, red blood
cells and platelets.
MOSAIC
An organism that consists of
cells of more than one genotype.
The strict definition requires
that the genotypically different
cells all derive from a single
zygote. The term mosaic is also
used more broadly to describe
any organism comprised of cells
of different genotypes.
COELENTERATE
Radially symmetrical
invertebrates, which include
corals, sea anemones and
jellyfish.
g
FLUOROPHORE
The core portion of a molecule
that is directly responsible for
absorbing photons.
THERMOSTABLE
Able to withstand moderate heat
without the loss of characteristic
properties, such as fluorescence.
QUANTUM YIELD
The probability of luminescence
occurring under given
conditions, which is expressed
as the ratio of the number of
photons emitted to the number
absorbed.
EMBRYONIC STEM CELLS
(ES cells). Stem cells have the
dual capacity to self-replicate
and differentiate into several
specialized derivatives. ES cells
are pluripotent cells that are
derived from pre-implantation
stage (usually blastocyst)
mammalian embryos. Mouse ES
cells can be propagated and
manipulated in vitro, yet still
retain their pluripotency.
HEMIZYGOTE
An animal with a transgene
insertion on one chromosome of
a homologous pair, rather than
on each of the two homologous
chromosomes (homozygote).
616
Figure 1 | Combinatorial fluorescent-protein-reporter
detection in live chimaeras and double transgenics.
Wide-field epifluorescence images illustrating dual-reporter
visualization in chimeric embryos (a–c) and adult organs (d–f).
Anterior view of an embryonic day (E) 7.5 double-tagged
chimaera, the epiblast (lower half of embryo) and its derivatives
are enhanced cyan fluorescent protein (ECFP)-positive,
whereas the extra-embryonic ectoderm, the visceral endoderm
and trophoblast are enhanced yellow fluorescent protein
(EYFP)-positive (upper half of embryo) (a–c). Dark-field images
were taken through an EYFP filter (a), an ECFP filter (b) and, for
the double-exposure image, consecutive use of ECFP and EYFP
filters (c). Non-invasive multiple-reporter visualization in chimeric
adult organs was generated by the aggregation of reportertagged diploid embryos and embryonic stem cells (d–f). All
images depict double exposures that were produced by the
consecutive use of ECFP and EYFP filters under dark-field
epifluorescence. Unlike in the heart (d) and pancreas (e),
there seems to be greater interspersion of cells in the liver (f),
which possibly reflects greater cell intermingling during the
development of this organ. The laser-scanning confocal image
(g) shows neurons in the brain of a Thy1-double-transgenic
mouse71 that has inherited different sets of fluorescently
expressing cells (ECFP versus EYFP) from each of its parents.
Panels a–d and f reproduced with permission from REF. 42 ©
(2002) Biomed Central.
| AUGUST 2003 | VOLUME 4
Because it is not uncommon for mouse mutant phenotypes to be complex, chimaeras that consist of both
wild type and mutant cells can be extremely helpful in
determining the site of action of a gene product or in
understanding the later consequences of a mutation
that also affects early development. However, a prerequisite for all chimeric experiments is the incorporation
of an independent genetic tag to distinguish mutant
and wild-type cells. In the past, markers such as a constitutively expressed lacZ reporter40 or high-copy-number
transgene integration41 have been used to mark one of
the cellular components in a chimaera, but these
could only be detected in fixed tissue. Fluorescentprotein-reporter technology now makes it possible to
identify both wild type and mutant components of a
chimaera in live embryos or tissues, using spectrally
distinct fluorescent proteins that mark each cell
type42 (FIG. 1).
Fluorescent tagging of cells in chimaeras has been
used in a number of studies, but none have fully exploited
the potential of dynamic cell imaging in vivo that is
offered by these vital reporters. Recently, a fluorescentlytagged chimeric approach has been used to investigate
the action of reduced levels of vascular endothelial
growth factor A (VEGFA), which has a pivotal role in
the first steps of the development of the yolk sac, as well
as in the establishment of the cardiovascular system.
Embryos that are homozygous for a lacZ-tagged hypomorphic allele of Vegfa die around mid-gestation owing
to abnormalities in yolk-sac vasculature and deficiencies
in the development of the dorsal aorta. An elegant set of
chimeric experiments showed that VEGFA expression
in the visceral endoderm is absolutely required for
the normal expansion and organization of both the
endothelial and haematopoietic lineages in the early
sites of vessel and blood formation43.
Non-invasive sexing. In one of the first strains of
transgenic mice to show constitutive expression
of enhanced green fluorescent protein (EGFP), the
transgene was inserted on the X chromosome. This
X-linked reporter presented the first opportunity to
use fluorescence to non-invasively sex embryos 44.
Because only female progeny inherit and express
paternally derived X-EGFP transgenes, this reporter
can be used for the fluorescence-based sexing of
pre-implantation-stage embryos more than ten days
before an overt morphological difference between the
sexes is observed44 (FIG. 2a).
X-inactivation in normal, mutant and cloned mice.
Females that are HEMIZYGOUS for an X-linked constitutively expressed EGFP reporter show imprinted transgene expression in extra-embryonic tissues, and the
transgene is silenced as a result of X-inactivation in
50% of their somatic cells45 (FIG. 2b,c). X-linked EGFP
mouse strains have been used to help investigate the
actions of genes that are involved in the regulation of
genomic imprinting46,47, because they allow the visualization of the status of X-linked gene expression in
living embryos.
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a
b
c
with a single null allele of Plxna3. In these experiments,
only the unlabelled cells showed the mutant phenotype,
whereas the EGFP-expressing neurons showed a wildtype phenotype, which indicates that mutations in
Plxna3 act cell autonomously48.
Over the past five years there has been an upsurge in
the number of successful attempts to clone animals, and
an increasing interest in the application of cloning as a
method of generating genetically matched stem cells for
therapeutic purposes49. One of the few biological insights
into this phenomenon used X-linked EGFP mice and
investigated whether cloning resets the epigenetic differences between the two X chromosomes in a female
somatic nucleus46. Somatic cells were pre-selected on the
basis of the X chromosome that was inactivated. As they
were alive, they could be used as donors to generate
cloned embryos in which X-chromosome activity was
monitored. Intriguingly, epigenetic marks are removed
and re-established on both X chromosomes during the
cloning procedure46.
Gene-expression reporters
Figure 2 | An X-linked fluorescent reporter. Wide-field epifluorescence images of a
constitutively expressed X-linked enhanced green fluorescent protein (EGFP) transgene44,45. This
reporter can be used for the non-invasive sexing of embryos before the appearance of overt
sexual dimorphism. Also, because the transgene is subject to X inactivation, as are the
endogenous genes on the X chromosome, it can be used for tracking X-inactivation. a | Live preimplantation (blastocyst stage) mouse embryos that were derived from a cross between a male
carrying an EGFP transgene on his X-chromosome and a wild-type female. Only female offspring
inherit the EGFP transgene and are marked by green fluorescence. b | Midbrain region from live
male (left) and female (right) embryonic day (E) 10.5 hemizygous transgenic embryos, both of
which carry an X-linked EGFP reporter. All cells in the male are fluorescent compared with ~50%
of the cells in the female, owing to random X inactivation (it is not clear which cells express the
fluorescent reporter, because of the low magnification that does not provide resolution at the
cellular level). c | Newborn offspring from a cross between a female carrying one copy
(hemizygous) of an X-linked EGFP transgene and a non-transgenic male. Transgenic male
pups (left) have ubiquitous green fluorescence in the skin owing to the presence of a single active
X chromosome. Transgenic female pups (right) have mosaic (tortoiseshell) green fluorescence in
the skin owing to random inactivation of one X chromosome. Non-transgenic pups (centre) are
not visible. Panel c reproduced with permission from REF. 45 © (2001) Wiley.
Recently, X-linked EGFP mice were used to investigate the defects in plexin-A3 mutant mice48. Plexin-A3
is a receptor for semaphorin ligands. Mutants show
defective pruning of specific axonal branches in the
developing brain. The natural mosaicism that is created
by the process of X-inactivation can be used for mutant
analysis. In such an experiment, X-linked EGFP mice
were crossed with mice that carried a targeted mutation
in the X-linked Plxna3 gene, so that one X chromosome contained the EGFP marker and a wild-type allele
of Plxna3, whereas the other was null for Plxna3. Because
X-inactivation creates a genetic mosaic, EGFP-expressing
cells were wild type and unlabelled cells were mutant
NATURE REVIEWS | GENETICS
Mouse lines can be generated with fluorescent-protein
reporters that reproduce the expression of specific
genes. To do this, a reporter construct that contains the
cis-regulatory elements from both the gene of interest
and the fluorescent protein is randomly inserted into
the genome, or, alternatively, the fluorescent protein is
targeted to a specific locus. A reporter construct can be
randomly integrated into the mouse genome by the
pronuclear injection of DNA into zygotes, or the introduction of DNA into ES cells, and these methods often
result in high-levels of reporter-gene expression.
However, this approach requires knowledge of the
cis-acting regulatory elements of the gene.
If minimal information is available about the
cis-acting regulatory elements that drive spatiotemporal
gene expression, a bacterial artificial chromosome (BAC)
transgenic approach can be adopted. In this strategy, a
fluorescent-protein reporter is introduced by recombination into the region of interest of a BAC vector. This large
construct, which probably contains all necessary regulatory sequences, is then randomly inserted into the mouse
genome50–52. A BAC-modification strategy using clones
that contain the regulatory elements of the Nkx2.5 gene53
was recently used to drive expression of a fluorescent
reporter in the embryonic heart. The resulting spatiotemporal pattern of green fluorescence was similar to that
obtained by RNA in situ hybridization.
Alternatively, a gene-targeting approach that uses
homologous recombination in ES cells can recapitulate
the expression of the gene of interest. However, this will
usually result in the incorporation of only a single copy
of the fluorescent reporter, which might not provide
adequate fluorescence intensity, particularly at loci that
are not strongly expressed. Gene-targeting strategies can
be designed to insert a fluorescent-protein reporter into
a locus so as to disrupt the function of the gene product
but leave its regulation intact. This knock-in strategy
provides a fluorescent marker for mutant cells with disrupted gene function, and allows the identification of
VOLUME 4 | AUGUST 2003 | 6 1 7
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Box 3 | Static-culture systems for time-lapse imaging
Similar to most mammals, mouse embryos develop in the relatively inaccessible environment of the maternal
reproductive system. Vital-imaging experiments require that protocols be modified to allow normal development to
take place in static cultures on the microscope stage. Existing protocols for the culture of whole embryos and
individual organs all require a robust temperature-controlled oxygenated environment for the duration of the
experimental period. Whole pre-implantation-stage embryos (from fertilization to embryonic day (E) 3.5) can be
easily grown in static culture and allow the observation of the intricacies of cleavage, compaction and cavitation to
form the blastocyst at E3.5 (see figure)123–125. For examples of whole embryos that have been imaged whilst being
cultured in vitro see supplementary movies 1 and 2 online.
Post-implantation
Pre-implantation
Ovary Oviduct
Uterus
E10.5–E12.5
Implantation
Cleavage
Ovulation
E2.5
E3.5
E2
E1.5
E4.5
Compaction and
cavitation
Fertilization
E8.5
Gastrulation
and neurulation
Hatching
Organogenesis
Culturing post-implantation mouse embryos is more difficult, and protocols for the static culture of these stages are
based on serum-rich media preparations. Methods for culturing early post-implantation embryos between E5.5–E8.5
(REFS 107,108) and E8–E10.5 (REF. 67) in serum-rich media are in use in several laboratories. Later-stage embryos
(E11.5–E12.5) can be grown in serum-free conditions126. Also, protocols for the explantation and in vitro culture of parts
of embryos, including germ layers from early gastrulating embryos127, are common and can be adapted to work on a
microscope stage. At later stages, whole embryos cannot be maintained in culture; however, their increasing size
makes them more opaque, so the culture of organ primordia is more effective for imaging experiments. Protocols for
the in vitro culture of several organ systems, such as ureteric buds61, lung buds128, limb buds129 and the neural
tube130,131, have been developed. Figure reproduced with permission from REF. 141.
cells in which the promoter of interest is active.
However, low levels of expression can be a complicating
factor for some loci. For example, it has been difficult
to recapitulate reporter expression under standard epifluorescent conditions when lacZ reporters are replaced
with fluorescent proteins (A.-K.H. and V.E.P., unpublished observations). This is usually because fluorescence
is a direct readout of gene activity, and is not detected
using a chromogenic signal-amplification reaction (as
with lacZ), so fluorescent reporters can be less sensitive.
The development of new and brighter fluorescentprotein reporters, for example the use of ‘venus’ 54
in contrast to enhanced yellow fluorescent protein
(EYFP)55,56 (BOX 2), should address this issue. Another
way to increase the sensitivity is by concentrating the
total available fluorescent protein using subcellularly
localized reporters (as discussed below).
Another limitation of many gene-based reporters is
PERDURANCE of the reporter protein. If the protein has a
long half-life it might not faithfully report the temporal
domain of the regulatory elements that drive its expression. A solution to this problem has been achieved by
fusing the fluorescent protein to the degradation
domains of other proteins to accelerate degradation57.
PERDURANCE
The ongoing stability and
activity of a protein in the
cellular environment.
EPIBLAST
An embryonic lineage that is
derived from the inner-cell mass
of the blastocyst, which gives rise
to the body of the fetus.
618
0 hours
24 hours
60 hours
84 hours
Figure 3 | Green fluorescence as a marker of branching morphogenesis in the developing kidney. Wide-field
epifluorescence images of an in vitro cultured embryonic kidney taken at various time-points during the same experiment.
Fluorescence is confined to the ureteric-bud derivatives of the kidney. The mouse embryo carried a HoxB7-EGFP transgene61.
The kidney primordium was dissected out of an embryonic day (E) 11.5 embryo and maintained in culture for 84 hours.
Reproduced with permission from REF. 61 © (1999) Wiley.
| AUGUST 2003 | VOLUME 4
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REVIEWS
Box 4 | Imaging fluorescent proteins using laser-scanning microscopy
In many of the examples described
a
b
Single-photon excitation
Multi-photon excitation
in this review, multiphoton
132
microscopy has been used for vital
and deep-tissue imaging. Introduced
over a decade ago, this form of laserscanning microscopy (LSM) uses
Laser illumination
Laser illumination
near-infrared light to excite
everywhere
everywhere
fluorochromes and results in
excitation only at the plane of focus
Excitation only
Excitation
(region of highest photon flux),
at highest photon
everywhere
flux
which eliminates noise that is
created by out-of-focus emission
photons. This is in contrast to
conventional wide-field fluorescence
microscopy and confocal LSM
(CLSM)133, which use visible light to
excite dyes. These techniques cause excitation of fluorescent dyes above and below the plane of focus, which results in
thick samples appearing blurred. Confocal microscopes reduce this problem and increase the resolution of the image
by using a pinhole in front of the detector to eliminate out of focus photons.
In multiphoton microscopy134,135, out of focus photons are never generated, which provides optical sectioning without a
pinhole. This is because fluorescence is only produced at the plane of focus. In standard fluorescence microscopy, a single
high-energy photon is absorbed by a fluorochrome and a photon of slightly lower energy is emitted when it returns to
ground state. For example, for a fluorescent protein such as GFP (panel a), a blue photon (blue arrows) is absorbed
and a green photon (green arrows) is emitted. In multiphoton excitation, the single high-energy photon is replaced
by the quasi-simultaneous absorption of two (two-photon excitation) or three (three-photon excitation) lower-energy
near-infrared photons. So, for GFP, rather than a single blue excitation photon, two near-infrared photons (red arrows)
can be used to excite the fluorochrome, which leads to the emission of a green photon (panel b). Fluorescence is generated
only at the point of focus, at the highest photon flux, because multiple photons must be absorbed simultaneously. Note
that the emission of photons (green arrows) is actually in all directions, although only those that can be collected by the
objective lens are shown here.
Multiphoton excitation has the advantage of less overall PHOTOBLEACHING and less damage to the tissue by repeated
illumination because, when creating a three-dimensional stack of images along the z-axis, each focal plane is excited only
once. By contrast, in confocal microscopy all focal planes are exposed to excitation light each time an optical plane is
collected. Confocal scanning can lead to extensive photobleaching and a greater chance of toxicity if multiple z-stacks are
taken repeatedly over time.
The use of near-infrared photons for excitation has a number of benefits. Near-infrared light penetrates deeper into the
sample than visible light, which allows the imaging of deeper structures than in confocal microscopy. Near-infrared light is
also less harmful to tissue in general, because few endogenous molecules absorb at near-infrared wavelengths, which greatly
limits the potential for tissue damage. This has been shown for pre-implantation hamster embryos136. Hamster embryos that
were imaged using confocal microscopy did not undergo normal cleavage, whereas embryos imaged using multiphoton
microscopy not only developed to the blastocyst stage, but could be implanted into a pseudo-pregnant host for further
development and, at least in one case, the imaged embryo gave rise to a healthy adult hamster.
PHOTOBLEACHING
The irreversible destruction of a
fluorophore that is under
illumination.
NATURE REVIEWS | GENETICS
Such reporters might provide a better temporal representation of promoter activity; however, there could
be a reduction in sensitivity if insufficient protein
accumulates to ensure robust fluorescence. At present,
few reports describe the use of destabilized GFP-variant
reporters in mice. One example is the successful use
of regions upstream of the Axin2 gene to direct the
tissue-specific expression of d2EGFP (an EGFP
fusion with a two hour half-life) in transgenic mouse
embryos58.
Both targeted and randomly integrated (injectionand ES-cell-based) transgenic reporters have been used
to generate strains of mice that express fluorescent proteins in a lineage-specific fashion. Next, we describe
some of the most recent studies that have used transgenic mice for multidimensional analyses of embryonic
and neuronal development.
Imaging primordial-germ-cell migration in embryos.
One of the first reports that exploited a fluorescentprotein reporter to follow the movement of cells in a
living mouse embryo (see BOX 3 for details of culturing
embryos) used a transgenic mouse line with EGFP
under the regulation of the Oct4 promoter (Oct4-EGFP).
This promoter is first active in the cells of the inner-cell
mass of the blastocyst and the EPIBLAST of the pregastrulation embryo, and is subsequently restricted to the
primordial germ cells (PGCs) of the post-implantation
embryo59,60. In this study, fluorescently labelled PGCs,
which are the founder cells of the gametes, were followed using time-lapse microscopy of living postimplantation-stage embryos. By imaging living
embryos, as well as investigating the shapes and positions of PGCs in fixed tissues, the site of origin of
PGCs was determined and these cells were shown to
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a
loop between the ureteric-bud epithelium and the stromal mesenchyme, and showed that vitamin A controls
epithelial–mesenchymal interactions through the
receptor tyrosine kinase Ret expression63. Time-lapse
imaging and three-dimensional (3D) reconstructions
of HoxB7-EGFP transgene expression were used to
show that ureter maturation depends on formation of
the ‘trigonal wedge’ — an epithelial outgrowth from the
base of the Wolffian duct — and that the urogenital
malformations observed in Rara–/–Rarb–/– mice probably
result from the failure of this process64.
b
H2B
EGFP
c
EGFP
d
myr
EGFP
tau
EGFP
Figure 4 | Fluorescent fusion proteins as markers of subcellular compartments.
Three-dimensional (3D) reconstructions of confocal laser-scanning-microscopy serial sections
of transgenic mouse embryonic stem (ES) cells that express various subcellularly-localized
fluorescent proteins. a | Native enhanced green fluorescent protein (EGFP), which labels the
entire cell. b | A fusion of EGFP with human histone H2B, which labels chromatin. c | A fusion
of EGFP with an N-terminally engineered myristoylation sequence (myr), which labels cell
membranes. d | A fusion of EGFP with the bovine microtubule-binding protein tau, which labels
microtubules (supplementary movies 3–6 online show 3D reconstructions of these images).
move by active locomotion from the primitive streak
(a region in the posterior epiblast) into the adjacent
endoderm, from where they migrate to the gonad.
Imaging branching morphogenesis. Urogenital abnormalities are common in human infants, and are often
caused by malformations of the kidneys or irregular
connections between the ureter and the bladder. Mice
present an excellent model for urogenital development,
and kidney primordia can be cultured in vitro (BOX 3)
where they recapitulate branching morphogenesis.
Fluorescent labelling of various cell types in the developing kidney allows dynamic imaging of these
events61,62 (FIG. 3). A HoxB7-EGFP transgene61 that fluorescently labels the developing ureteric bud was used in
a series of experiments to investigate the phenotype of
Rara –/–Rarb –/– mice, which, as a result of impaired
retinoid (vitamin A) signalling, develop syndromic
urogenital malformations that are similar to those seen
in humans. This led to the identification of a signalling
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Quantifying blood-flow dynamics in live embryos.
Traditional protocols for the in vitro development of
post-implantation mouse embryos involve rotating cultures65,66. These techniques have been recently adapted to
enable fluorescence time-lapse imaging of embryos up to
mid-gestation, which are maintained in static culture on
the microscope stage67 (BOX 3).
In mouse embryos, blood cells, along with endothelial cells, first form at embryonic day (E)7.5 in the yolk
sac, which is an extra-embryonic structure that surrounds the embryo. This site is the sole source for bloodcell production until definitive blood cells are produced
in the embryo itself several days later. The heart begins
beating at E8.5 and a notable remodelling of the vasculature, especially in the yolk sac, takes place over the next 24
hours. Using a transgenic mouse line that expresses GFP
under the control of the embryonic globin (ε-globin)
promoter68, the formation and flow of newly formed
blood cells in live intact mouse embryos have been
studied by confocal67 (single-photon) and multiphoton
microscopy (BOX 4). Because the flow of blood is
dynamic, such studies would have been impossible in
fixed samples. Brightly labelled spherical blood cells first
appear at E7.5 (REF. 68) and the expansion of these cells,
which are initially located in the blood islands, has been
followed through time-lapse microscopy67.
A large number of mutant mice have various defects
in the development of the cardiovascular system.
Through genetic manipulation, many genes have been
found to have a role in early cardiovascular development. As blood and endothelial-cell development occur
in concert with blood flow, there is need for a model system to study the interplay between physical and genetic
forces in the early cardiovascular system in mice. Using
laser-scanning microscopy and fluorescent reporters,
accurate measurements of blood-cell velocities can be
determined in early embryos as soon as circulation
begins (E. A. Jones, M. H. Baron, S.E.F and M.E.D.,
unpublished observations). By measuring velocity, and
other important parameters that have been made accessible using GFP-marked blood cells, the forces between
blood cells and surrounding cells can be determined in
both wild type and mutant backgrounds. This methodology represents a significant advance in studying quantitative blood flow in mammalian embryos. Other
techniques that are used to study blood flow in mouse
embryos, such as Doppler ultrasound69 and Doppler
optical-coherence tomography70, do not offer enough
spatial or temporal resolution to study microcirculation
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Box 5 | Simultaneous imaging of several fluorescent proteins
Relative intensity
When several genetically-encoded fluorescent Acquire a λ-stack
proteins are combined in the same imaging
λ
experiment, more information can be
x
obtained from the same image session and
the relationship between different cells or
different processes can be discovered.
y
However, many fluorescent-protein variants
have similar excitation and emission
Requires special hardware,
such as the Zeiss LSM 510 META
properties, so it can be difficult to
unambiguously separate the signals from
Select reference spectra from
the spectral database
each reporter. In fluorescence microscopy,
emission signals are usually separated using
1.0
glass filters, but in situations of significant
0.8
spectral overlap, glass filters are ineffective. In
0.6
much the same way, excitation curves are also
0.4
highly overlapping, which makes it difficult to
choose excitation wavelengths that would
0.2
excite only single variants. So, spectral
0.0
cross-talk between most genetically
460 480 500 520 540 560 580 600 620
encoded fluorescent proteins is inevitable.
Emission wavelength (nm)
However, there are mathematical
Apply linear unmixing algorithms to create
cross-talk-free multichannel images
approaches that can be used to separate
spatially and spectrally overlapping signals,
the most effective of which is linear
unmixing137–139.A series of images are
collected from the sample at different
wavelengths using a specialized detector to
generate a λ-stack. Linear unmixing makes use of the fact that variations in the intensity of
fluorescence spectra are specific to each fluorochrome. Therefore, reference spectra can be
selected from a special database. This information is used to convert the data from the
λ-stack into a multichannel image, with channels that contain the signal from each
fluorescent component. This approach has been used with great success to separate
spectrally and spatially overlapping fluorescent-protein signals and can be used by
either sampling emission spectra or excitation spectra.
at the onset of development, and can only provide
information about large vessels or blood flow through
the heart. Methods that are based on laser-scanning
microscopy (BOX 4) are especially attractive as they make
it possible to combine quantitative blood-flow analysis
with the study of other cell types simultaneously, by
using other fluorescent-protein variants or vital dyes
Vital imaging of neurons in mice. A growing number of
studies have used transgenic mice that express different
fluorescent proteins42 (BOX 2), in particular those under
the control of the Thy1 promoter, which is active in
neurons71. Interestingly, specific lines express fluorescent proteins in different subsets of neurons (expression mosaics in contrast to genetic mosaics), and
‘designer’ mice that have different combinations of
neurons labelled with spectral variants can be generated by crossing specific strains (FIG. 1g). These mice are
frequently used because many populations of cells can
be studied using different spectral combinations.
Mice that express fluorescent proteins that are driven by the Thy1 promoter have been used to explore
the formation and plasticity of synapses. For this analysis, the same neuromuscular junction was repeatedly
imaged every 24 hours using multiphoton microscopy
NATURE REVIEWS | GENETICS
to visualize dynamic changes in innervation and synapse
elimination. These studies take full advantage of mice
that express genetically-encoded fluorescent proteins,
and have given insights into the guidance of neurons
during injury and the mechanisms of neuronal branching and synapse maturation. For example, axons that are
damaged by crushing will make new synapses on the
same cells that they originally innervated, but if the
nerve is cut, this specificity is lost72.
Time-lapse multiphoton imaging has also provided
new information about axon-branch trimming at the
synapse. Although single muscle cells are originally
innervated by several motor axons, these connections
are refined as the synapse matures and many axons are
retracted, leaving, in most cases, a single axon. By watching the same synapses over time, it is clear that there is
no spatial or temporal bias to branch elimination, which
indicates that local competitive interactions are involved
in the elimination of one branch and the stabilization of
the other73. Using neurons that are labelled with different fluorescent-protein colour variants, it is clear that
the retraction of one axon is accompanied by the expansion of another, which explains the increase in synaptic
strength that is shown by the ‘winning’ axon74.
Recently, Thy-1 fluorescent reporter constructs in
mice have been used to examine synaptic plasticity in the
cortex75,76. In these studies, the brain of a living animal is
directly imaged using multiphoton microscopy (BOX 4).
The structures of dendritic arbors were imaged over the
course of days and even months, which shows that there
is considerable long-term stability of synaptic connections in adult mice, although there were dynamic changes
in the synapses of younger mice. These results indicate a
mechanism for long-term memory and experience. Such
approaches have not only been used in brain imaging77,78,
but also for the in vivo imaging of tumour formation79.
Fusion proteins allow subcellular resolution. A popular
application of fluorescent-protein reporters in cell biology is their incorporation into fusion proteins, which
can be used as vital labels for subcellular compartments80
or for tracking protein dynamics81. Also, to provide subcellular resolution, fluorescent-protein fusions can help
to concentrate the reporter, which increases its sensitivity and allows visualization of lower levels of reporter
protein. However, at present, the use of subcellularlylocalized fluorescent reporters is limited to a handful of
published reports that describe mice with constitutive
and lineage-restricted expression of a bovine tau–EGFP
fusion82–84, constitutive expression of a GPI-anchored
EGFP fusion85 and expression of EGFP with nuclearlocalization sequences86 (FIG. 4).
Subcellularly localized fluorescent-protein reporters
provide superlative resolution compared with other
genetically-encoded reporters and vital stains. Moreover,
fusion of a fluorescent protein to a nucleosomal component, such as a histone, offers a higher level of resolution
compared with a standard nuclear-localization sequence.
When samples from transgenic embryos that express a
nuclear-localized fluorescent reporter are imaged using
confocal or multiphoton microscopy (BOX 4), individual
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cells can be unequivocally identified in a group of cells,
and cells in mitosis can easily be discerned from cells in
interphase, as can those undergoing cell death. Nuclearlocalized reporters also allow nuclear morphology to be
visualized, and as this is specific to different cell types it
can be used to identify cell lineages. This raises the possibility of confocal or multiphoton ‘histology’ of live
specimens and paves the way for the development of
high-resolution multidimensional histological atlases.
Furthermore, subcellularly localized fluorescent proteins are more amenable to fixation and preservation of
the fluorescent signal (A.-K.H. and V. E. P., unpublished
observations).
Aa
Ab
Ac
Subcellularly localized reporters allow the visualization of dynamic subcellular events in an organismal
context. For example, cytoskeletally localized fluorescent proteins (such as actin, tubulin and tau fusions)
allow the dynamic visualization of changes in cell
shape. By contrast, nuclear-localized fluorescent proteins (such as those with a nuclear-localization
sequence or fusions to nuclear proteins such as
histones), allow the unequivocal identification and
‘tracking’ of individual cells in a field/population of
cells that all express the same fluorescent-protein
reporter. Nuclear-localized fluorescent proteins also
allow the vital imaging of active chromatin, such that
interphase nuclei can be distinguished from nuclei in
mitosis and from cellular debris (FIG. 4b). Histone–GFP
fusion proteins were first reported in cultured cells, in
which their use is now widespread87. Histone fusions
have also been used for high-resolution imaging in
whole-organism systems, including C. elegans 88,89,
Drosophila90,91 and zebrafish92,93.
Looking to the future
Ad
Ae
B
Af
0 µm
C
20 µm
40 µm
60 µm
80 µm
100 µm
Figure 5 | Towards a high-resolution multidimensional atlas of a living mouse. Multiphoton
microscopy images of a live embryonic day (E) 6.5 mouse embryo constitutively expressing a
histone–enhanced green fluorescent protein (EGFP) fusion protein and maintained in an in vitro
culture. This reporter labels active chromatin and facilitates the high-resolution visualization of cell
nuclei including mitotic cells and fragmenting nuclei of dying cells. Every tenth section is depicted
from a sequential optical z-stack comprising 60 optical sections taken at 2 µm intervals of the
distal end of a living mouse embryo, starting at the surface (Aa) and penetrating into the specimen
(Ab–f). The embryo is cup shaped and comprises two cell layers: an outer layer of visceral
endoderm cells surrounding an inner layer of epiblast cells. Deep to superficial (B) and superficial
to deep (C) colour-coded three-dimensional (3D) reconstructions generated from the z-stack
encompassing the entire image series. The colours of the scale bar denote fluorescence at
different depths within the embryo (red being outermost, and blue being deepest). This is one way
to present 3D data in two-dimensions (supplementary movies 7–9 online show z-stacks and 3D
images from live embryos). Panel c reproduced with permission from REF. 140.
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The continued evolution of genetically-encoded fluorescent-protein reporters, coupled with advances in opticalimaging modalities, will make reporter-expressing mice
an essential tool for the multidimensional analysis and
understanding of biological processes in wild type,
mutant and pathological contexts.
Technological advances. In the field of fluorescentprotein-reporter development, new spectral variants
that mature faster with increased fluorescence, and
improved excitation and emission spectra, will continue to become available as will methodologies that
allow the separation of proteins with similar spectral
profiles (BOX 5). These improvements will facilitate the
increased use of technicolour mice that express multiple
fluorescent reporters. So far, few published reports
illustrate combinatorial imaging with dual fluorescentprotein-reporter detection in individual embryos42 or
adult mice42,71, but there are already several lines of
mice that have been specifically designed for anticipated dual-tagging experiments29. Cross-talk between
fluorescent proteins has limited multicolour experiments to specific combinations (for example, enhanced
cyan fluorescent protein (ECFP) and EYFP). However,
recent advances in imaging technology that use both
excitation and emission fingerprinting to separate
closely overlapping markers, such as linear unmixing
(BOX 5), allow signal from different fluorescent
reporters to be resolved. This opens the door to
increased multiple fluorochrome experiments in the
future.
As imaging technology improves, there is a general
trend towards making imaging hardware more
portable, increasing the types of cell that can be imaged
in situ. The progress in fibre-optic confocal imaging
(FOCI) and two-photon fibre-optic imaging, together
with recent advances in implantable devices78, such as a
miniature head-mounted multiphoton microscope94,
raises the possibility of monitoring body functions
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MUSHROOM BODIES
The region of the Drosophila
brain that is required for
olfactory learning and memory.
through internalized miniature surveillance cameras.
These technologies combine the benefits of highconstrast high-resolution light microscopy with the
intravital advantages that are offered by other less invasive imaging technologies, such as magnetic-resonance
imaging (MRI ) (see links in online links box).
Also, to make progress in imaging techniques, the
development of reporter proteins with special features,
including photoactivatable proteins and biochemical
sensors, is anticipated to expand the use of fluorescent
mice. Recently, fluorescent reporters have emerged that
can be modulated photochemically. When irradiated
with light of the appropriate wavelength, these proteins
fluoresce at a specific wavelength, so irradiated cells and
their progeny can be visualized. These fluorescent highlighters include ‘PA-EGFP’, which is a GFP variant95, and
‘kaede’96, which was cloned from Trachyphyllia geffroyi.
There are no reports, at present, of the use of fluorescent
highlighters in mice; however, this class of reporter is
particularly attractive because it could be used in fatemapping experiments in which photo-activated
reporters could label cells non-invasively and be followed in situ.
One ‘final frontier’ in real-time imaging of live
specimens is a reporter acting as a direct read-out of a
physiological process. Several functional fluorescentprotein-based biochemical-sensor reporters have been
developed97–100, which include sensors for heterotrimeric
G-protein activity101, phosphoinositide102 and calcium103,104 signalling. Calcium sensors (often called
camaroos) have recently been used in Drosophila to
measure signalling in the MUSHROOM BODIES of the brain105
and to decipher the logic of odour perception through
the use of two-photon microscopy (BOX 4) for the analysis of odour-evoked patterns of neural activity106. It is
only a matter of time until biological-sensor reporters
are used in mice.
Applications. Developments in fluorescent-reporter
technologies should lead to the widespread incorporation of technicolour transgenics into functional genomic
approaches in the mouse, including phenotype-driven
screens (such as N-ethyl-N-nitrosourea (ENU) mutagenesis2,3,5,6) or expression-driven screens (such as genetrap mutagenesis4). This should help focus screens by
making it easier to identify mouse mutants that disrupt
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Acknowledgements
We thank F. Costantini, L. Jones and J. Lichtman for generously
providing figures. We apologize to the authors of many important
findings and strains of mice that are not discussed here owing to
space limitations. Our work is supported by grants from the
National Institutes of Health, National Science Foundation and
Muscular Dystrophy Association. A.-K.H. is a fellow of the
American Heart Association.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink
Axin2 | Nkx2.5 | Plxna3 | Rara | Rarb | Thy1 | Vegfa
FURTHER INFORMATION
BD Biosciences Inc.:
http://www.clontech.com/gfp/index.shtml
Bio-Rad Microscience: http://microscopy.bio-rad.com/
products/live_cell.htm
C. elegans movies:
http://www.bio.unc.edu/faculty/goldstein/lab/movies.html
Carl Zeiss Advanced Imaging Microscopy:
http://www.zeiss.de/us/micro/home.nsf
Coherent Inc.: http://www.cohr.com
Drosophila atlases: http://pbio07.uni-muenster.de
Drosophila brain atlas: http://flybrain.unifreiburg.de/Flybrain/html
FishScope: http://depts.washington.edu/fishscop
Leica Microsystems: http://www.leicamicrosystems.com/website/lms.nsf
Mouse MRI Atlas at the Biological Imaging Center:
http://quad.bic.caltech.edu/mouseatlas
NCBI mouse genome resources:
http://www.ncbi.nlm.nih.gov/genome/guide/mouse
Scott Fraser’s Laboratory: http://bioimaging.caltech.edu
Spectra-Physics: http://www.spectraphysics.com
The Edinburgh Mouse Atlas Project (EMAP):
http://genex.hgu.mrc.ac.uk
The Jackson Laboratory Induced Mutant Resource:
http://www.jax.org/imr/index.html
The Jackson Laboratory Mouse Genome Database (MGD):
http://www.informatics.jax.org
Virginia Papaioannou’s laboratory:
http://cpmcnet.columbia.edu/dept/genetics/papaioannoulab.html
Zebrafish vascular anatomy atlas:
http://mgchd1.nichd.nih.gov:8000/zfatlas/Intro%20Page/
intro1.html
Access to this interactive links box is free online.
VOLUME 4 | AUGUST 2003 | 6 2 5
ONLINE
Anna-Katerina Hadjantonakis is interested in understanding how the
mammalian body plan is established during embryogenesis, and in the
development and use of tools for genome alterations and biological imaging in mice. Her graduate work at Imperial College, London, focused on
the identification of the Celsr gene family, which are the vertebrate homologues of the Drosophila planar-cell-polarity gene flamingo. As a postdoctoral researcher at the Samuel Lunenfeld Research Institute, Toronto, she
generated some of the first strains of fluorescent-protein-expressing mice
and showed combinatorial reporter detection in living embryos. She is
now working with Virginia Papaioannou at Columbia University, New
York, using vital imaging as a tool for unravelling the genetic basis of
mouse embryonic development. She will be joining the Program in
Developmental Biology at the Sloan-Kettering Institute, New York, at
the start of 2004.
Mary E. Dickinson is developing new optical-imaging methodologies and
applying imaging techniques to the study of mouse and chicken embryology. She trained as a mouse embryologist, first as an undergraduate at
Vanderbilt University with Brigid Hogan, then as a graduate student at the
Roche Institute, New Jersey, and Harvard University with Andy
McMahon, where she worked on Wnt signalling. She came to the
California Institute of Technology to work with Scott Fraser to combine
advanced imaging technology with vertebrate development. She is now a
Member of the Professional Staff in the Biological Imaging Center, at the
California Institute of Technology, where she has been developing new
imaging modalities for studying vertebrate development and using vitalimaging studies to characterize dynamic aspects of early mouse embryogenesis.
Scott E. Fraser is the Anna L. Rosen Professor of Biology at the California
Institute of Technology. He has a long-standing interest in following
important lineage and migration events in embryos as they develop. For
the past decade he has directed the Biological Imaging Center at the
California Institute of Technology, which is dedicated to the application of
advanced optical and magnetic resonance microscopies to intravital imaging in systems that range from sea urchin and fish embryos to mouse
models of disease. Together with an active set of collaborators, he has
applied his training in physics and biophysics to challenges such as imaging the patterns of cell migration in the developing cerebellum and testing
the role of blood flow in the shaping of the developing heart. He has been
active in science education, previously serving as the Co-director of the
embryology course at the Marine Biological Laboratory in Woods Hole,
Massachusetts.
• Optical-imaging technologies provide a way to perform live-cell analyses in an organismal context. Continued improvements in fluorescence-based imaging technologies allow deeper imaging and better
spectral separation of fluorescent-protein reporters in living specimens.
• Genetically-encoded fluorescent proteins that are expressed in normal
and mutant mice represent high-resolution high-contrast multicolour
vital markers for monitoring gene activity and protein function. They
pave the way for the multidimensional multispectral imaging of living
specimens and the generation of multidimensional atlases of model
organisms.
• An increasing number of fluorescent-protein reporters are available for
use in mice. These fall into four main categories: spectral-variant
reporters, subcellularly localized reporters, photo-activatable reporters
and reporters that act as biochemical sensors.
• The development of static in vitro culture systems for mammalian
embryos and explanted tissues promotes normal ex vivo growth, which
is necessary for vital-imaging experiments.
• Researchers now possess a powerful toolbox that offers the potential to
visualize and quantify biological processes at the cellular and subcellular
level, both in vitro and in intact living organisms.
LocusLink
LocusLink
http://www.ncbi.nlm.nih.gov/LocusLink
Axin2
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=12006
Nkx2.5
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=18091
Plxna3
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=18846
Rarb
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=218772
Thy1
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=21838
Vegfa
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=22339
Further Information
Virginia E. Papaioannou is a Professor in the Department of Genetics and
Development at Columbia University. She has been interested in the
genetic control of early mammalian development and the nature of
embryonic stem cells since her graduate and postdoctoral studies at the
Universities of Cambridge and Oxford in the UK, where she began using
micromanipulation to study cell fate and cell lineage. As a faculty member
of Tufts University, Boston, she applied the technique of gene targeting to
mutate genes that are expressed during early embryonic development. She
has been involved in the study of the T-box family of transcription factor
genes since their discovery, and has generated and studied mutations in
many of these genes, which have profound effects on development.
• The mouse is the premier mammalian model organism and provides
an unparalleled platform for modelling mammalian development and
disease. Comprehensive functional annotation of the mouse genome
relies on an integrative approach, using an array of tools for unravelling
the molecular and cellular basis of normal and mutant phenotypes.
BD Biosciences Inc.
http://www.clontech.com/gfp/index.shtml
Bio-Rad Microscience
http://microscopy.bio-rad.com/products/live_cell.htm
C. elegans movies
http://www.bio.unc.edu/faculty/goldstein/lab/movies.html
Carl Zeiss Advanced Imaging Microscopy
http://www.zeiss.de/us/micro/home.nsf
Drosophila atlases
http://pbio07.uni-muenster.de
Drosophila brain atlas
ONLINE
http://flybrain.uni-freiburg.de/Flybrain/html
FishScope
http://depts.washington.edu/fishscop
Leica Microsystems
http://www.leica-microsystems.com/website/lms.nsf
Coherent Inc.
http://www.cohr.com
Mouse MRI Atlas at the Biological Imaging Center
http://quad.bic.caltech.edu/mouseatlas
NCBI mouse genome resources
http://www.ncbi.nlm.nih.gov/genome/guide/mouse
Scott Fraser’s Laboratory
http://bioimaging.caltech.edu
Spectra-Physics
http://www.spectraphysics.com
The Edinburgh Mouse Atlas Project (EMAP)
http://genex.hgu.mrc.ac.uk
The Jackson Laboratory Induced Mutant Resource
http://www.jax.org/imr/index.html
The Jackson Laboratory Mouse Genome Database (MGD)
http://www.informatics.jax.org
Virginia Papaioannou’s laboratory
http://cpmcnet.columbia.edu/dept/genetics/papaioannou-lab.html