Download Imaging fertilization in flowering plants, not so

Journal of Experimental Botany, Vol. 62, No. 5, pp. 1651–1658, 2011
doi:10.1093/jxb/erq305 Advance Access publication 15 October, 2010
REVIEW PAPER
Imaging fertilization in flowering plants, not so abominable
after all
Frédéric Berger1,2,*
1
2
Temasek LifeScience Laboratory, 1 Research Link, National University of Singapore, Singapore 117604
Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543
* E-mail: [email protected]
Received 2 August 2010; Revised 7 September 2010; Accepted 9 September 2010
Abstract
Although the discovery of double fertilization in flowering plants took place at the end of the nineteenth century little
progress had been made in understanding the cellular and molecular mechanisms involved until the end of the
twentieth century. After attempts to study fertilization with isolated male and female gametes, researchers turned to
Arabidopsis thaliana as a model for genetic analysis and in vivo imaging. The development of confocal imaging and
fluorescent proteins, coupled with new molecular insights into cell fate specification of plant gametes, allowed the
development of robust markers for cells participating in double fertilization. These markers enabled the imaging of
double fertilization in vivo in Arabidopsis. These studies have been coupled with the identification and molecular
characterization of genes controlling fertilization in Arabidopsis. Live imaging has already provided new insights on
sperm cell delivery, the equivalence of the fate of the sperm cells, gamete fusion, and re-initiation of the zygotic life.
This review covers these topics and outlines many important aspects of double fertilization that remain unknown.
Key words: Arabidopsis, fertilization, gametes, imaging.
Introduction
Microscopic descriptions of fertilization in flowering plants
marked the end of the nineteenth century (Guignard, 1899;
Nawaschin, 1898). Two parallel ‘fertilization events’ are
required to produce a viable embryo, a trait unique to
flowering plants. Hence the term ‘double-fertilization’ is
used to describe the sexual reproductive process in flowering
plants. Double-fertilization involves two sperm cells delivered by the pollen tube. The discharge of the sperm cells
by the pollen tube is triggered at the entrance of the embryo
sac, which contains the egg cell and the central cell, usually
referred to as female gametes. The embryo sac is the
haploid female gametophyte that also contains three
antipodals of unknown function and two synergids involved
in pollen tube guidance and in sperm cell discharge.
Fertilization of the egg cell produces a zygote, which further
develops as the plant embryo. Fertilization of the central
cell triggers the development of the endosperm, which
nurtures embryo development but does not participate in
the next plant generation. The history of the discovery of
double-fertilization in angiosperms has been reviewed
(Faure and Dumas, 2001; Friedman, 2001) and this review
will focus on the experimental investigations of doublefertilization using live imaging. Gametogenesis, pollen tube
growth, and pollen tube attraction precede the sperm cells
discharge that initiates double-fertilization and these events
are not reviewed here. Isolation of the male and female
gametes from maize (Zea mays) allowed imaging of gamete
fusion in vitro (Kranz et al., 1991; Faure et al., 1994; Kranz
and Lörz, 1994; Kranz and Dresselhaus, 1996) and recording of early electrical events (membrane potential and
calcium signal) that are triggered in the egg cell by fusion
with a sperm cell (Antoine et al., 2000; Digonnet et al.,
1997). However, these early studies used plant gametes
isolated before full maturity and took place outside of the
physiological context (in vitro fertilization was performed in
liquid medium). Thus researchers attempted to achieve live
imaging of fertilization in planta. Torenia fournieri produces
‘naked’ embryo sacs devoid of ovule integuments at the
ª The Author [2010]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1652 | Berger
micropylar pole where the pollen tube delivers the sperm
cells. An experimental set-up including isolated embryo sacs
on a medium favourable for in vitro pollen tube germination
allowed the recording of the release of the pollen tube
content (including sperm cells and cytoplasm) and of the
first series of syncytial nuclei division in the endosperm after
fertilization of the central cell (Higashiyama et al., 1997,
2000). However, DIC microscopy applied to the imaging of
fertilization in isolated Torenia fournieri ovules did not
allow clear images of the fusion of the sperm cells with the
egg or the central cell and subsequent karyogamy to be
obtained (gamete nuclei fusion).
New confocal microscopy protocols adapted to the
Arabidopsis female gametophyte (Christensen et al., 1997)
led to the first detailed description of double-fertilization
including the documentation of karyogamy in this species
(Faure et al., 2002). These studies performed with fixed
material indicated the time-course of the events taking place
during double-fertilization and paved the way for further
studies in vivo. With the identification of promoters that
express fluorescent markers in male and female gametes it
became possible to image in vivo double-fertilization in
Arabidopsis. These recent research developments are
reviewed here together with the results of combining in vivo
imaging with genetic studies. Although significant progress
has been made, new tools are required to assess how sperm
cells are discharged and fuse, the timing of cell cycle events,
how a second fertilization is prevented, how gamete nuclei
migration take place before karyogamy, the details of
mechanisms leading to karyogamy, and the events leading
to reactivation of the zygotic life.
Marking Arabidopsis male gametes
The discovery of fluorescent proteins from marine invertebrates and their derivative has been essential for research in
developmental and cell biology (Chalfie et al., 1994; Tsien,
1998; Haseloff, 1999; Campbell et al., 2002; Giepmans
et al., 2006). Identification of suitable promoters enabling
a robust and specific expression in plant gametes or
associated cells was achieved in recent years as a result of
a large community effort (Table 1). The isolation of sperm
cells from different plant species provided access to the
transcriptome of this cell type. cDNA and EST sequences
have been reported from generative cells of Lilium longiflorum (Okada et al., 2006), and from sperm cells of
Nicotiana tabacum (Weterings et al., 1992) Zea mays (Engel
et al., 2003, 2005) and Plumbago zeylanica (Gou et al., 2009)
and, eventually, an Arabidopsis thaliana sperm cell transcriptome was obtained (Borges et al., 2008).
The identification of the promoter LAT52 from tomato
pollen was the first step towards imaging the discharge of
the pollen tube during plant fertilization (Twell et al., 1991).
In Arabidopsis the promoter LAT52 drives expression in
the microspores before the first pollen division and later in
the vegetative cell (Eady et al., 1994). After the microspore
unequal division, pLAT52-GFP expression becomes confined
to the vegetative cell (Eady et al., 1994) (Fig. 1A). In mature
pollen pLAT52-GFP expression is very high in the mature
pollen vegetative cell (Cheung, 2001) (Fig. 1B). When pollen
germinates and produces a pollen tube the bright fluorescence from GFP expression driven by pLAT52 enables
observation of pollen tube growth and gamete delivery
in vivo (Rotman et al., 2003; Feijo and Moreno, 2004).
Identification of the first gene to be expressed only in
sperm cells originated from studies of the male gametophytic mutant duo1 that produces a single sperm cell
(Durbarry et al., 2005). This led to the identification of the
gene DUO1 that is expressed only in the male germ lineage
in Arabidopsis (Rotman et al., 2005). DUO1 encodes
a MYB domain protein, which activates the expression of
genes involved in cell cycle control and sperm cell fate
specification (Rotman et al., 2005; Brownfield et al., 2009).
Studies of genes expressed in lily pollen led to the
identification of the Arabidopsis gene GENERATIVE CELL
SPECIFIC 1 (GCS1) (Mori et al., 2006) (also named
HAPLESS2; von Besser et al., 2006), which is expressed
only in the sperm cell lineage including the generative cell
and sperm cells (Brownfield et al., 2009). Although DUO1
and GCS1 promoters drive the expression of fluorescent
markers in the sperm cells, the transgenic lines expressing
fluorescent proteins under the control of these promoters
did not provide sufficient signal to perform live imaging of
fertilization.
Table 1. Genes used for live imaging of double-fertilization
Gene name
AGI ID number
HTR10/AtGMH3 AT1G19890
LAT52
Promoter derived from
Tomato LAT52 promoter
EC1
AT1G76750
FWA
AT4G25530
FIE
AT3G20740
MYB98
CENH3
ATLIGASE1
AT4G18770
AT1G0130
AT1G08130
Cell type labelled
Step of fertilization studied
Sperm cells
Pollen vegetative cell
and pollen tube
Egg cell
Central cell
Egg cell and central cell
(non-specific)
Synergids
All
All
Sperm cell discharge, migration and karyogamy Ingouff et al., 2007, 2009; Aw et al., 2010
Pollen tube guidance and discharge
Eady et al., 1994; Rotman et al., 2003;
Sandaklie-Nikolova et al., 2007
Karyogamy
Ingouff et al., 2009
Karyogamy
Kinoshita et al., 2004; Aw et al., 2010
Karyogamy
Ingouff et al., 2007
Pollen tube discharge
Activation of transcription and translation
Paternal genome expression
Reference
Sandaklie-Nikolova et al., 2007
Aw et al., 2010
Ingouff et al., 2009
Imaging double fertilization | 1653
Fig. 1. Fluorescent markers of the male and female gametes,
which can be used for live imaging of fertilization in Arabidopsis.
(A) Bicellular pollen grain with expression of pLAT52-GFP in the
vegetative cell (v) and pHTR10-HTR10-RFP in the generative cell
(g). (B) Tricellular pollen grain with expression of pLAT52-GFP in
the vegetative cell (v) and pHTR10-HTR10-RFP in the sperm cells
(s). (C) Embryo sac with expression of pEC1-HISTONE2B-RFP in
the egg cell (ec) and pFWA-GFP in the central cell (cc). Bars
represent 10 lm.
A germline-specific HISTONE 3 encoded by the gene
HISTONE THREE RELATED 10 (HTR10; also known as
AtGMH3) eventually provided a marker of sperm cells that
fluoresces at levels required for in vivo live imaging (Okada
et al., 2005, 2006; Ingouff et al., 2007). The expression of
HTR10 fused to monomeric RFP under the control of its
own promoter provides a strong fluorescent signal only in
sperm cells in mature pollen. HTR10-RFP is not expressed
in microspores. HTR10 expression is initiated after the
unequal division leading to bicellular pollen and remains
confined in the generative cell and the sperm cells (Fig. 1).
HTR10 is only expressed in pollen and not in other cells
than the generative cell and sperm cells (Okada et al., 2005;
Ingouff et al., 2007). The HTR10 promoter can be used to
drive expression of other genes in the sperm cell lineage
(Brownfield et al., 2009).
Marking the female gametophyte in
Arabidopsis
A systematic search for genes expressed in the female
gametophyte has led to the identification of promoters
causing expression in all or only some of the four cell types
that constitute the female gametophyte (Steffen et al., 2007;
Wang et al., 2010). Most promoters identified do not cause
a specific expression in the gametophyte, nor in a single
gametophytic cell type. The promoters of the genes
corresponding to the AGI At5G01860 (Wang et al., 2010)
and At5G40260 (Pagnussat et al., 2007) allow expression in
all gametophytic cell types and are not active after
fertilization. Such promoters can be used to test the impact
of gene activity during gametophytic life and to label both
the egg cell and the central cell. Several promoters,
including the promoter of the gene MYB98 (Steffen et al.,
2007), cause a fairly specific expression in the synergids,
which can be useful for monitoring pollen tube arrival.
Amongst several promoters, the promoter of the imprinted
genes FWA confers a central cell-specific expression
(Kinoshita et al., 2004).
Egg cells have been isolated from various species including maize (Cordts et al., 2001; Yang et al., 2006), wheat
(Triticum cereale) (Sprunck et al., 2005), and Arabidopsis
(Wuest et al., 2010). EST sequencing and microarray
analyses have been used to identify transcripts from isolated
egg cells and have yielded some insights into the biology of
the angiosperm female gamete. Surprisingly, only a few
genes have been identified as being expressed specifically in
the egg cell and not in other cells from the female
gametophyte and the sporophytic tissues (Gross-Hardt
et al., 2007; Steffen et al., 2007; Alandete-Saez et al., 2008;
Ingouff et al., 2009; Wang et al., 2010). Transgenic lines
expressing the fluorescent HISTONE2B-RFP under the
control of the EC1 promoter enables specific and robust
labelling of the egg cell nucleus (Fig. 1C) (Ingouff et al.,
2009).
Imaging double fertilization
The small size and transparency of Arabidopsis reproductive
organs are suitable for imaging double fertilization. An
experimental set-up has been developed for imaging
in planta (Rotman et al., 2003). Four hours after pollination,
the pistils are placed on double-sided tape and one ovary
wall is removed. A joint of silicon grease around the
double-sided tape supports a coverslip, thus defining a small
humid chamber. Fertilization and early endosperm and
zygote development can be observed using confocal microscopy in vivo for up to 24 h after dissection. This set-up
preserves as accurately as possible the normal conditions of
fertilization since no liquid is in contact with the ovule and
fertilization takes places inside the ovary. This set-up was
used for imaging the discharge of the pollen tube contents
in the female gametophyte (Rotman et al., 2003) and might
be used to monitor sperm cell fusion and karyogamy. One
disadvantage of imaging in planta is that it involves an air
interface between the ovule and the coverslip, leading to
relatively poorer optical resolution compared with when
water immersion is employed in another in vivo imaging setup described hereafter. The in vivo imaging set-up was
adapted from the method developed with Torenia fournieri
(Higashiyama et al., 1998; Palanivelu and Preuss, 2006) and
consists of excised ovules placed on medium on which
pollen tubes grow (Sandaklie-Nikolova et al., 2007). This
system enables higher resolution but does not reflect so
1654 | Berger
precisely in vivo conditions because the ovules are excised
and placed on semi-liquid medium and pollen tubes do not
grow all the way inside maternal tissues. This set-up was
used to image the delivery of sperm cell mitochondria
labelled with GFP tagged with the N-terminal targeting
signal from the gene encoding a mitochondrial F1-ATPase d
subunit (Matsushima et al., 2008). This method also
allowed imaging of sperm cell discharge, and karyogamy
using sperm cells tagged with HTR10-RFP (Ingouff et al.,
2007). In vivo imaging was performed using a disc-scan
confocal scanning laser microscope equipped for rapid
recording of z-stacks, and a prism to monitor two colours
at the same time. Images could be recorded at a rate of one
per 15 s or 30 s. The chromatin of nuclei of male gametes
was labelled with HTR10-RFP and the female gametes
were labelled with GFP expressed under the promoter of
FERTILIZATION INDEPENDENT ENDOSPERM. It was
possible to observe the discharge of sperm cells, followed by
the observation of karyogamy (Fig. 2A).
These series of experiments allowed the dynamics of the
paternal chromatin upon double-fertilization to be monitored in vivo. Karyogamy results from the fusion between
the male and female gamete nuclei and takes place about
1 h after release of the gametes (Fig. 2A). The spermcontributed HISTONE 3 (H3) variant HTR10 is actively
Fig. 2. Double fertilization in Arabidopsis. (A) Karyogamy upon
fertilization of an embryo sac marked with GFP expression under
the FWA promoter in the central cell (cc) 7 h after pollination with
tricellular pollen with sperm cells marked by the expression of
pHTR10-HTR10-RFP. The paternal chromatin in red mixes with
the maternal chromatin in the central cell nucleus and in the egg
cell nucleus (ec). (B) Activation of transcription and translation
marked by the expression of CENH3-GFP from the paternal allele
after fertilization of an embryo sac marked by expression of pEC1HISTONE2B-RFP in the egg cell. The maternal chromatin in the
zygote nucleus (z) is marked in red with green dots corresponding
to centromeres. Centromeres are also marked by de novo synthesis
of CENH3-GFP in endosperm (ed) nuclei. Bars represent 10 lm.
removed within a few hours from the zygotic nucleus
(Ingouff et al., 2007). Replacement of paternally inherited
HTR10 does not occur after fertilization of the central cell,
suggesting that the two fertilization events are not equivalent in terms of chromatin dynamics (Ingouff et al., 2007).
Using the ubiquitously expressed centromeric histone
variant H3 (CENH3), in vivo imaging enabled the earliest
timing of zygotic activation to be determined (Aw et al.,
2010) (Fig. 2B). CENH3-GFP is removed from the zygote
with dynamics similar to that of HTR10 (F Berger,
unpublished data). The absence of CENH3-GFP at the end
of karyogamy allows recording of the onset of the activity
of the transcription and translation machinery after fertilization. In developing seeds resulting from crosses between
wild type pollen and ovules of a transgenic line expressing
CENH3-GFP, CENH3 is expressed in the zygote from the
maternal allele as early as 8 h after fertilization (Aw et al.,
2010). This indicates that transcription and translation are
intiated in the zygote earlier than previously thought (Pillot
et al., 2009).
New insights beyond description
In planta and in vivo imaging protocols not only provide
a more accurate description of double-fertilization, but also
allows the phenotype of mutants defective for fertilization
to be analysed in depth. Hence, live imaging provides
mechanistic insights into double-fertilization.
Studies of mutants that impair the dialogue between the
synergids and the pollen tube and regulates sperm cell
release have identified a new signalling pathway responsible
for the control of sperm discharge. In vivo imaging showed
that, in the mutant sire`ne, the pollen tube grows and coils
in the female gametophyte without releasing its content
(Rotman et al., 2003). The gene affected in the mutant
sire`ne was later cloned from the allele feronia and shown to
encode a receptor-like kinase (RLK), which accumulates on
the synergid plasma membrane (Escobar-Restrepo et al.,
2007). Other mutations causing a phenotype similar to
sire`ne and feronia have been characterized. ANXUR1 and
ANXUR2 are paralogues of the RLK FERONIA and are
expressed in the pollen tube. ANXUR proteins are present
in the plasma membrane and might participate in signalling
events in the FERONIA pathway (Miyazaki et al., 2009)
indicating that male and female gametes exchange signals
regulating pollen tube arrest and gamete release. The female
gametophytic mutants lorelei, scylla, and sire`ne share the
same phenotype (Capron et al., 2008; Rotman et al., 2008).
The gene SCYLLA has not been identified. LORELEI
encodes a GPI-anchored G protein, which could be involved
in signalling events downstream of FERONIA (Capron
et al., 2008). The mutant abstinence by mutual consent (amc)
(Boisson-Dernier et al., 2008) is characterized by a feronia
phenotype but shows both female and male gametophytic
effect. The role played by the AMC peroxin and the function
of peroxysomes in fertilization remains unclear.
In a few species double fertilization involves dimorphic
female gametes and dimorphic sperm cells, while in most
Imaging double fertilization | 1655
species sperm cells are not distinguished by their morphology (Lord and Russell, 2002). Still it was proposed that
molecular functions are distinct between the two sperm cells
and that one sperm cell is fated to fuse with the central cell
while the other is fated to fuse with the egg cell (Lord and
Russell, 2002). This hypothesis was tested in Arabidopsis
using ovules of the mutants eostre (Pagnussat et al., 2007)
and retinoblastoma related (rbr) (Ingouff et al., 2006), which
produce two egg cells. It was possible to monitor directly
the fusion of each sperm cell with each egg cell of the rbr
mutant ovule (Ingouff et al., 2009) and to show that two
embryos are produced (Ingouff et al., 2009), thus leading to
the conclusion that each sperm cell is able to fuse with an
egg cell. It was also shown, using live markers, that mutants
in the CHROMATIN ASSEMBLY FACTOR pathway
(Chen et al., 2008) and for the CYCLIN DEPENDENT
KINASE A;1 (CDKA;1) (Aw et al., 2010) produce a fraction
of pollen with a single sperm cell. The cdka;1 single sperm
cell is able to fertilize either the central cell or the egg cell,
which supports the idea that both sperm cells are equivalent. All the above results have been obtained using either
mutant male or mutant female gametes. Such mutations
may suppress the hypothetical mechanisms that may
distinguish each sperm cell within the male germ unit. To
know whether the two sperm cells are identical in Arabidopsis will ultimately require a protocol involving both
wild-type male and female gametes.
Initial studies of the cdka;1 phenotype had concluded
that the mutant pollen produces a single sperm cell, that
fertilizes the egg cell preferentially, causing the development
of an embryo, which would produce a signal activating the
central cell division (Iwakawa et al., 2006; Nowack et al.,
2006). Live imaging applied to the analysis of the impact of
cdka;1 on sexual reproduction has showed that, in a large
fraction of cdka;1 pollen, the single sperm cell divides
during pollen tube growth. Hence a majority of cdka;1
pollen tubes deliver two sperm cells. When cdka;1 pollen
fertilizes wild-type ovules, one sperm cell fuses with the egg
cell and the other sperm cell fuses with the central cell.
Karyogamy is successful in the egg cell but not in the
central cell. Sperm entry triggers cell division in the central
cell, but after a few nuclei divisions, the endosperm-like
structure aborts (Aw et al., 2010). These results indicate that
the initiation of development of an endosperm-like structure
by pollination with cdka;1 pollen does not result from
a signal from the embryo as initially proposed (Nowack
et al., 2006), but from an event associated with sperm entry.
In addition, the absence of paternal genome expression in
the endosperm leads to early abortion, which does not
support the functional significance of a potential delay in
paternal genome expression (Vielle-Calzada et al., 2000).
Unanswered questions
How exactly does sperm cell discharge take place?
From the live imaging of pollen tube growth to the point of
gamete release it is now clear that the pollen tube discharge
does not cause synergid death (Rotman et al., 2003;
Sandaklie-Nikolova et al., 2007). Still it remains unclear
what causes synergid death and whether male gametes are
delivered inside a degenerated synergid or if the gametes are
discharged between one synergid and the egg cell. Sperm
cell discharge occurs in less than 30 s. To answer these
questions it will be necessary to develop imaging technology
that allows sampling high definition images every 5 s, which
should be possible with the latest generation of confocal
microscopes and transgenic plant lines that combines
several markers for the male and female gametes.
How do sperm cells migrate?
Dynamic observations suggest that after gamete fusion, the
sperm cell nuclei migrate actively toward the nucleus of
each female gametes. This migration may involve the
actin cytoskeleton as suggested by immunolocalizations in
tobacco and Torenia (Huang and Russell, 1994; Fu et al.,
2000). Live imaging using newly developed markers of actin
Lifeact-GFP (Era et al., 2009) and GFP-TUBULINa6
microtubules (Oh et al., 2010) will allow the dynamic
aspects of the cytoskeleton after gamete fusion and its
potential association with nuclei migration to be precisely
determined.
What prevents polyspermy?
If the two sperm cells have an equivalent capacity to fuse
with the egg cell and the central cell, there must be
a mechanism preventing both sperm cells fusing with only
one of the two female gametes. A limited number of studies
in maize and Arabidopsis have suggested that the fertilization of the central cell causes a polyspermy block that is
relatively inefficient in contrast to the fertilization of the egg
cell (Scott et al., 2008; Spielman and Scott, 2008). These
studies were based on mutant pollen that delivers additional
sperm cells and it is still unknown what happens in the wildtype background. The lack of fluorescent markers for the
plasma membrane hampers the establishment of the precise
timing of gamete fusion. The site of gamete fusion is also
not well defined. Live imaging with faster acquisition rates
and higher optical resolution will be required to know
whether the two sperm cells fuse simultaneously with
each female gamete or if the fusions occurs sequentially,
a prerequisite to understand the need for a polyspermy
prevention mechanism.
The regulation of the cell cycle during double-fertilization
and in the zygote
We still do not know at what stage of the cell cycle
fertilization takes place. Convincing evidence has indicated
that sperm cells are discharged after the S phase (Durbarry
et al., 2005). The fact that mitosis is initiated immediately
after karyogamy with the central cell nucleus also suggests
that the central cell has reached the G2 phase at the time of
fertilization. However, the regulation of the cell cycle in the
zygote remains unclear. The 12 h delay between karyogamy
1656 | Berger
and zygotic mitosis suggests two scenarios. (i) The male and
female gametes all undergo DNA replication and karyogamy takes place in the G2 phase. The requirement for
zygotic activation of transcription and translation accounts
for the 12 h that elapses between karyogamy and the first
mitosis in the fertilized egg. The fertilized central cell is
transcriptionaly active immediately after fertilization (Aw
et al., 2010) and is thus able to undergo mitosis immediately. (ii) The sperm cell and the central cell fuse after DNA
replication while the egg cell does not undergo DNA
replication. However, it is difficult to conceive how, after
gamete nuclei fusion, the maternal genome would undergo
S phase while the male genome has already duplicated in
the zygotic nucleus. Both male and female gametes may
undergo the S phase prior to fertilization and both gamete
fusion events take place in G2 phase. The delayed mitosis in
the zygote (relative to the immediate mitosis onset in
the central cell) might be associated with a slight delay
in transcriptional activation (estimated to a few hours), the
acquisition of polarity, and directional elongation of the
zygote. Investigations of these questions require the development of fluorescent markers that accurately reflect the
dynamics of the cell cycle regulators, which has not currently
been achieved.
Acknowledgement
FB’s research is funded by Temasek Lifescience Laboratory.
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