Developmentally regulated expression of the regulator of G-protein signaling gene 2 (Rgs2) in the...
Transcript of Developmentally regulated expression of the regulator of G-protein signaling gene 2 (Rgs2) in the...
Developmentally regulated expression of the regulator of G-protein
signaling gene 2 (Rgs2) in the embryonic mouse pituitary
L.D. Wilsona,b, S.A. Rossa, D.A. Leporea, T. Wadac, J.M. Penningerc, P.Q. Thomasa,*
aMurdoch Childrens Research Institute, Royal Childrens Hospital, Melbourne, Vic. 3052, AustraliabDepartment of Paediatrics, University of Melbourne, Melbourne, Vic. 3010, Australia
cInstitute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr Bohrgasse 3-5, Vienna A-1030, Austria
Received 20 July 2004; received in revised form 22 October 2004; accepted 22 October 2004
Available online 8 December 2004
Abstract
During the development of the anterior pituitary gland, five distinct hormone-producing cell types emerge in a spatially and temporally
regulated pattern from an invagination of oral ectoderm termed Rathke’s Pouch. Evidence from mouse knockout and ectopic expression
studies indicates that 12.5 days post coitum (dpc) to 14.5 dpc is a critical period for the expansion of the progenitor cell pool and the
determination of most hormone-secreting cell types. While signaling proteins and transcription factors have been identified as having key
roles in pituitary cell differentiation, little is known about the identity and function of proteins that mediate signal transduction in progenitor
cells. To identify genes that are enriched in the embryonic pituitary gland, we compared gene expression in 14.5 dpc pituitary and 14.5 dpc
embryo minus pituitary tissues using the NIA 15K microarray. Analysis of the data using the R program revealed that the Regulator of G
Protein Signaling 2 (Rgs2) gene was 3.9-fold more abundant in the 14.5 dpc pituitary. In situ hybridisation confirmed this finding, and
showed that Rgs2 expression in midline tissues was restricted to the pituitary and discrete regions of the nervous system. Within the pituitary,
Rgs2 was expressed in undifferentiated cells, and was downregulated at the completion of the hormone cell differentiation. To investigate
Rgs2 function in the pituitary, we examined hormone cell differentiation in Rgs2 null neonate mice. Pituitary cell differentiation and
morphology appeared normal in the Rgs2 mutant animals, suggesting that other Rgs family members with similar activities may be present in
the developing pituitary.
q 2004 Elsevier B.V. All rights reserved.
Keywords: Rgs2; Pituitary; Wnt signaling; G protein; Microarray
The transformation of an embryo into a mature organism
requires massive expansion and directed differentiation of
progenitor/stem cell populations. Identification of genes that
control this process is one of the main objectives of
developmental biology. Many components of the genetic
program that direct organogenesis of the murine pituitary
have already been identified, and have been shown to have
evolutionarily conserved functions (Scully and Rosenfeld,
2002; Dattani and Robinson, 2000). These data provide a
framework for the understanding of additional genes that are
1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.modgep.2004.10.005
* Corresponding author. Address: Pituitary Research Unit, Murdoch
Childrens Research Institute, Royal Childrens Hospital, Melbourne, Vic.
3052, Australia. Tel.: C61 3 8341 6285; fax: C61 3 9348 1391.
E-mail address: [email protected] (P.Q. Thomas).
implicated in this process, and their possible involvement in
the aetiology of birth defects.
The pituitary gland controls a wide range of fundamental
bodily activities, including growth, reproduction, thyroid
function and the ability to cope with stress. These functions
are mediated by hormones secreted from five distinct cell
types, all of which differentiate during embryogenesis. The
anterior and intermediate lobes of the pituitary gland develop
from a midline invagination of oral ectoderm known as
Rathke’s Pouch (RP), which in murine embryos is formed
around 9.0 days post coitum (dpc). By 12.5 dpc, RP extends
dorsally and expands to form a flattened epithelium, which is
closely associated with the infundibular recess of the
diencephalon. At this time, many of the anterior pituitary
progenitor cell types are believed to be specified, in a process
that involves Wnt and other signaling proteins emanating
Gene Expression Patterns 5 (2005) 305–311
www.elsevier.com/locate/modgep
Fig. 1. RT-PCR validation of Pit and E-Pit samples. RT-PCR analysis of
E-Pit (lanes 1, 3 and 5) and Pit (lanes 2, 4 and 6) samples for expression of
the ubiquitous gene GAPDH (lanes 1 and 2), and the pituitary genes Hesx1
(lanes 3 and 4) and Prop1 (lanes 5 and 6). Note the absence of Hesx1 and
Prop1 PCR products in the E-Pit sample.
L.D. Wilson et al. / Gene Expression Patterns 5 (2005) 305–311306
from the pituitary and neighbouring tissues (Treier et al.,
1998; Cha et al., 2004; Kioussi et al., 2002; Ericson et al.,
1998). Progenitor cell differentiation is also evident from this
stage, with the appearance of the alpha Glycoprotein Subunit
(aGSU)-positive rostral tip thyrotrophs in the ventro-anterior
region. At 14.5 dpc, the anterior pituitary primordium has
expanded further, and contains a mixture of Prop1-positive
progenitor cells and Pomc1-positive corticotrophs. Terminal
differentiation of the remaining hormone-secreting cells
occurs prior to birth and is marked by the expression of
luteinizing hormone (LH) and follicle-stimulating hormone
(FSH) in gonadotropes, growth hormone (GH) in somato-
trophs, and prolactin (PRL) in lactotrophs.
While the importance of signaling proteins for organo-
genesis is well established, in most cases little is known about
the molecular events that occur in response to receptor–
ligand interaction. One group of proteins, which mediate
signal transduction in a broad range of cellular contexts are
the Regulator of G Protein Signaling (RGS) family. The RGS
family consists of 25 members all of which share a common
domain implicating them in heterotrimeric GTP-binding
protein (G proteins) signal termination. Heterotrimeric G
proteins are intracellular signaling molecules that are
activated by seven transmembrane receptors (Hamm and
Gilchrist, 1996; Neer, 1995) and are composed of a, b and gsubunits. Many neurotransmitters and hormones exert their
effect on target tissues by activating receptors that are
coupled to G proteins (Bourne et al., 1990). Activation of
these receptors results in exchange of GTP for GDP on Gasubunits and the dissociation of GTP-bound active Gasubunits from the Gbg heterodimers, resulting in the
activation of downstream effector pathways. The function
of RGS family members is to promote the intrinsic GTPase
activity of the a subunit, resulting in the rapid deactivation
and reformation of the inactive Gabg heterotrimer (Druey
et al., 1996; Hunt et al., 1996; Watson et al., 1996). Given the
importance of signaling in many developmental processes,
including pituitary organogenesis, RGS proteins may also
function during development. However, to date, the
expression and function of RGS genes in the developing
pituitary had not been examined.
In this report, we use a microarray screening strategy to
identify genes that have enriched expression in the murine
pituitary. We show for the first time that Rgs2 is
developmentally regulated in the embryonic pituitary, and
is downregulated after birth. We also investigate the
function of Rgs2 in the pituitary using Rgs2 null animals.
1. Results
1.1. Identification of Rgs2 as a pituitary-enriched
transcript using microarray
A critical time-point for the commitment and differen-
tiation of murine pituitary progenitor cells is 14.5 dpc.
To identify genes that are selectively expressed in the
developing pituitary at this time, we used a microarray-
based screening strategy. Our approach was based on
the assumption that transcripts that are pituitary-specific
(or pituitary-enriched) will have greater relative abundance
in mRNA pools derived from 14.5 dpc pituitary tissue
versus 14.5 dpc whole embryos without pituitary tissue.
RNA was prepared from 14.5 dpc pituitary glands (Pit) and
14.5 dpc whole embryos from which the pituitary gland had
been removed (E-Pit). The integrity and authenticity of the
samples was established by RT-PCR analyses of the
pituitary genes Prop1 and Hesx1 (Fig. 1). To avoid
the possibility of identifying false positives due to non-
linear amplification of mRNA, we generated sufficient
mRNA from 14.5 dpc embryonic pituitary tissue for direct
labeling and hybridisation analysis. Fluorescently labeled
cDNA probes from each sample were cohybridised to the
mouse NIA 15K array (Kargul et al., 2001; Tanaka et al.,
2000), and relative expression calculated using the R
program. Of the 15,247 genes on the array, Rgs2 (Regulator
of G protein signaling 2) exhibited the greatest enrichment
score in the Pit sample (3.9-fold expression difference,
P!0.05, BZ12). In this report, we will focus on the
expression and function of the Rgs2 gene. Analyses of
other putative pituitary-enriched genes will be presented
elsewhere (manuscript in preparation).
1.2. Rgs2 is highly expressed and developmentally
regulated in the embryonic pituitary
To validate the results of the microarray experiment, we
performed in situ hybridisation analysis (ISH) of Rgs2 on
14.5 dpc whole embryo sagittal sections (Fig. 2). Rgs2
expression was most prominent in the developing pituitary
and discrete regions within the fronto-nasal mass and
hindbrain, confirming that Rgs2 is a pituitary-enriched
transcript (Fig. 2A). Within the anterior pituitary primor-
dium, Rgs2 expression was restricted to the ventro-posterior
Fig. 2. Rgs2 expression in the 14.5 dpc mouse embryo. (A) Sagittal section showing strong Rgs2 expression in the anterior lobe of the developing
pituitary (arrow). Expression is also evident in the dorsal hindbrain, hindbrain flexure and fronto-nasal mass (arrowheads). (B) Magnified view of the pituitary
shown in (A). (C) No signal is detected using an Rgs2 sense control probe.
L.D. Wilson et al. / Gene Expression Patterns 5 (2005) 305–311 307
quadrant, and was expressed by most cells within this
domain, including those that line the residual lumen of
Rathke’s Pouch. Low-level Rgs2 expression was also
evident in the infundibulum (Fig. 2B). To further charac-
terise the Rgs2-positive cell population, we compared Rgs2
expression with the pituitary markers Prop1 and aGsu.
Prop1 is a progenitor cell marker and is expressed by the
dorsal periluminal lining cells and occasional cells in the
ventro-posterior quadrant. Conversely, aGsu is expressed in
differentiated cells of the thyrotrope and gonadotrope
lineages which occupy the ventro-anterior domain of the
anterior pituitary at 14.5 dpc. No significant overlap in the
expression domains of Rgs2 and aGsu was detected
(Fig. 3A,B), indicating that Rgs2 is not expressed in
differentiated cells. In contrast, the Prop1 and Rgs2 zones
of expression partially overlap, suggesting that a subset of
Fig. 3. Rgs2 expression in the pituitary is restricted to a subset of undifferentiat
to compare Rgs2 (A), aGSU (B) and Prop1 (C) expression domains. (A) Rgs2
(B) Expression of the differentiated cell marker aGSU is restricted to the antero-ve
of Rgs2 and the progenitor cell marker Prop1 is evident in the ventro-anterior
Orientation is indicated by the arrows in panel A. Abbreviations: Dor, dorsal; Po
Prop1 cells express Rgs2 (Fig. 3A,C). A notable exception
is the dorsal Prop1Cluminal lining cells, which clearly do
not express Rgs2. These data suggest that Rgs2 is most
active in cells at an early stage of differentiation and is
downregulated prior to the onset of hormone gene
expression.
To investigate the developmental regulation of Rgs2 in
the embryonic and post-natal pituitary, we examined its
expression in 10.5, 12.5, 14.5, 16.5 dpc embryos and in 3
day neonates. At 10.5 dpc, Rgs2 is expressed at low levels in
the infundibulum and no expression is evident in Rathke’s
Pouch (RP; Fig. 4A). By 12.5 dpc, Rgs2 expression
is activated in the presumptive anterior lobe, where it is
highest in the progenitor cells that occupy the ventral half of
the primordium (Fig. 4B). The aGSU-positive rostral tip
thyrotropes which first appear at this time are negative for
ed cells. In situ hybridisation of serial 14.5 dpc sagittal sections was used
expression is confined to the ventro-posterior quadrant of the pituitary.
ntral region, and does not overlap with Rgs2 expression. (C) Co-expression
domains and not in the dorsal Prop1-positive cells that line the lumen.
s, posterior; Ven, ventral; Ant, anterior.
Fig. 4. Rgs2 is developmentally regulated throughout pituitary development and downregulated after birth. Rgs2 expression in midsagittal sections of the
developing pituitary at 10.5 dpc (A), 12.5 dpc (B), 14.5 dpc (C), 16.5 dpc (D) and post-natal day 3 (P3, E). (A) Rgs2 is not detected in Rathke’s pouch, and is
present at low levels in the infundibulum. (B) Rgs2 expression is highest to the ventral cells of Rathke’s Pouch. (C) Rgs2 is expressed in the undifferentiated
cells of the ventro-posterior domain. (D) At 16.5, Rgs2 expression is restricted to two patches of periluminal cells (arrows), which may correspond to residual
progenitor cells. (E) No expression of Rgs2 is evident at P3, indicating that Rgs2 is downregulated after birth. Sections are orientated as described for Fig. 3.
L.D. Wilson et al. / Gene Expression Patterns 5 (2005) 305–311308
Rgs2 expression (data not shown). Rgs2 expression
continues in the ventral most part of the presumptive
anterior pituitary gland at 14.5 dpc, and by 16.5 dpc is most
abundant in the periluminal cells of the ventral and medial
regions (Fig. 4C,D). Three days after birth (P3), the pituitary
is fully differentiated, and contains all of the hormone-
secreting lineages (Scully and Rosenfeld, 2002). At this
time-point, Rgs2 was not detected, indicating that the
activity of this gene is restricted to the cell specification and
differentiation phase of pituitary development (Fig. 4E).
1.3. Rgs2 is not required for pituitary cell differentiation
To explore the function of Rgs2 in pituitary develop-
ment, we examined gland morphology and hormone-
secreting lineage differentiation in Rgs2 null neonate
mice (Oliveira-Dos-Santos et al., 2000). Corticotropes,
somatotropes and gonadotropes/thyrotropes were identified
by expression of Pomc1, GH and aGSU, respectively. Cells
belonging to each of these lineages were present in the
mutant pituitaries, and no difference in their number or
position was apparent when compared to wildtype or
heterozygous littermates (Fig. 5 and data not shown).
Pituitary gland morphology was also normal in the Rgs2
mutants. These data indicate that Rgs2 is not absolutely
required for pituitary cell differentiation.
2. Discussion
Microarray analysis has proven to be a powerful method
for the identification of genes which regulate their
expression in response to changing physiological circum-
stances and developmental contexts (Luo et al., 2003;
Ramalho-Santos et al., 2002; Buttitta et al., 2003). We have
used this technology to screen for genes, which are
developmentally regulated in the embryonic pituitary, by
comparing the transcriptional profiles of 14.5 dpc pituitary
versus 14.5 dpc embryo minus pituitary. Rgs2 exhibited
the highest fold-difference in relative gene expression and
was 3.9 times more abundant in the Pit mRNA sample.
Using ISH analysis, we show for the first time that Rgs2
expression in midline tissues is restricted to the 14.5 dpc
ventral pituitary, and a few additional sites including the
CNS and fronto-nasal mass. These expression data indicate
that the 3.9-fold expression difference indicated by the array
is probably a reasonable estimate of the relative abundance
of Rgs2 transcripts in the Pit versus E-Pit samples. We
therefore conclude that the screening strategy that we have
used is a useful method for identifying novel developmen-
tally regulated genes in the pituitary, and could be readily
applied to other embryonic tissues.
Rgs2 has previously been implicated as having a role in
the deactivation of the Gq family members but has limited
Fig. 5. Rgs2 is not required for pituitary hormone cell differentiation. In situ hybridisation of wild-type (A, C and E) and Rgs2 null (B, D, and F) neonate
transverse sections did not reveal any difference in number or location of the somatotropes (A, B), thyrotropes/gonadotropes (C, D) or corticotropes (E, F).
L.D. Wilson et al. / Gene Expression Patterns 5 (2005) 305–311 309
activity towards the other subunit family members
(Heximer et al., 1997a; Heximer et al., 1999). In vitro
studies also indicate that Rgs2 is able to regulate signaling
from different G protein Coupled Receptors in a variety of
cell types (Kehrl and Sinnarajah, 2002). Post-natally, Rgs2
is expressed in many tissues including the immune system,
brain and heart (Ingi and Aoki, 2002; Chen et al., 1997;
Heximer et al., 1997), suggesting that this gene may have a
functional role in multiple tissues. This is supported by the
phenotype of Rgs2 null mice, which exhibit impaired
function of hippocampal neurons, increased anxiety
responses, decreased aggression in males, impaired T-cell
function and hypertension (Olivera-dos-Santos et al., 2000;
Heximer et al., 2003; Tang et al., 2003). While these data
have established a role for Rgs2 in adult mice, little is
known about the possible developmental role of this gene.
We have shown that Rgs2 is expressed in the embryonic
pituitary gland and is downregulated post-natally,
suggesting that this gene may have a functional role in
pituitary organogenesis. Within the anterior pituitary
primordium, Rgs2 expression is restricted to a subset of
progenitor cells which appear, in some cases, not to express
Prop1, suggesting that they are at an early stage of
differentiation. These data suggest that in the pituitary,
Rgs2 may be involved in the regulation of signal
transduction pathways that modify progenitor cell behavior
in response to environmental cues. While is it not clear at
present which signal transduction pathways may be
regulated by Rgs2 in the pituitary, Xenopus microinjection
experiments performed by Wu et al. (2000) may provide a
clue. These authors showed that global overexpression of
RGS2 and rRgs4 in Xenopus results in marked inhibition of
trunk development, phenocopying the effects of dominant
negative Xwnt-8 or frzb microinjection. In addition, RGS4
was shown to block the ability of XWnt8 to induce axis
duplication and mesoderm ventralisation. These data
indicate that Rgs2/4 inhibit Wnt signaling, possibly by
promoting the GTPase activity of Gq proteins associated
with frizzled receptors. Interestingly, genes encoding Wnt
signaling components are expressed in the embryonic
pituitary, and functional studies in mice have demonstrated
the importance of this signaling pathway in pituitary
morphogenesis and differentiation of the hormone-secreting
lineages (Cha et al., 2004; Kioussi et al., 2002; Treier et al.,
1998). Taken together, these data suggest that Rgs2 may
play a role in developmental events that require Wnt
signaling in the pituitary.
To investigate the function of Rgs2 in the pituitary, we
analysed hormone cell differentiation in Rgs2 null neonates.
The somatotroph, gonadotroph, thyrotroph and corticotroph
L.D. Wilson et al. / Gene Expression Patterns 5 (2005) 305–311310
lineages all appear to differentiate normally in the absence
of Rgs2, consistent with the normal growth and fertility of
these mutant mice (Olivera-dos-Santos et al., 2000). This
apparent lack of pituitary phenotype in the Rgs2 null mice
may be due to their genetic background, which has been
shown to modify the phenotypic penetrance of other
developmental genes (e.g. Dixon and Dixon, 2004). Another
possibility is that Rgs2 shares functional redundancy with
other RGS family members that are expressed in the
embryonic pituitary. To date, Rgs2 is the only RGS gene
that has been shown to be expressed in the pituitary.
However, many Rgs family members are expressed in the
adult mouse pituitary (Chen et al., 1997), including Rgs16
which has been identified in a 14.5 dpc pituitary library
(http://www.ncbi.nlm.nih.gov/UniGene/). Full understand-
ing of the developmental role of Rgs2 in the murine pituitary
will therefore require gain-of-function studies, including
ectopic and overexpression analysis in transgenic animals.
3. Experimental procedures
3.1. Mouse embryo dissection
Pituitary glands were dissected from 139 HSDola-Swiss
14.5 dpc mouse embryos, generated from the breeding
colony at Melbourne University, Australia. The embryo
minus pituitary RNA was prepared from a single 14.5 dpc
embryo from which the pituitary gland was removed. All
tissue samples were dissected under a microscope and were
processed immediately.
3.2. RNA isolation and RT-PCR
Total RNA was isolated from dissected tissues using a
Qiagen RNeasy Kit. RNA was quantified by A260/A280
absorbance ratios. Approximately 30 mg of total RNA was
isolated from the 139 embryos dissected, and the one whole
embryo minus the pituitary gland. The integrity of RNA
samples including the 18S and 28S ribosomal RNA
bands were assessed by agarose gel electrophoresis. RT-
PCR analysis was performed using Epicenter buffers and
the following primer sequences: Prop1 5 0gagctggggaga-
cctaagctttgcc, 5 0gctgggtgcaaggtggggtacca; Hesx1 5 0tttca-
gcctccgaaacacgctct, 5 0ctctgatgtcaatgccagggtagca; GAPDH
5 0acccagaagactgtggatgg, 5 0cttgctcagtgtccttgctg.
3.3. Probe preparation and microarray hybridisation
7.5 mg of total RNA was reverse-transcribed using
Superscript (( RT kit (Gibco-BRL, USA) and AncT primer
as instructed by the manufacturer. Indirect labeling of the
cDNA was performed by adding amine-modified amino
allyl dNTPs (dUTP containing allyl link; Sigma) during the
reverse transcription reaction. RNA was hydrolysed with
0.25 M NaOH (BDH) and 0.5 M EDTA at 65 8C for 15 min
followed by the addition of 0.2 M acetic acid (BDH) to stop
the hydrolysis. The cDNA was then purified using a
MiniElute column (Qiagen) according to manufacturer’s
directions and coupled to Cy3 or Cy5 reactive dye
(Amersham, UK) resuspended in 0.1 M sodium bicarbonate
buffer pH.9 (BDH) by incubation for 1 h in the dark. The
coupled cDNA samples were then purified on MiniElute
columns (Qiagen) and combined with 4 mg/ml tRNA
(Sigma), 10 mg/ml Cot-1 DNA (Invitrogen) and 8 mg/ml
Poly dA oligonucleotide (Geneworks, Australia). The
labeled probe was combined with 5! SSC, 50% deionised
formamide (Merck), 1.25% SDS and denatured at 95 8C for
5 min. The probe was then incubated for a further 30 min at
45 8C and put on ice for 30 s. The microarray slides were
pre-heated on a heat block for 2 min and placed back-to-
back. The labeled probe mix was then added to the edge of
the microarray slides, allowed to diffuse into the slides
and incubated at 42 8C for 16 h in a humidified chamber.
Post-hybridisation, slides were washed for 1 min in
0.5! SSC/0.01% SDS, 3 min in 0.5! SSC and 3 min in
0.06! SSC before being rinsed in sterilised water and spin
dried at 800 rpm in a 5810 R centrifuge (Eppendorf,
Germany) and scanned immediately. The experiment used
four microarrays in two dye-swap pairs. Since the slides
were printed in duplicate, each clone was represented eight
times in the analysis.
3.4. Data analysis
The microarrays were scanned with a GenePix 4000B
microarray scanner (Axon Instruments, Amersham Pharma-
cia Biotech) and the data analysed using the ‘R’ program, a
language and environment for statistical computing and
graphics, developed by John Chambers et al. at Bell
Laboratories (University of Toronto). The information
generated from the ‘R’ program analyses included the fold
change (M-value), the ordinary t-statistic, the moderated
t-statistic and the B-statistic for each gene. The data
generated was normalised using print-tip loess normal-
isation using the LIMMA package (Smyth, 2003). Print-tip
loess normalisation, normalises each M-value by subtract-
ing from it the corresponding value of the tip group loess
curve. This method corrects the M-values for both sub-array
spatial variation and for intensity-based trends. Between
arrays normalisation is a simple scaling of the M-values
from a series of arrays so that each array has the same
median absolute deviation (Smyth, 2003).
3.5. In situ hybridisation
Mouse embryos were fixed for 18 h at 4 8C in
paraformaldehyde (4% (w/v) in PBS) and then placed in
sucrose (20% (w/v) in PBS) until they sank. After
embedding in OCT (TissueTek), 16-mm cryostat frozen
sections were prepared and mounted onto Superfrost-Plus
slides. Gene expression was detected by hybridising
L.D. Wilson et al. / Gene Expression Patterns 5 (2005) 305–311 311
digoxigenin-labeled antisense riboprobes as described
previously (Dunwoodie et al., 1997). The Rgs2 riboprobe
spans nucleotides 1–1627 bp (Accession No. NM_009061),
and all other probes were described previously (Raetzmann
et al., 2004).
3.6. Rgs2 mutant analysis
Rgs2 null mice were genotyped as described previously
(Oliveira-Dos-Santos et al., 2000) and were maintained on a
C57BL/6J genetic background. A total of six null neonates
were sectioned and examined by in situ analysis.
Acknowledgements
We thank Ms Kelly Roeszler for technical assistance, and
Dr Katrina Bell and Mr Michael Hildebrand for assistance
with the microarray experiments. P.T. is an Australian
National Health and Medical Research Council R.D. Wright
Fellow.
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