Developmental expression of Pod 1 in Xenopus laevis.

Abstract

The basic helix-loop-helix transcription factor, Pod 1, has been shown to be expressed in the mesenchyme of many developing mouse organs, including the heart, lungs and gut. In the kidneys of developing mice, Pod 1 is highly expressed in the condensing metanephric mesenchyme, differentiating and late stromal cells and in developing podocytes. We have obtained an EST (CF270487) which contains the Xenopus laevis Pod 1 sequence. Conceptual translation of the Xenopus laevis Pod 1 sequence shows approximately 85% similarity to other vertebrate homologues. RT-PCR indicates that expression is initiated at stage 13 and increases differentially in the developing pronephros compared to the whole embryo. RT-PCR of a kidney dissection at stage 42 shows higher expression in the glomus than in the tubule or duct. In situ hybridisation analysis at tail bud stages shows the anterior-most branchial arch and pronephric glomus are intensely stained. At stage 40, staining persists in the glomus and in the epicardium region of the heart. Adult organ analysis shows expression is highest in the rectum and the spleen, with significant expression in the duodenum, heart, kidney, lungs, pancreas, skin, liver and muscle.

Full text

Page 1

Developmental Expression Pattern
Developmental expression of Pod 1 in Xenopus laevis
SUBREENA SIMRICK, KARINE MASSÉ and ELIZABETH A. JONES*
Molecular Physiology, Department of Biological Sciences, Warwick University, Coventry, U.K.
ABSTRACT The basic helix-loop-helix transcription factor, Pod 1, has been shown to be expressed
in the mesenchyme of many developing mouse organs, including the heart, lungs and gut. In the
kidneys of developing mice, Pod 1 is highly expressed in the condensing metanephric mesenchyme,
differentiating and late stromal cells and in developing podocytes. We have obtained an EST
(CF270487) which contains the Xenopus laevis Pod 1 sequence. Conceptual translation of the
Xenopus laevis Pod 1 sequence shows approximately 85% similarity to other vertebrate homo-
logues. RT-PCR indicates that expression is initiated at stage 13 and increases differentially in the
developing pronephros compared to the whole embryo. RT-PCR of a kidney dissection at stage 42
shows higher expression in the glomus than in the tubule or duct. In situ hybridisation analysis at
tail bud stages shows the anterior-most branchial arch and pronephric glomus are intensely
stained. At stage 40, staining persists in the glomus and in the epicardium region of the heart. Adult
organ analysis shows expression is highest in the rectum and the spleen, with significant
expression in the duodenum, heart, kidney, lungs, pancreas, skin, liver and muscle.
KEY WORDS: Pod 1, epicardin, capsulin, transcription factor 21, glomus
During development, basic helix-loop-helix transcription factors
are involved in cell lineage commitment and organogenesis. They
bind to E box regions (CANNTG) (Murre et al., 1989) and are
classified into two groups, ubiquitous class A and tissue specific
class B (Lassar et al., 1991). Pod 1, also known as Epicardin,
Capsulin, Transcription factor 21, is a class B basic helix-loop-helix
transcription factor (Hidai et al., 1998, Quaggin et al., 1998, Robb
et al., 1998; Lu et al., 1998). In murine development Pod 1 is first
expressed at 9.5 days post coitus (dpc) with high levels in the
presumptive epicardium and the mesenchymal cells surrounding
the gut, lung and kidney (Hidai, et al., 1998). Expression then
extends to the mesenchyme of the lung, metanephros, gonads and
the gut at 13.5 dpc (Lu et al., 1998, Robb et al., 1998).
In the adult mouse, RT-PCR reveals expression in the lung,
ovary, spleen, kidney, intestine and uterus (Hidai et al., 1998). In
situ hybridisation analysis, however, indicates that expression is
restricted to the podocytes lining the glomerulus, lungs and gut (Lu
et al., 1998).
Pod 1 has been shown to have multiple roles in vertebrate
development. The Pod 1 null mouse dies from multiple organ
failure. The kidneys show severe branching defects, lacking ma-
ture glomeruli and the lungs are deficient in alveoli (Quaggin et al.,
1999). Chimeric null Pod 1 mice show that although Pod 1 is not
required for the specification of glomeruli, podocytes and stromal
cells, it is required for their terminal differentiation and branching
morphogenesis (Cui et al., 2003). Pod 1 is also required for
Int. J. Dev. Biol. 49: 59-63 (2005)
doi: 10.1387/ijdb.051982ss
*Address correspondence to: Dr. Elizabeth A. Jones. Molecular Physiology Department of Biological Sciences, Warwick University, Coventry, CV4 7AL, U.K.
Fax: +44-24-7652-3701. e-mail: Elizabeth.Jones@warwick.ac.uk
Abbreviations used in this paper: BLAST, basic local alignment search tool; RT-
PCR, reverse transcription-polymerase chain reaction; UF, unfertilised.
0214-6282/2005/$25.00
© UBC Press
Printed in Spain
www.intjdevbiol.com
capillary remodelling, chimeric null Pod 1 mice having large,
dilated and poorly organised vascular structures (Cui et al., 2003).
Pod 1 appears to have a role in regulating differentiation and
proliferation of a variety of tissues. Pod 1 is required for the initial
differentiation and proliferation of the splenic lineage, where the
absence of Pod 1 results in apoptotic cell death (Lu et al., 2000).
In skeletal muscle, exogenous expression of Pod 1 inhibits the
terminal differentiation of C2C12 myoblasts (Funato et al., 2003).
Pod 1 knock-down using antisense oligonucleotides, inhibits the
differentiation of stratified cells to differentiated epithelia in stom-
ach tissue cultures in the presence of 1 µM hydrocortisone
(Andersson et al., 2001). Studies using Pod 1 null/GFP chimeric
mice reveal that the loss of Pod 1 results in an increase of
metanephric condensing mesenchyme and a failure of stromal
cells to differentiate (Cui et al., 2003). Pod 1 is also involved in
gonadogenesis. Pod 1 transcriptionally represses steroidogenic
factor 1, a regulator of sexual differentiation (Tamura et al., 2001,
Cui et al., 2004).
As a group B basic helix loop helix transcription factor, Pod 1 is
expected to either form homodimers or heterodimerise with class
A basic helix-loop-helix transcription factors. Although a number of
studies have been carried out, it is unclear which is the case in vivo.

Page 2

60 S. Simrick et al.
Gel mobility shift assays using E-box sequences as the probe have
shown that Pod 1 alone does not bind to DNA, but a heterodimer
of Pod 1 and E12 does bind to DNA (Lu et al., 1998). Yeast two-
hybrid screens have identified other possible Pod 1 heterodimer
complexes, HEB, HEB-s and ITF-2 (Miyagishi et al., 2000a,
Miyagishi, et al., 2000b). However, sequence analysis and in vitro
experiments have shown that Pod 1 alone is capable of binding
cognate E-box consensus sequences and activating
transcription (Hidai et al., 1998).
Sequencing and cloning
Using the BLAST programme (Basic Local Align-
ment Search Tool) (Altschul et al., 1990) and the
mouse Pod 1 homologue, O35437, we have identified
an EST, Expressed Sequence Tag, containing the full
Xenopus laevis Pod 1 sequence. This EST CF270487,
was obtained from the IMAGE consortium (http://
image.llnl.gov, IMAGE No. 5512805) and used in this
study. The published NCBI nucleotide sequence (http:/
/mgc.nci.nih.gov/) of this clone fails to identify a trans-
lational start site. The open reading frame runs from
23bp to 560bp, encoding a 179 amino acid protein that
is approximately 25kDa when separated on a 10%
SDS polyacrylamide gel (data not shown). In house
sequencing corrected the sequencing error by identi-
fication of the translational start site at an equivalent
point to that of the mouse sequence (AF029753).
Conceptual translation of the Xenopus laevis Pod 1
sequence showed 100% conserved identity in the
basic helix-loop-helix region between the vertebrate
Pod 1 homologues (Fig. 1A). A similarity of approxi-
mately 85% among the Pod 1 vertebrate homologues
was identified over the whole length of the protein (Fig.
1B).
Temporal expression of Xenopus laevis Pod 1
RT-PCR was performed on unfertilised Xenopus
laevis eggs and whole Xenopus laevis embryos at
stages 9, 13, 16.5, 21, 26.5, 30 and 35. This temporal
RT-PCR analysis indicated that Pod 1 expression was
initiated by stage 13 after which there was a gradual
increase in Pod 1 expression from stages 16.5 to 26.5
and maintenance at this concentration through the stages 30 and
35 (Figs. 2,3A). The input cDNA was approximately equalised
using the ubiquitously expressed gene ODC and a linearity using
doubling dilutions of stage 35 cDNA was performed. The Xenopus
laevis temporal expression pattern is comparable to that observed
in mouse embryos. Detection of Pod 1 in mouse embryos by
Northern blot analysis shows first expression at 8.5 dpc in the
branchial arches, after which there is an increase to 15.5 dpc where
it peaks at and persists to 17.5 dpc (Hidai et al., 1998). By
comparing the developmental stages at the start of neurulation,
stage 13 in Xenopus laevis, 8-9 dpc in mouse, the onset of Pod 1
expression is temporally similar.
Fig. 1. Alignment of related Pod 1 amino acid sequences. (A) Alignment of the
predicted X. laevis amino acid sequence (conceptually translated from CF270487), H.
sapiens Pod1 (NP_003197), M. musculus Pod 1 (NP_035675) and potential R.
norvegicus homologue Cor1 (XP_341738). Black shading indicates identical amino
acids, whereas the gray shading specifies the same amino acid family. (B) Percentage
identity (%). The table indicates paired percentage identity as given by BLAST (Basic
Local Alignment Search Tool) (Altschul et al., 1990).
-R-R
N
A
N
A
Pod 1 Pod 1
ODCODC
991313U
F
U
F
16½
(-) (-)0.25
0.125 0.50.5
21 21
30 30
26½
1.0 1.035 35 -R-R
TT
StagesStages Neg. controls Neg. controlsLinearity Linearity
16½
0.25
0.125
26½
Fig. 2. Temporal expression pattern of Pod 1 in X. laevis. RT-PCR was
performed on unfertilised Xenopus laevis eggs (UF) and whole Xenopus
laevis embryos at stages 9, 13, 16.5, 21, 26.5, 30 and 35, using Pod 1
primers. Equalisation was carried out using ODC as a loading control and
linearity was performed with doubling dilutions of input cDNA from stage
35 embryos to reduce the risk of a PCR plateau. Negative controls were
carried out as described in the methods. RT-PCR analysis shows that Pod
1 expression is not initiated until the beginning of neurulation, stage 13
followed by a gradual increase in expression until stage 35.
X.laevis
-
87
85
85
H.sapiens
-
-
96
97
M.musculus
-
-
-
99
R.norvegicus
-
-
-
-
X.laevis
H.sapiens
M.musculus
R.norvegicus
X.laevis
H.sapiens
M.musculus
R.norvegicus

MSTGSLSDVEDFQDMEMLECDGIKLDPNKEFGISNDSNEESSTCDNGSPK
MSTGSLSDVEDLQEVEMLECDGLKMDSNKEFVTSNESTEESSNCENGSPQ
MSTGSLSDVEDLQEVEMLDCDSLKVDSNKEFGTSNESTEEGSNCENGSPQ
MSTGSLSDVEDLQEVEMLDCDSLKVDSNKEFGTSNESTEEGSNCENGSPQ

Basic Helix
X.laevis
H.sapiens
M.musculus
R.norvegicus

KGRGTSGKRRKASSKKSPLGTINQEGKQVQRNAANARERARMRVLSKAFS
KGRGGLGKRRKAPTKKSPLSGVSQEGKQVQRNAANARERARMRVLSKAFS
KGRGGLGKRRKAPTKKSPLSGVSQEGKQVQRNAANARERARMRVLSKAFS
KGRGGLGKRRKAPTKKSPLSGVSQEGKQVQRNAANARERARMRVLSKAFS

Loop Helix
X.laevis
H.sapiens
M.musculus
R.norvegicus

RLKTTLPWVPPDTKLSKLDTLRLASSYIAHLRQILANDKYENGYIHPVNL
RLKTTLPWVPPDTKLSKLDTLRLASSYIAHLRQILANDKYENGYIHPVNL
RLKTTLPWVPPDTKLSKLDTLRLASSYIAHLRQILANDKYENGYIHPVNL
RLKTTLPWVPPDTKLSKLDTLRLASSYIAHLRQILANDKYENGYIHPVNL


X.laevis
H.sapiens
M.musculus
R.norvegicus

Conserved

TWPFMVAGKPENDLKEVVSTSRLCGPTAS
TWPFMVAGKPESDLKEVVTASRLCGTTAS
TWPFMVAGKPENDLKEVVTANRLCGTTAS
TWPFMVAGKPESDLKEVVTASRLCGTTAS
Same amino acid family
A
B
Temporal expression of Xenopus laevis Pod 1 in the proneph-
ros
Pronephric tissue or presumptive pronephric tissue was dis-
sected from Xenopus laevis embryos at stages 12.5, 15, 20, 28
and 35 (Brennan et al., 1998) and RT-PCR was performed. This
temporal RT-PCR of the whole embryo and kidney dissections
revealed an increased expression of Pod 1 in the developing
pronephros compared to the whole embryo (Fig. 3A). No expres-
sion was seen in stage 12.5 whole embryos or dissected proneph-
ros, thus confirming the initiation of Pod 1 expression at stage 13,
the start of neurulation. The input cDNA was approximately equalised

Page 3

Pod 1 in Xenopus laevis 61
in the glomerulus, peritubular interstitial cells, pericytes in the renal
vessels and adventitial cells in the blood vessels (Cui et al., 2003).
Spatial expression of Xenopus laevis Pod 1
In situ hybridisation analysis was performed on albino Xenopus
laevis embryos from stage 22 through to stage 40.5. The in situ
hybridisation analysis gave little staining at stage 22 with either the
anti-sense probe or the control sense probe, even when visualised
after clearing the embryos (Fig. 4A-B and data not shown). The first
indication of specific staining is seen initially in the anterior bran-
chial arch at stage 26 (data not shown) and in the branchial arch
and glomus at stage 28 (Fig C-D). Between stages 28 to 40.5 the
anterior branchial arch, followed by the remaining arches, are
Fig. 3. Whole embryo and dissected kidney temporal expression of
Pod 1 in X. laevis. (A) Pronephric tissue or presumptive pronephric tissue
was dissected from Xenopus laevis embryos at stages 12.5, 15, 20, 28 and
35 (Brennan, et al., 1998) and RT-PCR was performed using specific Pod
1 primers. The RT-PCR was equalised using ODC and linearity and
negative controls carried out as described in Experimental Procedures.
Expression of Pod 1 in whole embryos (W) was compared with that of the
kidney or kidney primordium dissections (K). The whole embryo temporal
expression pattern confirms the profile shown in Fig 2. The Pod 1
expression in the kidney dissections, however, is proportionally greater
than that of the whole embryo, first appearing during neurulation, stage 15
and increasing as the embryo develops. (B) The pronephros from a
Xenopus laevis stage 42 embryo was dissected into the glomus, tubules
and duct regions and RT-PCR using Pod 1 primers was carried out. Equalisation was carried out using EF1α and linearity and negative controls carried
out as described in Experimental Procedures. A fine dissection of the pronephros of a stage 42 X. laevis tadpole reveals the spatial expression of Pod1
within the pronephros. Pod 1 appears to be most expressed in the glomus, less in the tubules and weakest in the duct.
using ODC and a linearity using doubling dilutions of stage 35
cDNA was performed. This increased expression in the kidney
compared to the whole embryo was also observed in the mouse,
where Northern blot analysis at 15.5 dpc revealed higher expres-
sion in the kidney, lung and intestine than in the whole embryo
(Hidai, et al., 1998).
The pronephros from a Xenopus laevis stage 42 embryo was
dissected into the glomus, tubules and duct regions and RT-PCR
was performed on mRNA extracted from the dissected pieces. The
pronephric dissection RT-PCR suggested a greater expression of
Pod 1 in the glomus compared to the tubules and duct (Fig. 3B).
This is comparable to mouse kidney expression, where Pod 1 is
expressed in several differentiated cell types including podocytes
Pod 1Pod 1Pod 1
ODCODCODC
KKKWWW
WWWWWW
KKK -R-R-R
NNN
AAA
0.25 0.125
0.5WWW
WWW
KKK
1.0
KKK
KKK
StagesStagesStages Neg. controlsNeg. controlsNeg. controlsLinearityLinearityLinearity
-R-R-R
TTT
(-)
12½12½12½ 151515 202020282828 353535
-R-R-R
NNN
AAA
0.25
0.125
0.51.0
Neg.
controlscontrolscontrols
LinearityLinearityLinearity
-R-R-R
TTT
(-)
EF1αEF1αEF1α
Pod 1Pod 1Pod 1
WWW
hole em
bryo
hole em
DDD
uct
Tubule
GGG
lom
us
lom
PronephrosPronephrosPronephros
0.250.125
0.5
1.0(-)
0.25 0.125
0.5
1.0 (-)
0.25
0.125
0.51.0
Neg. Neg.
(-)
bryo
hole em
uct
Tubule
us
lom
0.25
0.125
0.51.0
(-)
bryo
uct
Tubule
us
A
B
C
J
H
A
B
D
EFG
I
Fig. 4. Whole mount in
situ hybridisation of Pod
1 in X. laevis. Wholem-
ount in situ hybridisation
with a Pod 1 DIG-labelled
antisense (A,C,D,F,G and
I) and sense (B,E,H and J)
RNA probe was performed
on embryos at the stage indi-
cated. Embryos in panels D, G, I
and J have been cleared in
Murray’s solution to facilitate the
observation of internal staining.
No staining above background
was present in embryos at stage
22 (A,B) as staining observed in
the eye placode, ep, and somites,
s, was present in the antisense and sense in situ hybridisations. (C,D) Glomus, g and
anterior branchial arch, b, staining is visible in uncleared and cleared stage 28-29
embryos. (E) Stage 29 sense RNA probed embryos do not show staining in the
branchial arch or the glomus. (F) Uncleared antisense stage 33 embryos show
discrete staining in the branchial arches, b and the glomus, g, which is also observed
in the cleared embryos (G). (H) Sense stage 33 embryos also have some weak
staining in the branchial arches. The staining observed in the eye is observed in both
antisense and sense stage 33 embryos and is considered to be background (F,H). The
late tailbud embryos are stained in the epicardium, h, of the heart and in all three components of the pronephros, the glomus, g, tubules, t and duct, d
(I). Non-specific staining is seen in the anterior region of late stage embryos in both sense and antisense hybridised embryos (J).

Page 4

62 S. Simrick et al.
EF1α EF1α
Pod 1Pod 1
EF1αEF1α
Pod 1Pod 1
Eye
IleumKK
idney
Liver
LungsOO
vary
RR
ectum
SkinBB
lood
BB
rain
DD
uodenum
HH
eart
MM
uscle
Pancreas
Spinal cord
Spleen
Stom
ach
Stom Testis
-R-R
NN
AA
0.25 0.125 0.51.0
Neg.
controlscontrols
LinearityLinearity
-R-R
TT
(-)(-)
Eye
Ileum
idney
Liver
Lungs
vary
ectum
Skin
lood rain
uodenum
eart
uscle
Pancreas
Spinal cord
Spleen
ach
Testis
0.250.1250.51.0
Neg.
Fig.5. Expression pattern of Pod 1 in the adult
organs of X. laevis. RT-PCR was performed on
mRNA isolated from the organs of an adult Xeno-
pus laevis. The RT-PCR was equalised using EF1α
and linearity and negative controls performed as
described in Experimental Procedures. Pod 1 ex-
pression in adult X. laevis organs appears to be
greatest in the spleen and rectum. Other, more
moderate expression is seen in the duodenum, heart, kidney, lungs, pancreas and skin. There are
also low levels in the liver, muscle, spinal cord and stomach.
intensely stained (Fig. 4C, D, F, G and I). As embryos progress
through the somite forming stages, non-specific staining is ob-
served in the head region of both sense and anti-sense probe
hybridised embryos (Fig. 4E, H and J). Hybridisation of anti-sense
probe to the developing glomus is first observed from stage 28
(Fig. 4C, D, F, G and I) and persists to stage 40.5, the last stage
tested. No hybridisation to the glomus is observed in any of the
negative control, sense hybridised embryos (Fig. 4E, H and J). At
stage 40.5 the presumptive epicardium and all components of the
pronephros show intense Pod 1 expression (Fig. 4I). At these late
stages non-specific staining with both sense and antisense probe
is observed in the anterior head region (Fig. 4J). In murine
development in situ hybridisation has revealed expression from
9.5dpc with high levels in the presumptive epicardium and the
mesenchymal cells surrounding the gut, lung and kidney (Hidai et
al., 1998). This may be comparable to the staining pattern in the
Xenopus laevis stage 40.5 embryos where expression is ob-
served in the heart and kidney, although anterior branchial arch
expression is identified earlier.
Expression of Xenopus laevis Pod 1 in adult organs
RT-PCR was performed on mRNA isolated from the organs of
an adult Xenopus laevis. The input cDNA was approximately
equalised using EF1α and a linearity using doubling dilutions of
kidney cDNA was performed. The RT-PCR indicated that Pod 1
expression is highest in the rectum and the spleen, with significant
expression in the duodenum, heart, kidney, lungs, pancreas, skin,
liver and muscle (Fig. 5). A similar adult expression pattern is
observed in the mouse. Northern blot experiments have shown
Pod 1 expression in the lung, kidney, heart liver, spleen and testis
(Lu et al., 1998 and Miyagishi et al., 2000). However, unlike the
Pod 1 expression in Xenopus laevis, there is no expression in the
muscle (Lu, et al., 1998 and Miyagishi et al., 2000).
We have identified the Xenopus laevis Pod 1 homologue within
the EST CF270487 in the NCBI database (http://mgc.nci.nih.gov/
) and have analysed the temporal and spatial expression patterns
of this gene. In summary, there is a high percentage of protein
sequence similarity of the Xenopus laevis Pod 1 homologue with
other vertebrates, especially within the basic helix-loop-helix
region suggesting a conserved DNA binding function. Temporal
expression pattern shows that Pod 1 expression is initiated at
stage 13 and is found to be higher in the kidney, specifically in the
glomus, compared to the whole embryo. Spatial expression analy-
sis identifies expression domains in the glomus, heart and bran-
chial arches. The adult organs show high expression in the rectum,
spleen, duodenum, heart, kidney, lungs, pancreas, skin, liver and
muscle.
This paper, therefore describes the distribution of a basic helix-
loop-helix transcription factor expressed very early in the develop-
ment of the pronephric glomus. Pod 1 is one of the few transcription
expressed at this early stage and now the functional role of this
gene in transcription hierarchy of glomus development can be
established. Xenopus laevis is particularly amenable to functional
analysis by mRNA over expression and morpholino oligonucle-
otide knock down. Preliminary data shows that Pod 1 plays a major
role at these early stages which cannot be easily analysed in the
mouse.
Experimental Procedures
Bioinformatics
The Xenopus laevis Pod 1 sequence was identified by a BLAST (Basic
Local Alignment Search Tool) (Altschul et al., 1990) search using the
murine Pod 1 homologue protein sequence, GenBank Acc. O35437. The
EST containing the Xenopus laevis Pod 1 sequence, GenBank Acc.
CF270487, was obtained from the IMAGE Consortium (http://image.llnl.gov,
IMAGE No. 5512805). Alignment has been carried out with CLUSTAL
(Thompson et al., 1994).
Xenopus embryo handling
Xenopus laevis females were induced to lay by hormone injections of
follicle stimulating hormone and human chorionic gonadotrophin. The eggs
were fertilized in vitro using dissected testes. The embryos were dejellied
in 2% cysteine pH 8, washed and incubated until the required stage in 1/10
Barth’s X at 12ºC. Embryos were staged according to Nieuwkoop and
Faber (1994).
The whole embryo and kidney dissections were prepared as detailed in
Brennan et al., (1998). For the glomus, pronephric tubule and duct
dissection, the pronephros was first removed from stage 42 embryos and
the glomus and the posterior pronephric duct dissected from the tubules.
This resulted in the anterior duct being included in the tubule preparation.
mRNA from the adult organ tissues was extracted
using TRIzol® (INVITROGEN) reagents in accor-
dance to the manufacturers instructions.
RT-PCR
The tissues were homogenised, total RNA was
extracted and cDNA synthesis was performed as
described in Barnett et al., (1998). The cDNA was
equalised using EF1α (Mohun et al., 1989) (or-
gans) or ODC (whole embryos) (Bassez et al.,
1990) as loading controls. Linearities were in-
cluded to ensure that the PCR signal fell within a
linear range using doubling dilutions of input cDNA
from an appropriate stage or organ. Negative
controls were carried out where no mRNA (-RNA),
no reverse transcriptase (-RT), or no cDNA (-) was
added to the PCR.
Pod 1 PCR was carried out using the Pod 1
forward primer, 5’ TCT CAG TGA TGT GGA GGA
CTT 3’ and the reverse primer, 5’ TGA CGC AGG
TGA GCT ATG TAA 3’, giving a product of 384 bp.

Page 5

Pod 1 in Xenopus laevis 63
The annealing temperature was 57ºC and the PCR was run for 22 cycles
in total.
In situ hybridisation
The in situ hybridisation was carried out on albino embryos previously
fixed in MEMFA and was performed using protocols adapted from Hemmati-
Brivanlou et al., (1990) and Harland (1991). The probes were prepared
using a Roche digoxygenin (DIG) labelling kit. The IMAGE Pod 1 open
reading frame was cloned into pCS2+ using Eco RI and Xho I (INVITRO-
GEN). This clone was then used to make an antisense probe by linearising
the IMAGE clone with Cla I (INVITROGEN) and transcribing with T7 RNA
polymerase (TAGN). Similarly, the sense in situ probe was made by
linearising with Xba I (INVITROGEN) and transcribing with SP6 RNA
polymerase (TAGN).
Acknowledgements
We would like to thank Robert Taylor and Paul Jarrett for kindly providing
the frogs. This work was supported by the BBSRC.
References
ANDERSSON, M., GIRAUD, A.S. AND FAMILARI, M. (2001). The role of capsulin in
the morphogenesis and differentiation of fetal rat gastric mucosa. Int. J. Dev. Biol.
45: 887-893.
ALTSCHUL, S.F., GISH, W., MILLER, W., MYERS, E.W. AND LIPMAN, D.J. (1990).
Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
BARNETT, M.W., OLD, R.W. AND JONES, E.A. (1998). Neural induction and
patterning by fibroblast growth factor, notochord and somite tissue in Xenopus.
Dev. Growth. Diff. 40: 47-57.
BASSEZ, T., PARIS, J., OMILLI, F., DOREL, C. AND OSBORNE, H.B. (1990). Post-
transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.
Development 110: 955-62.
BRENNAN, H.C., NIJJAR, S. AND JONES, E.A. (1998). The specification of the
pronephric tubules and duct in Xenopus laevis. Mech. Dev. 75: 127-137.
CUI, S., SCHWARTZ, L. AND QUAGGIN, S.E. (2003). Pod 1 is required in stromal
cells for glomerulogenesis. Dev. Dyn. 226: 512-522.
CUI, S., ROSS, A., STALLINGS, N., PARKER, K.L., CAPEL, B. AND QUAGGIN, S.E.
(2004). Disrupted gonadogenesis and male-to-female sex reversal in Pod1
knockout mice. Development 131: 4095-4105.
FUNATO, N., OHYAMA, K., KURODA, T. AND NAKAMURA, M. (2003). Basic helix-
loop-helix transcription factor epicardin/capsulin/Pod-1 suppresses differentiation
by negative regulation of transcription. J. Biol Chem. 278: 7486-7493.
HARLAND, R.M. (1991). In situ hybridisation – an improved whole mount method for
Xenopus embryos. Method. Cell Biol. 36: 685-695.
HEMMATI-BRIVANLOU, A., FRANK, D., BOLCE, M.E., SIVE, H.L. AND HARLAND,
R.M. (1990). Localisation of specific mRNAs in Xenopus by wholemount in situ
hybridisation. Development 110: 325-330.
HIDAI, H., BARDALES, R., GOODWIN, R., QUERTERMOUS, T. AND
QUERTERMOUS, E.E. (1998). Cloning of capsulin, a basic helix-loop-helix factor
expressed in progenitor cells of the pericardium and the coronary arteries. Mech.
Dev. 73: 33-43.
LASSAR, A.B., DAVIS, R.L., WRIGHT, W.E., KADESCH, T., MURRE, C., VORONOVA,
A., BALTIMORE, D. AND WEINTRAUB, H. (1990). Functional activity of myogenic
HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.
Cell 66: 305-315.
LU, JIANRONG., RICHARDSON, J.A. AND OLSON, E.N. (1998). Capsulin: a novel
bHLH transcription factor expressed in epicardial progenitors and mesenchyme of
visceral organs. Mech. Dev. 73: 23-32.
LU, J., CHANG, P., RICHARDSON, J.A., GAN, L., WEILER, H. AND OLSON, E.N.
(2000). The basic helix-loop-helix transcription factor capsulin controls spleen
organogenesis. Proc. Natl. Acad. Sci. USA. 97: 9525-9530.
MIYAGISHI, M., NAKAJIMA, T. AND FUKAMIZU, A. (2000b). Molecular
characterisation of mesoderm-restricted basic helix-loop-helix protein, POD-1/
Capsulin. Int. J. Mol. Med. 5: 27-31.
MIYAGISHI, M., HATTA, M., OHSHIMA, T., ISHIDA, J., FUJII, R., NAKAJIMA, T. AND
FUKAMIZU, A. (2000a). Cell type-dependent transactivation or repression of
mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin. Mol. Cell.
Biochem. 205: 141-147.
MOHUN, T. J., TAYLOR, M.V., GARRETT, N AND GURDON J.B. (1989). The CArG
promoter sequence is necessary for muscle specific transcription of the cardiac
actin gene in Xenopus embryos. EMBO J. 8: 1153-1161.
MURRE, C., McCAW, P.S., VAESSIN, H., CAUDY, M., JAN, L.Y., JAN, Y.N.,
CABRERA, C.V., BUSKIN, J.N., HAUSCHKA, S.D. AND LASSAR, A.B. (1989).
Interactions between heterologous helix-loop-helix proteins generate complexes
that bind specifically to a common DNA sequence. Cell 58: 537-544.
NIEUWKOOP, P.D. AND FABER, J. (1994). Normal Table of Xenopus laevis, Garland
Publishing, New York, NY.
QUAGGIN, S.E., SCHWARTZ. L., CUI. S., IGARASHI, P., DEIMLING, J., POST, M.
AND ROSSANT, J. (1999). The basic-helix-loop-helix protein Pod1 is critically
important for kidney and lung organogenesis. Development. 126: 5571-5783.
QUAGGIN, S.E., VANDEN HEUVEL, G.B. AND IGARASHI, P. (1998). Pod-1, a
mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and
glomerular epithelial cells in the developing kidney. Mech. Dev. 37-48.
ROBB, L., MIFSUD, L., HARTLEY, L., BIBEN, C., COPELAND, N.G., GILBERT, D.J.,
JENKINS, N.A. AND HARVEY, R.P. (1998). Epicardin: A novel basic helix-loop-
helix transcription factor gene expressed in epicardium, branchial arch myoblasts
and mesenchyme of developing lung, gut, kidney and gonads. Dev. Dyn. 213: 105-
113.
TAMURA, M., KANNO, Y., CHUMA, S., SAITO, T. AND NAKATSUJI, N. (2001). Pod-
1/Capsulin shows a sex- and stage-dependent expression pattern in the mouse
gonad development and represses expression of Ad4BP/SF-1. Mech Dev. 102:
135-144.
THOMPSON, J. D., HIGGINS, D. G. AND GIBSON, T. J. (1994). CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22: 4673-80.
Received: September 2004
Reviewed by Referees: October 2004
Modified by Authors and Accepted for Publication: January 2005
Edited by: Thomas Sargent