Yonago Acta medica 2015;58:15–22

Original Article

P19 Cells Overexpressing Lhx1 Differentiate into the Definitive Endoderm
by Recapitulating an Embryonic Developmental Pathway
Akira Hasegawa and Yasuaki Shirayoshi
Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute ofRegenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Yonago 683-8503, Japan

ABSTRACT

leads to identification of LIM homeobox 1 (Lhx1) as oneof the key regulators of epiblast development (Hasegawa
et al., submitted). Lhx1-null embryos displayed headtruncation due to disruption in germ layer morphogenesis.5 Since Lhx1 is required for the movement of theanterior visceral endoderm (AVE) at the onset of gastrulation, it is one of the gastrula organizers for specifying
anterior parts of embryos along with A-P axis.6, 7
Mouse embryonal carcinoma (EC) P19 cells were
derived from a teratocarcinoma formed by transplantation of E7.5 embryo into the testis.8, 9 P19 cells exhibit
typical flat compact colonies and express pluripotencyrelated genes such as Oct3/4 and Sox2, but not Rex1.
Differentiation of P19 cells can be effectively inducedby various drugs at non-toxic concentrations. Retinoic
acid and DMSO induce their differentiation to neuronalcells10 and cardiomyocytes,11 respectively. Thus P19 cells
are considered equivalent to epiblasts in blastocysts, andsuitable as an experimental environment for studying
Lhx1 function in epiblasts. Here we examined the role ofLhx1 on developmental processes of epiblasts using P19
cells as a model system.

Background

Epiblasts occur at the last pluripotent
stage of embryonic development and are important inelucidating how the three germ layers are formed. However, little is known of the molecular mechanisms of
their development. We have shown that LIM homeobox
1 (Lhx1) was involved in epiblast development in embryonic stem cells, especially meso- and endodermal differentiation. However, since epiblasts in embryoid bodies
spontaneously develop into a further stage, it is difficult
to study their development in this system.
Methods Mouse embryonal carcinoma P19 cells
which have properties similar to those of epiblasts provided new avenues of investigation into the regulatory
mechanism of epiblasts.
Results Overexpression of Lhx1 in P19 cells induced
expression of organizer marker genes (Cer1, Gsc) andendoderm marker genes (Gata6, Foxa2, Sox17) but not
extra-embryonic endoderm marker genes (Sox7 or Hnf4alpha).
Conclusion This study suggested that Lhx1 overexpression caused P19 cells to differentiate into an endodermal lineage. Thus, P19 cells and their derivatives canbe a useful model system to study how the three germlayers are formed.

MATERIALS AND METHODS
Cell culture and differentiation

P19 and HEK293T cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Wako Pure Chemical, Osaka, Japan), supplemented with 10% fetal bovine
serum (FBS; Corning Life Sciences, Tewksbury, MA)
and penicillin-streptomycin-L-glutamine solution (PSG;

Key words

cell differentiation; epiblast; germ layer;organizer; P19 cells

Early mammalian developmental processes such as organizer formation and primitive streak invagination, occur at epiblasts in blastocysts and lead to differentiationof cells in the three germ layers.1 These developmental
processes are extensively investigated in primitive vertebrate species. Little is known of the molecular mechanisms of epiblast development in mammals, primarily
because it is difficult to directly observe or experimentally manipulate these cells that exist in the uterus. Embryonic stem (ES) cells in embryoid bodies (EBs) differentiated in vitro to epiblasts and then further to cells
in the three germ layer lineages.2, 3, 4 This differentiation
does not happen in ES cells in a monolayer culture.
Comparison of gene expression profiles between ES
cells cultured in EBs and those cultured in a monolayer,

Corresponding author: Yasuaki Shirayoshi, PhD
yshirayo@med.tottori-u.ac.jp
Received 2014 December 8
Accepted 2014 December 18
Abbreviations: ADE, anterior definitive endoderm; APS, anteriorprimitive streak; AVE, anterior visceral endoderm; DE, definitive
endoderm; DMEM, Dulbecco’s modified Eagle’s medium; EB,embryoid body; EC, embryonal carcinoma; ES, embryonic stem;
ExEn, extraembryonic endoderm; FBS, fetal bovine serum; HA,
hemagglutinin; HRP, horseradish peroxidase; IgG, immunoglobulin G; KD, knockdown; Lhx1, LIM homeobox 1; MEF, mouseembryonic fibroblast; NEAA, MEM non-essential amino acidsolution; O/E, overexpression; PBS, phosphate-buffered saline;
PS, primitive streak; PSG, penicillin-streptomycin-L-glutamine
solution; qPCR, quantative PCR; RT, reverse transcription; SBE,
Smad binding element; TBS-T, Tris-buffered saline with Tween
20; TRC, the RNAi Consortium

15

A. Hasegawa and Y. Shirayoshi.
Table 1. Primer sequences in RT-PCR
Gene

GAPDH
Rex1
Pecam1
Oct3/4
Sox2
Nanog
Fgf5
Otx2
Sox7
Bmp2
Ttr
Hnf4alphaForward 5'-3'TGAACGGGAAGCTCACTGG
TTCACGGAGAGCTCGAAACTGCTTGGCAGCGAAACACTAAATTCTCGAACCTGGCTAAGCTGGCAGCTACAGCATGATGCAGGAGCCACCCACCCATGCTAGTCTT
CTGTACTGCAGAGTGGGCATCGG
CACTTCGGGTATGGACTTGC
CTTCAGGGGACAAGAGTTCGGAGGCGAAGAAAAGCAACAG
TTCACAGCCAACGACTCTGG
CGAACAGATCCAGTTCATCAAG

Wako. Stock solution diluted 1:100). AB1 mouse ES
cells were cultured on mouse embryonic fibroblasts(MEFs) in ES cell medium comprising DMEM supplemented with 20% FBS and PSG (Stock solution diluted
1:100) and MEM non-essential amino acid solution(NEAA; Wako. Stock solution diluted 1:100) and 0.1
mM 2-mercaptoethanol (Sigma, St. Louis, MO) and1000 U/mL ESGRO (Millipore, Milford, MA). MEFs
were obtained from ICR-strain E13.5 fetuses and maintained in DMEM supplemented with 10% FBS andPSG (Stock solution diluted 1:100). MEFs were treatedwith mitomycin C and seeded as feeders. EB formationin ES cells was achieved by the hanging drop method.
After ES cells were adjusted to 2.5 × 104 cells/mL, 20
μL aliquots were added to a 100-mm petri dish. The differentiation medium was comprised of DMEM, supplemented with 20% FBS and PSG (Stock solution diluted
1:100) and NEAA (Stock solution diluted 1:100) and 0.1
mM 2-mercaptoethanol, which was added 2 days later.
ES cells were seeded onto 0.1% gelatin-coated dishes at
a density of 1 × 102/cm2 in the differentiation medium toform a monolayer.

Reverse 5'-3'TCCACCACCCTGTTGCTGTA
CCATCCCCTTCAATAGCTCATGCGATGGTGTATAACGTCAATGGTGGTCTGGCTGAACACCTTTTGGAGTGGGAGGAAGAGGTA
ACCCTCAAACTCCTGGTCCTGACTTCTGCGAGGCTGCGACAGGCTCTCCCTTCGCTGTTTCC
GCTTGCCTTGTTTCTTCCTG
GGGGAAGCAGCAACACTAGA
AATGCTTCAGGGCATCTTCC
ATGTGTTCTTGCATCAGGTGAG

F-5'-TATGGTACCATGGACCTCTGAAACAAGTAAAACATA-3', R-5'-TATAAGCTTGCTTCCCAGAGACTGAGGTT-3',12 The Smad binding element (SBE)
sequence was produced by artificial gene synthesis(IDT, Coralville, IA). Each fragment was inserted into
pNluc1.3 (Promega, Madison, WI). Cells (1 × 104) wereseeded in a 96-well plate and incubated with 0.15 μgreporter plasmid, 0.15 μg effector plasmid, 0.015 μg internal control plasmid pSEAP2-control vector (Clontech,
Palo Alto, CA), and FuGENE HD (Promega) to transfectthese plasmids as per the manufacturer’s protocol. Luciferase assays were performed 24 h later using the NanoGlo Luciferase Assay System (Promega) and the Great
EscAPe SEAP Chemiluminescence Kit 2.0 (Clontech).
RNA preparation, RT-PCR, and RT-qPCR analysis

Total RNA was extracted from the cells using an
RNeasy Mini Kit (Qiagen, Valencia, CA) The PrimeScript RT reagent Kit with gDNA Eraser 1 (Takara Bio,
Shiga, Japan) was utilized to synthesize the cDNA. The
PCR cycling conditions were as follows: one cycle of
94 ˚C for 2 min, 22 to 30 cycles of 98 ˚C for 10 s, 60 ˚Cfor 30 s and 72 ˚C for 30 s, followed by 4 ˚C. GAPDH
was used as the invariant control. The primer sequences
are described in Table 1. Reverse transcription (RT)quantative PCR (qPCR) was performed with the appropriate ABI TaqMan Gene Expression Assays in a 10 μL
mixture containing cDNA and specific probes and primers, and the EXPRESS qPCR Supermix with Premixed
ROX (Invitrogen). Probes and primers were selectedusing web-based assay design software (Probefinder:
https://qpcr.probefinder.com/organism.jsp). The PCR cycling conditions were as follows: one cycle of 95 ˚C for
20 s, 45 cycles of 95 ˚C for 1 s and 60 ˚C for 20 s. The
results were expressed as the ratio between the gene ofinterest and the beta-actin reference gene. The primer
sequences are described in Table 2.

Transfection

The pCAG IRES Puro vector DNA (2 μg) containing Lhx1 or Gata6 cDNA was complexed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and added
to adherent P19 cells and ES cells in 24-well plates. The
cells were trypsinized and re-seeded into 60-mm culture
dishes 3 to 8 h after transfection. Puromycin (1.5 μg/mL)
was added 24 h later to select transfected cells.
Luciferase analysis

DNA fragment, including the sequence immediately at
5' end of the coding region, was PCR-amplified to construct the luciferase assay vector from ES cell gDNA
using the following oligonucleotide primers: Cer1:
16

Lhx1 induces definitive endodermal cells
Table 2. Primer sequences in RT-qPCR
Gene

Beta-actin
Gsc
Cer1
Gata6
Foxa2
Sox17
Fgf5
Otx2
Gata4
Lhx1

Forward 5'-3'CTAAGGCCAACCGTGAAAAGGAGACGAAGTACCCAGACGTG
GACTGTGCCCTTCAACCAGGGTCTCTACAGCAAGATGAATGG
AAATAATGTAAGAGTCTGGTGTACCGCACAACGCAGAGCTAAGCAA
AAAACCTGGTGCACCCTAGAAAATCAACTTGCCAGAATCCA
GGAAGACACCCCAATCTCGCAGGAGACTGGCCTCAACAT

Immunofluorescence

Reverse 5'-3'ACCAGAGGCATACAGGGACA
GCGGTTCTTAAACCAGACCTCAGCAGTGGGAGCAGAAGCTGGCACAGGACAGTCCAAG
CCATGTCCAGAATGGGATGT
CGCTTCTCTGCCAAGGTCCATCACATTCCCGAATTAAGC
GGCCTCACTTTGTTCTGACCCATGGCCCCACAATTGACGTTTCATCCTTCGCTCCTTG

Lentiviral transfection to generate P19 Lhx1 knockdown cells

The cells were fixed in 4% paraformaldehyde for 15 minat room temperature. Furthermore, they were rinsed
in phosphate-buffered saline [PBS (−), Wako] and incubated with 0.1% Triton X-100 in PBS for 15 min,then blocked in 5% skim milk in Tris-buffered salinewith Tween 20 (TBS-T) for 2 h. Primary antibody wasapplied using 0.1% bovine serum albumin in TBS-T
overnight at 4 °C. The cells were washed and secondary
antibody was applied for 1 h at room temperature, followed by counterstaining with DAPI (Molecular Probes,
Sunnyvale, CA). The primary antibodies used were antiLhx1 (Millipore), anti-hemagglutinin (HA) (Wako), antiOct3/4 (Santa Cruz Biotechnology, Santa Cruz, CA),anti-Pecam1 (anti-CD31; BD Biosciences, San Diego,
CA). The secondary antibodies used were Alexa Fluor
546 anti-mouse immunoglobulin G (IgG), 488 antirabbit IgG and 568 anti-rat IgG (Molecular Probes).

We used the RNAi consortium (TRC) Lentiviral
Mouse L h x1 sh R NA ( T RC N0 0 0 0 0 70525 a nd
TRCN0000070527; GE Healthcare) and the TRC Lentiviral pLKO.1 Empty Vector Control (GE Healthcare).
The lentivirus was produced in HEK293T cells using
the Trans-Lentiviral shRNA Packaging Kit with theCalcium Phosphate Transfection Regent (Thermo Scientific). The cell culture medium containing the virus
particles was collected and filtered through a 0.45 μm
filter 72 h after transfection. The viral particles were
further concentrated using PEG-it Virus Precipitation
Solution (SBI, Mountain View, CA). The pellets were
resuspended in DMEM containing 25 mM HEPES. P19
cells were seeded at a density of 5.0 × 104 to 1.0 × 105
cells/well in a 24-well plate and infected with lentivirus
to express either Empty or Lhx1 shRNA. The cells weretrypsinized 24 h after infection and re-seeded into 100mm culture dishes. Puromycin (1.5 μg/mL) was added to
select the infected cells 72 h after infection.

Western blotting

Cells were lysed in sample buffer (62.5 mM Tris-HCl,
2% sodium dodecyl sulfate, 25% glycerol, 0.01% bromophenol blue) and boiled for 10 min at 95 °C. The
samples were separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to amembrane (Millipore). The membranes were blockedwith 5% skim milk for 2 h and then incubated withprimary antibody for 1 h at room temperature. Afterwashing the membranes, the blots were incubated withhorseradish peroxidase (HRP)-coupled secondary antibody and developed using ECL-Plus regents (Thermo
Scientific, Rockford, Il). The primary antibodies usedwere anti-HA (Wako), anti-actin (CP01; Calbiochem,
San Diego, CA). The secondary antibody was the ECL
Mouse IgG HRP-linked whole antibody (GE Healthcare,
Piscataway, NJ). Western blotting for Nodal-Smad signaling was conducted with the Phospho-Smad Antibody
Sampler Kit (Cell Signaling Technology, Danvers, MA).

Alkaline phosphatase staining

The cells were washed with PBS twice, and 3.6% formaldehyde was added for 15 min at room temperature.
The cells were washed in PBS twice again and incubated with PBS and 1.5 M Tris-HCl and naphthol AS-BI
phosphate (Sigma) and Fast Red Violet LB salt (Sigma)
for 5 to 10 min at room temperature. After incubation,
the cells were washed with PBS.
RESULTSP19 cells express low levels of Lhx1 and have epiblastic properties

P19 cells showed the flat compact colonies (Fig. 1A). P19
cells expressed pluripotent cell markers Oct3/4 and Sox2
and Nanog like ES cells, but not ICM markers Rex1 andPecam1. Importantly, they expressed high levels of an
epiblast marker Fgf5 (Fig. 1B). Immunostaining showed17

A. Hasegawa and Y. Shirayoshi.

Fig. 1. The characteristics of embryonal carcinoma P19 cells.
A: Typical P19 cells morphology. Bar = 100 μm.
B: RT-PCR to determine the properties of P19 cells.
C: Immunofluorescence and alkaline phosphatase staining of ES cells and P19 cells. Bar = 100 μm.
D: Lhx1 mRNA expression in ES cells and the EBs on day 3 and P19 cells was evaluated by RT-qPCR. Relative to beta-actin.
ALP, alkaline phosphatase staining; EB, embryoid body; qPCR, quantitative PCR; RT, reverse transcription.

uniform expression of Oct3/4 but no expression of Pecam1, and a much lower level of alkaline phosphatasethan in ES cells (Fig. 1C). P19 cells expressed much less
Lhx1 than EBs on day 3 (as a positive control), whereas
undifferentiated ES cells did not express Lhx1 (Fig. 1D).
Taken together, these results reveal that P19 cells possesscharacteristics similar to those of epiblasts and slightly
express Lhx1.

overexpression (O/E) P19 cells] was used in the current
study. After several passages with drug selection, Lhx1
O/E P19 cells maintained high and uniform expressionof exogenous Lhx1 (Fig. 2B).
Lhx1 O/E P19 cells showed increased expression oforganizer markers Cer1 and Gsc (Fig. 2Cb), and persistent expression of Oct3/4 and Nanog (data not shown).
The gastrula organizer is divided into some centers: theanterior primitive streak (APS) and the node and theAVE. Since these structures are barely distinguishable
from each other at the molecular level, we could not
determine which one was induced from Lhx1 O/E P19
cells. Interestingly, the epiblast marker Fgf5 was upregulated by Lhx1 approximately 10-fold, whereas anotherepiblast marker Otx2 was downregulated (Fig. 2Ca). It isnotable that Cer1 mRNA expression increased 50-fold in
Lhx1 O/E P19 cells. To verify whether this induction ofCer1 was due to transcriptional activation by Lhx1, we
measured Cer1 promoter activity by reporter assay. Cer1

Lhx1 induces expression of organizer and endoderm marker genes

To investigate the effect of Lhx1 during epiblast celldifferentiation, P19 cell lines overexpressing Lhx1 were
established in which Lhx1 was tagged with the influenza HA epitope at the C-terminus. After selection withpuromycin, more than thirty colonies morphologicallydifferent from wild-type were picked up. The highest
level of exogenous Lhx1 expression was detected in celllines #4 and #5 (Fig. 2A). Therefore, this #5 line [Lhx118

Lhx1 induces definitive endodermal cells

Fig. 2. The establishment of P19 cell lines overexpressing Lhx1.
A: Western blotting of P19 cell lines overexpressing Lhx1 #4 and #5.
B: Immunofluorescence staining of Lhx1 O/E P19 cells. Bar = 100 μm.
C: Gene expression levels determined by RT-qPCR are indicated. (a) epiblast marker, (b) organizer marker, (c) endoderm marker.
*P < 0.05, **P < 0.01, versus W. Relative to beta-actin.
O/E, overexpression (Lhx1 O/E P19 cells); PC, positive control; W, wild type P19 cells.

Lhx1 activates Nodal signaling to induced differentiation of P19 cells into the definitive endoderm

promoter activity was increased 20-fold in Lhx1 O/E
P19 cells (Fig. 2D), suggesting that organizer induction
was a direct consequence of Lhx1 O/E.
The morphology of Lhx1 O/E P19 cells in normal
culture condition medium was similar to that of endoderm-like cells (Fig. 2B). This morphological change in
Lhx1 O/E P19 cells to endodermal cells was confirmed
by RT-qPCR as they expressed pan-endodermal markers
Gata4 and Gata6 and Foxa2 (Fig. 2Cc). Taken together,
Lhx1 O/E lead P19 cells to differentiate into endodermal
cells.

The morphological change of Lhx1 O/E P19 cells wasinvestigated in more detail. Early embryonic endodermal cells are divided into two categories: definitive
endoderm (DE) and extraembryonic endoderm (ExEn).
Marker expression was examined by RT-PCR to determine which endodermal lineage was induced by Lhx1.
The primitive endodermal marker Sox7 and visceralendoderm markers Bmp2 and Ttr and Hnf4alpha were19

A. Hasegawa and Y. Shirayoshi.

Fig. 3. The investigation of definitive endoderm via Nodal signaling in P19 cell lines overexpressing Lhx1.
A: RT-PCR analysis of extra-embryonic endoderm marker genes. ES cells ML day 4 are shown as a positive control of early primitive
endoderm. Gata6 O/E ES cells are a strong positive control of extra-embryonic endoderm.
B: SBE activity levels determined by the luciferase assay. *P < 0.05, versus Lhx1 (−).
C: Western blotting of Smad family in Lhx1 overexpression P19 cell lines (upper panel). Lower panels show P-Smad2/Smad2 (left)
and Smad4/Actin (right) staining intensity.
Lhx1 O/E #4, Lhx1 overexpression P19 cell line #4; Lhx1 O/E #5, Lhx1 overexpression P19 cell line #5; ML, monolayer; SBE, smad
binding element.

Lhx1 is not required for the maintenance of P19
cells

not detected in Lhx1 O/E P19 cells (Fig. 3A). The lackof ExEn marker expression indicated that Lhx1 induced
DE differentiation. This gene expression pattern wasconsistent with the results of RT-qPCR, which demonstrated increased expression of DE markers Cer1 andGsc and Sox17 in Lhx1 O/E P19 cells (Fig. 2Cb and Cc).
Since DE differentiation is triggered by Nodal signaling,
we hypothesized that Lhx1 facilitated endodermal differentiation by activating Nodal signaling. This signaling
pathway is activated by binding of Smad transcriptional
activator to SBE. Luciferase reporter assay in P19 cells
showed transcriptional activation through SBE 24 h after Lhx1 O/E (Fig. 3B). However, only a slight increase
in Smad2 phosphorylation and Smad4 was observed in
Lhx1 O/E P19 cells (Fig. 3C), which may reflect induction of Foxh1 expression in these cells (data not shown).
These results suggest that Lhx1 activates Nodal signaling to induce differentiation of P19 cells into DE.

Two types of Lhx1 shRNA (#25, #27) were introduced
into P19 cells to determine whether Lhx1 was necessary
for maintenance and differentiation of P19 cells. Two
Lhx1 knockdown (KD) P19 cell lines KD-#25 and KD#27 showed reduced expression levels of Lhx1, respectively (Fig. 4A). No substantial changes in morphology
or self-renewing activity were detected in the two KD
P19 cell lines compared to those in P19 cells (data notshown). The KD-#25 and KD-#27 P19 cells exhibitedgene expression profiles similar to that of wild-type cells(Fig. 4B). These KD cells did not differentiate spontaneously in normal culture media. These results suggest that
Lhx1 is not involved in the maintenance of P19 cells,
which is consistent with the low expression level of Lhx1
in these cells.

20Lhx1 induces definitive endodermal cells

Fig. 4. The establishment of Lhx1
knockdown P19 cell lines.
A: Lhx1 expression levels (relative to beta-actin), as determined by RT-qPCR, areshown for the Lhx1 KD P19
cell lines. *P < 0.05 versus
CTR.
B: RT-PCR of pluripotent andepiblast marker genes in the
Lhx1 KD P19 cell lines.
CTR, control (P19 cells infected
Empty vector); KD, knockdown.

phology and marker expression in Lhx1 O/E P19 cells,
the various Lhx1 expressing cells at anterior parts of PS
stage embryos were possible candidates as induced tissue by Lhx1 O/E. At present, we could not specify thewhole signature of Lhx1 O/E P19 cells.
Lhx1 deficiency leads to abnormal morphogenesisof the visceral endoderm and primitive streak and nascent mesoderm and the mesendoderm derivatives of thegastrula organizer in the gastrula embryo, which resultin head truncation. Amot acts downstream to Lhx1, anda loss of Amot activity impairs the movement of AVE
from the anterior region of embryo to the extraembryonic sites.16 This disruption impeded anterior migrationof DE in Lhx1-deficient mice. Thus Lhx1 is indirectly
implicated in the production of definitive endoderm anda primary function of Lhx1 in early embryos is to enableproper cell movement during gastrulation. In this study,
most Lhx1 O/E P19 cells showed endodermal morphology with DE and/or AVE marker expression such as Cer1
and Foxa2 and Gsc and Sox17, suggesting that Lhx1 O/
E caused P19 cells to differentiate directly into DE and/or AVE from mesendodermal cells like APS. In addition
to the regulation of morphogenetic movement and cell
behavior in gastrulation, Lhx1 could regulate the fate of
epiblasts or anterior primitive streak.
In accordance with very low expression of Lhx1
in P19 cells, Lhx1 KD did not affect the state of P19
cells, indicating that Lhx1 was not involved in the pluripotency of epiblasts. However, Lhx1 overexpression
induced Fgf5 expression in P19 cells. Although Fgf5
is a marker for epiblasts,17 neither pluripotent stem cellmarkers like Oct3/4, Sox2 and Nanog nor the populationof stem like cells was increased. Therefore increased expression of Fgf5 was not directly involved in the maintenance or propagation of epiblasts. Recently ADE cells
were generated and isolated from differentiating ES cell
cultures.18 The production of ADE in culture required
FGF signaling in addition to activin and BMP signaling.

DISCUSSION
Here we showed that Lhx1 O/E induced differentiationof P19 cells into DE. DE arises at gastrulation from epiblasts in the APS, which later gives rise to a specialized
structure called the node, the organizer for posterior
parts of embryos. DE specification is regulated by Nodal
signaling and induction of the Sox17 transcriptional
factor in primitive streak (PS) stage embryos. Lhx1 O/
E P19 cells expressed Sox17 and their Nodal signaling
pathway was activated with pan-endodermal markers
Gata4 and Gata6 and Foxa2. Together with DE marker
expression, the expression of the organizer related genes
Cer1 and Gsc remained detectable in Lhx1 O/E P19
cells. Thus the differentiation of DE in Lhx1 O/E P19
cells recapitulated an embryonic developmental pathway
for DE formation.
Genes uniquely upregulated in Lhx1 O/E P19 cells
included anterior endodermal markers such as Cer1 andGsc and Foxa2 and Sox17. Lhx1–/– cells in the endodermal layer of the null-mutant embryo did not express
Sox17 and Foxa2 and Shh and Cer1 appropriately.13
These results suggested that anterior endodermal marker
genes could be the downstream targets of Lhx1 as indicated by our Cer1 promoter assay. There are two distinct
endodermal lineages, AVE and anterior definitive endoderm (ADE) in the distal parts of PS stage embryos.14,15
AVE is extra-embryonic tissue, but has quite a uniquecapacity as a gastrula organizer for specifying anterior parts of embryos including the head region. AVE
showed a gene expression profile similar to ADE as wellas the node. Therefore Lhx1 O/E P19 cells could contain
AVE in addition to DE. Basically, the identity of anterior
parts of cells was defined based on the allocation in anembryo at a specified stage and showed very similar
expression profiles to marker genes. Mouse Lhx1 is expressed in both the AVE and in primitive streak-derived
tissues including the node and the mesodermal wings
and the anterior mesendoderm. Considering their mor21

A. Hasegawa and Y. Shirayoshi.

Although Fgf5 does not promote the inductive processesof ADE, the active induction of Fgf5 in Lhx1 O/E P19
cells might suggest an unknown function of Fgf5 for
ADE and/or AVE formation.
Organizers form intricate regulatory networks forthe patterning of the anterior-posterior and dorsal-ventral
axes in developing gastrula. These processes are regulated by secreted factors from the organizer that induce the
TGF-beta pathway and inhibit the BMP and WNT pathway, which is accomplished by secreting proteins suchas Nodal related genes and Dkk1 and Noggin/Chordin.
Cer1, another marker for the organizer, is an inhibitor of
all three signaling pathways. Since Lhx1 O/E P19 cells
expressed Cer1 and Dkk1 and Noggin (data not shown)with activated nodal signaling, all essential factors fororganizer activities might be provided in Lhx1 O/E P19
cells. Moreover, the fundamental transcriptional factors
for organizer function such as Gsc and Foxa2 and Lhx1
itself were all expressed in Lhx1 O/E P19 cells. Taken
together, Lhx1 O/E P19 cells might show organizer
function in vitro so that Lhx1 O/E P19 cells could be avery good cell source for organizer factors, which were
essential for gastrulation, subsequent specification and /or differentiation of organizer tissues and their induced
anterior tissues. Since Lhx1 O/E P19 cells could serve asa model for AVE and/or ADE, P19 cells and their Lhx1
expressing derivatives give us a useful model system forstudying the underlying mechanisms of head formationand three germ layer cell specification.

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Acknowledgments: We thank the members of I.H. laboratory forhelping with some experiments and T.N. laboratory for advice on
manipulating the lentivirus.
This work was performed in part under the Cooperative Research Program of Institute for Frontier Medical Sciences, Kyoto
University, Japan and was supported in part by Grand-in-Aid for
Scientific Research (C) to Y.S. (26501003), Grand-in-Aid for JSPS
Fellows to A.H. (256969). A.H. was supported as a Research Fellow of the Japan Society for the Promotion of Science.
The authors declare no conflict of interest.
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