To isolate soluble factors expressed in early phases of hematopoietic differentiation, we applied the signal sequence trap method to the in vitro murine hematopoietic differentiation system, in which ES cells are cocultured with OP-9 stroma cells. This strategy allowed us to isolate cDNA for a secreted protein, ESOP-1, of 160 amino acids, the sequence of which shows 64% identity with human ESOP-1/MD-2. ESOP-1 mRNA was highly expressed in the mouse embryos at 7.5 days after coitus. Expression of the ESOP-1 mRNA and protein was shown in the embryonic and adult hematopoietic system. In addition, the ESOP-1 protein was found in the yolk sac–blood islands, the developing nervous system, and the adult reproductive system. These results suggest that ESOP-1 may play some roles in the development or maintenance of hematopoietic, nervous, and reproductive systems.

To understand the complex molecular mechanisms for mesoderm induction and early hematopoiesis of mouse embryos, several attempts were performed, and some genes were isolated,1-3 from in vitro differentiation systems that form embryoid bodies and generate hematopoietic cells from embryonic stem (ES) cells. More recently, the in vitro system to yield hematopoietic cells was further refined to avoid the formation of embryoid bodies by coculturing ES cells with the OP9 stromal cell line4 or by plating ES cells on collagen IV plates with the enrichment of differentiated cells by cell sorting.5Such techniques allowed the identification and characterization of the hemangioblasts, the common precursor of both endothelial cells and hematopoietic cells.6,7 In addition, several membrane receptors and their ligands have been shown to be involved in the regulation of hematopoietic stem cell differentiation. The best examples are c-Kit and its ligand stem cell factor (SCF), Flk-1/vascular endothelial growth factor (VEGF),8,9 and Tie2/angiopoietin-1.10,11 

Because these factors are too limited to explain the complex regulation of the in vivo hematopoietic pathway(s), it is likely that there are other soluble factors involved in the early phases of hematopoiesis.

By combining the ES-OP9 hematopoietic differentiation system4 and the signal sequence trap method,13we attempted to isolate cDNA for secreted proteins and membrane proteins expressed when the hematopoietic progenitor cells appear, and we isolated a candidate cDNA.14 Here we report the structure and expression of the cDNA-encoded secreted protein, named ESOP-1 (ES-OP9 coculture clone-1).

Isolation of ESOP-1 cDNA

As described previously,4 mRNA was extracted from 5-day differentiation-inducted ES cells. A signal sequence trap cDNA library was constructed as described,13 with modifications described below. First-strand cDNA was synthesized with a random nonamer-SalI-linker primer as follows.

5′-GAGACGGTAATACGATCGACAGTAGGTCGACNNNNNNN- NN-3′. The second strand was synthesized according to Gublerr and Hoffman's method. A double-stranded EcoRI adaptor was ligated to both ends of the double-stranded cDNA, as follows:

5′-CCGCGAATTCTGACTAACTGATT-3′

3′-CAGGCGCTTAAGACTGATTGACTAA-5′

After the cDNA was size-fractionated from 400 to 600 bp in length, 25 cycles of polymerase chain reaction (95°C for 30 seconds, 55°C for 1 minute, 72°C for 1 minute) was performed with the primers 5′-CCGCGAATTCTGACTAACTGATT-3′ and 5′-GACGGTAATACGATCGACAGTAGG-3′. To obtain the full-length ESOP-1 cDNA, the 3′ RACE system (GIBCO-BRL) was used.

Anti-mESOP-1 antibody preparation

Two polypeptides, ESEKQQWFCNSSD and AEAIAGDTEEKLF, were synthesized and used as antigens for production of the antibodies AK612 and AK641, respectively (Biologica, Nagoya, Japan). Antibodies were then affinity purified with each polypeptide.

Whole-mount immunohistochemistry

Specimens were fixed with 2% paraformaldehyde. Immunostaining was performed on whole embryos or after they were sliced into 0.5-mm thick sections, and were visualized using 0.025%p-dimethylaminoazobenzene (Dojin, Tokyo, Japan), 0.08% NiCl2.6H2O, and 0.0075% H2O2.

Murine ESOP-1 (mESOP-1) cDNA encodes a protein of 160 residues with an N-terminal hydrophobic region (16 residues) that was predicted to be the signal sequence for secretion.15 In fact Western blot analysis showed that 5% to 10% of the total ESOP-1 produced was secreted into the culture supernatant of 293T cells transfected by ESOP-1 cDNA (data not shown). The human ESOP-1 (hESOP-1) cDNA was also isolated using mESOP-1 cDNA as probe14; hESOP-1 is composed of 160 residues and is 64% identical to mESOP-1 (Figure1A). A database search indicated that hESOP-1 is identical to MD-2.16 

Fig. 1.

Alignment of mouse and human ESOP-1 and mESOP-1 expression.

(A) Alignment of amino acid sequences of murine and human ESOP-1. Identical residues are indicated by asterisks. Putative cleavage site by the signal peptidase is indicated by an arrowhead.17 The 7 cysteine residues conserved are boxed. (B) mESOP-1 mRNA expression during mouse embryogenesis. Clontech mouse embryo multiple-tissue Northern blot was probed with mESOP-1 cDNA. (C) mESOP-1 expression among adult mouse organs; 20 μg total RNA was isolated from mouse tissues and blotted onto the nylon membrane, then hybridized with labeled mESOP-1 cDNA. The expression of mESOP-1 mRNA in adult tissues was detected strongly in spleen, bone marrow, decidua of 8.5 dpc, liver, kidney, and weakly in thymus, ovary, testis, small intestine, and skin.

Fig. 1.

Alignment of mouse and human ESOP-1 and mESOP-1 expression.

(A) Alignment of amino acid sequences of murine and human ESOP-1. Identical residues are indicated by asterisks. Putative cleavage site by the signal peptidase is indicated by an arrowhead.17 The 7 cysteine residues conserved are boxed. (B) mESOP-1 mRNA expression during mouse embryogenesis. Clontech mouse embryo multiple-tissue Northern blot was probed with mESOP-1 cDNA. (C) mESOP-1 expression among adult mouse organs; 20 μg total RNA was isolated from mouse tissues and blotted onto the nylon membrane, then hybridized with labeled mESOP-1 cDNA. The expression of mESOP-1 mRNA in adult tissues was detected strongly in spleen, bone marrow, decidua of 8.5 dpc, liver, kidney, and weakly in thymus, ovary, testis, small intestine, and skin.

Close modal

By Northern blot analysis, mESOP-1 mRNA expression was induced 5 days after the differentiation induction of ES cells in vitro (data not shown). During embryogenesis, mESOP-1 mRNA expression was detected strongly on 7.5 dpc embryos (Figure 1B). The expression of mESOP-1 mRNA in adult tissues was detected in hematolymphoid tissues (spleen, bone marrow, and thymus), reproductive organs (ovary, decidua of 8.5 dpc, and testis), liver, kidney, small intestine, and skin (Figure1C) at variable expression levels.

To know the distribution of mESOP-1 during embryonic development, whole-mount immunohistochemical analysis was performed. Both anti-mESOP-1 polyclonal antibodies, AK612 and AK641, always gave the same results in Western blot and immunostaining analyses (Figure2A,B). In 7.5 dpc embryos, mESOP-1 was expressed in the yolk sac–blood islands (Figure 2Bi). Expression of mESOP-1 was seen in the spreading vascular network derived from blood islands (Figure 2Bii, 2Biii). By high-power magnification of the 9.0 dpc yolk sac, endothelial cells of the vascular plexus was found to be ESOP-1 positive (Figure 2Biv). Flat-mount view of the 9.0 dpc yolk sac and a cross-section of 8.5 dpc yolk sac showed that mESOP-1 was expressed in blood cells and endothelial cells (Figure 2Bv, 2Bvi). This immunostaining pattern in the yolk sac partially overlapped those of c-Kit, Flk-1, and Tie2.12,17 Because ESOP-1 was expressed in both blood and endothelial cells in blood islands, ESOP-1 may be also expressed in their common progenitor cells, hemangioblasts, and may regulate their differentiation. These remain to be tested.

Fig. 2.

Anti-mESOP-1 antibodies and mESOP-1 protein expression.

(A) Analysis of the specificity of anti-mESOP-1 antibodies. The coding region of mESOP-1 with FLAG tag at the 3′ end was recloned into the expression vector pEF-BOS (pEF-BOS-mESOP-1-FL). 293T cells were transfected with pEF-BOS-mESOP-1-FL (lanes 2, 4, 6) or vacant pEF-BOS vector (lanes 1, 3, 5) as a negative control using lipofectamine. Lysates of 293T cells were probed by either anti-FLAG antibodies (lanes 1, 2) or anti-mESOP-1 antibodies AK612 (lanes 3, 4) or AK641 (lanes 5, 6). (B) Immunohistochemical detection of mESOP-1 protein expression in yolk sac (i-vi), developing nervous system (vii, viii), and adult genital organs (ix, x). Both AK612 and AK641 gave the same results. Whole-mount staining of 7.5 dpc (i, ×30), 8.5 dpc (ii, ×25) embryo, and 9.0 dpc yolk sac (iii, ×12) . Magnified view (iv, ×80) and flat-mount view (v, ×200) of 9.0 dpc yolk sac, Cross-section of ii (vi, ×600). Arrowheads in a show blood islands. Arrowheads in iv show ESOP-1–positive endothelial cells in yolk sac. Spaces between the pairs of facing arrowheads indicate lumen of vascular plexus. Arrows in e show endothelial cells that expressed ESOP-1 protein. Transverse (vii, ×45) and parasagittal (viii, ×12) slices of 12.5 dpc embryos show ESOP-1 expression in the nervous system. Arrowheads show the hair follicles of vibrissae. Staining of adult ovary (ix, ×45) and testis (x, ×30) shows that all follicles, from small follicles to large, maturated follicles, oocytes (arrowheads), and granulosa cells (arrows) are ESOP-1 positive, and the signals become stronger as follicles develop. In testis, the cells in seminiferous tubules (asterisks) are stained. They may be spermatogonia or spermatocytes. VV, vitelline vessel; Br, brain; NT, neural tube; DRG, dorsal root ganglia; SN, sympathetic nerve; TN, trigeminal nerve; SF, small follicles; MF, mature follicles.

Fig. 2.

Anti-mESOP-1 antibodies and mESOP-1 protein expression.

(A) Analysis of the specificity of anti-mESOP-1 antibodies. The coding region of mESOP-1 with FLAG tag at the 3′ end was recloned into the expression vector pEF-BOS (pEF-BOS-mESOP-1-FL). 293T cells were transfected with pEF-BOS-mESOP-1-FL (lanes 2, 4, 6) or vacant pEF-BOS vector (lanes 1, 3, 5) as a negative control using lipofectamine. Lysates of 293T cells were probed by either anti-FLAG antibodies (lanes 1, 2) or anti-mESOP-1 antibodies AK612 (lanes 3, 4) or AK641 (lanes 5, 6). (B) Immunohistochemical detection of mESOP-1 protein expression in yolk sac (i-vi), developing nervous system (vii, viii), and adult genital organs (ix, x). Both AK612 and AK641 gave the same results. Whole-mount staining of 7.5 dpc (i, ×30), 8.5 dpc (ii, ×25) embryo, and 9.0 dpc yolk sac (iii, ×12) . Magnified view (iv, ×80) and flat-mount view (v, ×200) of 9.0 dpc yolk sac, Cross-section of ii (vi, ×600). Arrowheads in a show blood islands. Arrowheads in iv show ESOP-1–positive endothelial cells in yolk sac. Spaces between the pairs of facing arrowheads indicate lumen of vascular plexus. Arrows in e show endothelial cells that expressed ESOP-1 protein. Transverse (vii, ×45) and parasagittal (viii, ×12) slices of 12.5 dpc embryos show ESOP-1 expression in the nervous system. Arrowheads show the hair follicles of vibrissae. Staining of adult ovary (ix, ×45) and testis (x, ×30) shows that all follicles, from small follicles to large, maturated follicles, oocytes (arrowheads), and granulosa cells (arrows) are ESOP-1 positive, and the signals become stronger as follicles develop. In testis, the cells in seminiferous tubules (asterisks) are stained. They may be spermatogonia or spermatocytes. VV, vitelline vessel; Br, brain; NT, neural tube; DRG, dorsal root ganglia; SN, sympathetic nerve; TN, trigeminal nerve; SF, small follicles; MF, mature follicles.

Close modal

In the nervous system, mESOP-1 was found in 12.5 dpc embryos (Figure2Bvii, 2Bviii). Neural tube, sympathetic nerves, dorsal root ganglia, brain, trigeminal nerves, and hair follicles of vibrissae were clearly stained. It was also found in olfactory bulb, optic nerve, and nipples (data not shown). Immunostaining also revealed that mESOP-1 was expressed in female and male genital organs of adult mice (Figure 2Bix, 2Bx). In ovary, mESOP-1 was present in oocytes and granulosa cells. In testis, mESOP-1 was expressed in the cells of seminiferous tubules. In these systems, ESOP-1 expression also overlaps with c-Kit expression.17,18 c-Kit/SCF is thought to act in synaptic connection, neuritis elongation, and maintenance of DRG cell differentiation.19 c-Kit/SCF also regulates the oocyte maturation and the development of follicles20and spermatocytes.21 These results, taken together suggest that ESOP-1 may play some roles in the development, maintenance, or both of the hematopoietic, nervous, and reproductive systems.

Recently, it was reported that MD-2, the human counterpart of mESOP-1, confers lipopolysaccharide responsiveness by physical association with Toll-like receptor 4 (TLR4).16 Toll was originally isolated as a gene for dorsoventral patterning of the Drosophilaembryo,22 and it was reported to be needed for proper motoneuron and muscle development, larval hematopoiesis, and oocyte development.23,24 Although the tissue distribution of ESOP-1 in embryo and adult mice does not contradict the hypothesis that ESOP-1/MD-2 collaborates with a member of the TLR family during embryogenesis, it is an open question whether ESOP-1 acts in association with TLR4 in embryonic development because TLR4-deficient mice showed the hyporesponsiveness to lipopolysaccharide but have no embryonic abnormality,25 and embryonic TLR4 expression is unknown.

We thank Dr S.-I. Nishikawa and Dr H. Yoshida for their kind gift of the anti-c-Kit and anti-Flk-1 monoclonal antibodies, their teaching on whole-mount immunohistochemistry, and the helpful discussions. We thank N. Tomikawa, M. Tanaka and M. Yamaguchi for their assistance in the experiments and in the preparation of the manuscript.

Supported in part by grants from the Ministry of Education, Science, and Culture of Japan (Monbusho) and by a fellowship from the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Genbank accession numbers for mouse and human ESOP-1 are AF168120 andAF168121, respectively.

Reprints:Tasuku Honjo, Department of Medical Chemistry, Faculty of Medicine, Yoshida, Konoe-cho, Sakyo-ku, Kyoto 606-8507, Japan; e-mail: honjo@mfour.med.kyoto-u.ac.jp.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Guimarães
MJ
Bazan
JF
Zlotnik
A
et al
A new approach to the study of haematopoietic development in the yolk sac and embryoid bodies.
Development.
121
1995
3335
3346
2
Choi
K
Wall
C
Hanratty
R
Keller
G
Isolation of a gene encoding a novel receptor tyrosine kinase from differentiated embryonic stem cells.
Oncogene.
9
1994
1261
1266
3
Shen
MM
Wang
H
Leder
P
A differential display strategy identifies Cryptic, a novel EGF-related gene expressed in the axial and lateral mesoderm during mouse gastrulation.
Development.
124
1997
429
442
4
Nakano
T
Kodama
H
Honjo
T
Generation of lymphohematopoietic cells from embryonic stem cells in culture.
Science.
265
1994
1098
1101
5
Nishikawa
S-I
Nishikawa
S
Hirashima
M
Matsuyoshi
N
Kodama
H
Progressive lineage analysis by cell sorting and culture identifies FLK-1+VE-cadherin+cells at a diverging point of endothelial and hematopoietic lineages.
Development.
125
1998
1747
1757
6
Nishikawa
S-I
Nishikawa
S
Kawamoto
H
et al
In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.
Immunity.
8
1998
761
769
7
Choi
K
Hemangioblast development and regulation.
Biochem Cell Biol.
76
1998
947
956
8
Shalaby
F
Rossant
J
Yamaguchi
TP
et al
Failure of blood–island formation and vasculogenesis in Flk-1-deficient mice.
Nature.
376
1995
62
66
9
Carmeliet
P
Ferreira
V
Breier
G
et al
Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.
Nature.
380
1996
435
439
10
Sato
TN
Tozawa
Y
Deutsch
U
et al
Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation.
Nature.
376
1995
70
74
11
Suri
C
Jones
PF
Patan
S
et al
Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis.
Cell.
87
1996
1171
1180
12
Takakura
N
Huang
XL
Naruse
T
et al
Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis.
Immunity.
9
1998
677
686
13
Tashiro
K
Nakamura
T
Honjo
T
The signal sequence trap method.
Methods Enzymol.
303
1999
479
495
14
Honjo T, Kato K, Tada H. Isolation of a new polypeptide and cDNA differentiated ES cells by SST method. International publication number WO 99/18205. April 15, 1999.
15
von Heijne
A new method for predicting signal sequence cleavage sites.
Nucl Acid Res.
14
1986
4683
4690
16
Shimazu
R
Akashi
S
Ogata
H
et al
MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.
J Exp Med.
189
1999
1777
1782
17
Yoshida
H
Takakura
N
Hirashima
M
et al
Hematopoietic tissues, as a playground of receptor tyrosine kinases of the PDGF-receptor family.
Dev Comp Immunol.
22
1998
321
332
18
Orr–Urtreger
A
Avivi
A
Zimmer
Y
Givol
D
Yarden
Y
Lonai
P
Developmental expression of c-kit, a proto-oncogene encoded by the W locus.
Development.
109
1990
911
923
19
Hirata
T
Morii
E
Morimoto
M
et al
Stem cell factor induces outgrowth of c-kit-positive neurites and supports the survival of c-kit-positive neurons in dorsal root ganglia of mouse embryos.
Development.
119
1993
49
56
20
Yoshida
H
Takakura
N
Kataoka
H
Kunisada
T
Okamura
H
Nishikawa
SI
Stepwise requirement of c-kit tyrosine kinase in mouse ovarian follicle development.
Dev Biol.
184
1997
122
137
21
Vincent
S
Segretain
D
Nishikawa
S
et al
Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis.
Development.
125
1998
4585
4593
22
Hashimoto
C
Hadson
KL
Anderson
KV
The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein.
Cell.
52
1988
269
679
23
Halfon
MS
Hashimoto
C
Keshishian
H
The Drosophila Toll gene functions zygotically and is necessary for proper motoneuron and muscle development.
Dev Biol.
169
1995
151
167
24
Qui
P
Pan
PC
Govind
S
A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis.
Development.
125
1998
1909
1920
25
Hoshino
K
Takeuchi
O
Kawai
T
et al
Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.
J Immunol.
162
1999
3749
3752
Sign in via your Institution