ESOP-1, a secreted protein expressed in the hematopoietic, nervous, and reproductive systems of embryonic and adult mice

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).

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.

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.

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 

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.

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.