earlyexpressionofendomucinonendotheliumofthe...

Early Expression of Endomucin on Endothelium of theMouse Embryo and on Putative Hematopoietic Clusters inthe Dorsal AortaGERTRUD BRACHTENDORF,1 ANNEGRET KUHN,1,2 ULRIKE SAMULOWITZ,1 RUTH KNORR,1

ERIKA GUSTAFSSON,3 ALEXANDRE J. POTOCNIK,4 REINHARD FASSLER,3 AND DIETMAR VESTWEBER1*1Institute of Cell Biology, ZMBE, University of Munster and Max-Planck-Institute of Physiological and Clinical Research,Bad Nauheim, Germany2Department of Dermatology, University of Dusseldorf, Dusseldorf, Germany3Department of Experimental Pathology, Lund University, Lund, Sweden4Basel Institute for Immunology, Basel, Switzerland

ABSTRACT Endomucin is a recently identi-fied sialomucin that is specifically expressed onendothelium of the adult mouse. Here, we haveanalysed the expression of endomucin during de-velopment of the vascular system by immunohis-tochemistry by using three monoclonal antibod-ies (mAb). We demonstrate that two of the mAb,V.5C7 and V.1A7, recognize epitopes on the non-glycosylated protein, because they recognize theantigen when it is synthesized as a bacterial fu-sion protein and when it is in vitro translated ina membrane-free reticulocyte lysate. During invitro differentiation of embryonic stem cells toendothelial cells, endomucin is expressed at day6 after onset of differentiation, 1 day later thanPECAM-1. During differentiation of the mouseembryo, endomucin is first detected at E8.0 in allembryonic blood vessels detectable at this stagebut is absent in blood islands of the yolk sac.Analysing the paraaortic-splanchnopleura (P-SP)region and the aorta-gonad-mesonephros (AGM)region as sites of intraembryonic hematopoiesis,we found that endothelium of the dorsal aorta isbrightly positive for endomucin at E8.5–9.0 andat E11.5. At later stages and in the adult aorta,endothelial staining is strongly reduced and con-fined to focal areas. Cell clusters associated withthe luminal surface of the endothelium of thedorsal aorta could be stained for endomucin andfor CD34. At a later stage (E15.5) single leuko-cytes in the lumen of large venules were stainedfor endomucin. We conclude that endomucin isan early endothelial-specific antigen that is alsoexpressed on putative hematopoietic progenitorcells. © 2001 Wiley-Liss, Inc.

Key words: sialomucin; hematopoisis; glycosyla-tion; endothelium

INTRODUCTION

Endomucin is an endothelial type 1 integral mem-brane protein that is densely substituted with sialy-lated O-linked carbohydrates (Morgan et al., 1999).

This extensive O-glycosylation is a characteristic struc-tural feature of mucins. Mucin-like glycoproteins aresubdivided into the epithelial mucins, encompassingMUC-1 to MUC-8, and the leukocyte/endothelial mu-cins, also called sialomucins. Prominent members ofthe sialomucins are the endothelial molecules CD34,mucosal addressin MAdCAM-1, glycosylation-depen-dent cell adhesion molecule-1 (GlyCAM-1), podoca-lyxin, reviewed by Vestweber (1999), and endoglycan(Sassetti et al., 2000), and the leukocyte moleculesCD43, CD45, reviewed by Barclay et al. (1997) andP-selectin glycoprotein ligand-1 (PSGL-1) (McEver,1997). This cell type specificity is not absolute, becauseMUC-1 is also found on hematopoietic cells (Brugger etal., 1999),whereas GlyCAM-1 is expressed by epithelialcells of the lactating mammary gland (Dowbenko et al.,1993), CD34 by bone marrow stroma (Simmons et al.,1992), and podocalyxin was originally identified onpodocytes (Kerjaschki et al., 1984).

Extensive O-glycosylation causes mucin domains toadopt an extended, rodlike structure. This event allowsmucins to protrude beyond the polysaccharide glycoca-lyx that surrounds the cell. The extended structure, thebulky glycosylation, and the negative charges allowsmucins to serve as repulsive barriers, as shown forCD43 on T cells (Stockton et al., 1998) and for podoca-lyxin on podocytes (Kerjaschki et al., 1984). In othercases, when specific receptors are present on the othercell, sialomucins can serve as ligands for the selectinsand thereby initiate cell–cell interactions. This findingis the case for the P-selectin ligand PSGL-1 or theL-selectin ligands GlyCAM-1 (Lasky et al., 1992), MAd-CAM-1 (Berg et al., 1993), CD34 (Baumhueter et al.,1993), and podocalyxin (Sassetti et al., 1998). In addi-

Grant sponsor: IZKF, Munster; Grant sponsor: the Craeford Foun-dation.

G. Brachtendorf and A. Kuhn contributed equally to this work.*Correspondence to: Dietmar Vestweber, Institute of Cell Biology,

ZMBE, University of Munster, Von-Esmarch-Str. 56, 48149 Muen-ster, Germany. E-mail: [email protected]

Received 30 May 2001; Accepted 20 July 2001Published online 3 October 2001; DOI 10.1002/dvdy.1199

DEVELOPMENTAL DYNAMICS 222:410–419 (2001)

© 2001 WILEY-LISS, INC.

tion to mediating cell adhesion, sialomucins probablyalso function as signal transducing molecules. How-ever, this has not yet been intensively analysed.

Interestingly, two of the endothelial sialomucins,CD34 (Civin et al., 1984; Katz et al., 1985; Andrews etal., 1986) and podocalyxin (Hara et al., 1999), are alsofound on hematopoietic progenitor cells. Both proteinsshow structural similarities beyond the fact of beingsialomucins. They share motifs in their cytoplasmicpart and contain a cysteine-containing, presumablyglobular domain within their extracellular part, whichmay fold in a manner similar to an immunoglobulin(Ig) -like domain (Barclay et al., 1997). CD34 is foundon multipotent hematopoietic progenitors but not onmature peripheral leukocytes (Civin et al., 1984; Katzet al., 1985; Andrews et al., 1986). Remarkably, CD34is expressed already on the earliest population of long-term reconstituting hematopoietic stem cells (HSCs) inthe AGM region at E11 (Sanchez et al., 1996). Thechicken ortholog of podocalyxin, thrombomucin, wasdescribed on hematopoietic progenitors and on throm-bocytes (McNagny et al., 1997). Podocalyxin was alsodescribed as a cell surface marker for hemangioblasts,the still controversially discussed precursor cells withthe potential to differentiate into hematopoietic as wellas into endothelial cells (Hara et al., 1999). CD34 andpodocalyxin are both broadly expressed on the luminalsurface of vascular endothelium (Horvat et al., 1986;Fina et al., 1990; Kershaw et al., 1995).

Another sialomucin, which is more broadly distrib-uted, is CD164. Different classes of epitopes on thisprotein show a different tissue distribution. Amongseveral other cell types CD164 is expressed on endo-thelia in various tissues (Watt et al., 2000). Originally,the protein was found on human bone marrow stromaand on subpopulations of CD341 hematopoietic cells,including clonogenic myeloid and erythroid progenitorsand the hierarchically more primitive precursors (Zan-nettino et al., 1998). Binding of hematopoietic progenitorcells to bone marrow stroma cells could be inhibited withantibodies against CD164. In addition, the antibody couldinterfere with proliferation (Zannettino et al., 1998).

The first rudiments of the vascular system form asaggregates of mesenchymal cells in the yolk sac knownas blood islands. These islands consist of erythro- andhematopoietic precursor cells as well as endothelialprecursors. Blood islands give rise to a primary vascu-lar plexus. Within the embryo, blood vessels are formedby vasculogenesis and angiogenesis. During vasculo-genesis, angioblasts differentiate to form a primarycapillary plexus that is rapidly remodelled either byfusion, to give rise to larger vessels, or by regressionand migration. Vascularization of some organs such askidney and brain is dependent on angiogenesis, forma-tion of new vessels by sprouting from preexisting ves-sels that invade the organ.

During embryogenesis, the development of the he-matopoietic system proceeds through two distinctphases, primitive and definitive hematopoiesis. In

mice, primitive hematopoiesis begins in the extraem-bryonic yolk sac around E7 to E7.5, whereas the defin-itive hematopoietic system originates from long-termrepopulating HSCs generated in the intraembryonicAGM region at E9.5 reviewed by (Dzierzak et al., 1998).The emergence of these cells is preceded by multipo-tential hematopoietic progenitors, including cells har-boring lymphoid potential, in the para-aortic splanch-nopleura (P-Sp) region of mouse embryos at E7.5–E8.5,an intraembryonic site preceding the AGM region. Af-ter their emergence in the AGM region, long-term re-constituting HSCs colonise the fetal liver as the mainsite for fetal hematopoiesis and subsequently the bonemarrow (Delasssus and Cumano, 1996).

CD34 and CD164 have been detected on cell clustersof presumptive hematopoietic cells associated to theventral side of the dorsal aorta of 32-day-old humanembryos (Watt et al., 2000). In addition, podocalyxinwas reported to be expressed on cells of the AGM regionof mouse embryos at stage E11.5. These cells wereshown to be able to generate endothelial cells as well ashematopoietic cells when cultured under appropriateconditions in vitro or when injected into busulfan-treated mice (Hara et al., 1999), suggesting that podo-calyxin could serve as a cell surface marker for heman-gioblasts.

Recently, we have identified endomucin as a novelsialomucin, that is specifically expressed on endotheliaof the adult mouse. Here, we have analysed the tissuedistribution and expression kinetics of endomucin dur-ing mouse development. We found endomucin to bespecifically expressed on all endothelia, and it wasexpressed on blood vessels as early as E8.0 of embryo-nal development. It was not detected in blood islands ofthe extraembryonic yolk sac at this stage, suggestingthat endomucin is absent on extraembryonic precur-sors of endothelial cells. Interestingly, endothelium ofthe dorsal aorta at stage E8.5–9.0 and E11.5 as well asputative hematopoietic cell clusters associated to theendothelial cell layer of the aorta were stained forendomucin. Thus, endomucin, although absent fromthe majority of mature leukocytes in the adult mousecould have a function on hematopoietic progenitors andis very early and specifically expressed on endotheliaduring mouse development.

RESULTS AND DISCUSSIONCharacterization of Endomucin Epitopes

We had originally identified endomucin with the helpof three mAb, V.1A7, V.5C7, and V.7C7 that we hadraised against mouse endothelial cells (Morgan et al.,1999). Each had been shown to immunoprecipitate a75-kDa major form and a 66-kD minor form of endo-mucin from surface biotinylated endothelial cells. Siali-dase treatment of the purified antigen destroyed bind-ing to each of the three mAb, suggesting that theabsence of sialic acid on endomucin affects antibodyrecognition. We now tested whether the three antibod-ies would indeed recognize epitopes provided by the

411ENDOMUCIN IN MOUSE EMBRYO DEVELOPMENT

carbohydrate side chains or whether the absence ofsialic acid would indirectly affect the formation ofepitopes by the polypeptide backbone of the antigen. Tothis end, we synthesized and radioactively labeled en-domucin with [35S]-methionine by coupled in vitrotranscription/translation reactions in reticulocyte ly-sates and tried to immunoprecipitate the nonglycosy-lated antigen with each of the three antibodies. Sur-prisingly, the two mAb V.1A7 and V.5C7 clearly boundto the antigen, whereas V.7C7 did not (Fig. 1A). Thisfinding demonstrates that at least the first two mAbrecognize protein epitopes. A similar result was ob-tained when bacterial fusion proteins consisting of glu-tathione-S-transferase (GST) and full-length endomu-cin were immunoblotted and tested for reactivity.Again V.1A7 and V.5C7 but not V.7C7 recognized thebacterial fusion protein (Fig. 1B), verifying that theformer two antibodies recognize epitopes on the nong-lycosylated backbone of endomucin. Whether theV.7C7 epitope is indeed a clean carbohydrate epitope ora mixed carbohydrate/protein epitope or only indirectlydependent on glycosylation is still unclear. However,that the expression pattern of the V.7C7 epitope in theadult (Morgan et al., 1999) as well as in the embryo (seebelow) is identical with the other two epitopes stronglysuggests that the epitope of V.7C7 is not defined bycarbohydrates alone.

Expression of Endomucin on EmbryonicStem Cells Differentiating in Vitro toEndothelial Cells

The endothelial specific expression of endomucin inthe adult organism prompted us to test whether andwhen endomucin would be expressed during in vitrodifferentiation of embryonic stem (ES) cells. To inducedifferentiation, ES cells were removed from the embry-onal fibroblast feeder layer and cultured for 2 days in“hanging drops” to initiate the formation of embryoidbodies. Subsequently, cell aggregates were cultured onbacterial Petri dishes. Embryoid bodies at differentstages were subjected to cryostat sectioning and stain-ing with V.5C7 and a mAb against PECAM-1. Asshown in Figure 2, PECAM-1 was expressed after 5days of culture, whereas endomucin was first detected1 day later. The expression kinetics of PECAM-1 are inagreement with a report of Vittet et al. (1996), whofound that the PECAM-1 protein was first detectedvery weakly at day 4 and more clearly at day 5 indifferentiating embryoid bodies. Interestingly, thesame report described that VE-cadherin, the endothe-lial-specific cadherin, becomes first detectable at theprotein level at day 6 of embryoid body differentiation,hence, at the same time as endomucin. We could repro-duce this expression kinetics by staining with the anti–VE-cadherin mAb 11D4 (not shown). VE-cadherin isalready expressed very early during embryo develop-ment in blood islands of the yolk sac of E7.5 mouseembryos. Our results establish that in vitro differenti-ating ES cells autonomously regulate the expression of

VE-cadherin and endomucin with similar expressionkinetics.

Specific Expression of Endomucin onEndothelial Cells During Mouse Development

We analysed the expression of endomucin in theembryo by immunohistochemistry of sections of paraf-

Fig. 1. Biochemical characterization of endomucin epitopes. A: Im-munoprecipitation of in vitro transcribed and translated endomucin. Invitro translation products obtained in the absence (lane 1) or in thepresence of an expression vector (lanes 2–5) were either directly loadedon the gel (lanes 1 and 2) or subjected to immunoprecipitations with theanti-endomucin monoclonal antibody (mAb) V.1A7, V.5C7, and V.7C7,as indicated. B: Reactivity of anti-endomucin mAb with a bacterial gluta-thione-S-transferase (GST) -endomucin fusion protein in immunoblots.Isolated GST (4 mg per lane) or GST-endomucin fusion protein (GST-EM,150 ng per lane) were electrophoresed, blotted, and the resulting filterwas analysed for reactivity with an anti GST antiserum (anti GST), or oneof the three monoclonal antibodies against endomucin, as indicated.Molecular mass markers (in kilodaltons) are indicated on the left.

412 BRACHTENDORF ET AL.

fin-embedded mouse embryos of various stages. No spe-cific staining for endomucin was detected in the embryoat stage E7.0, whereas blood vessels in the uterus wereclearly stained (Fig. 3A). A similar result was obtainedfor CD34 (Fig. 3B). First signs of expression of endo-mucin were detected in embryonic blood vessels atE8.0. Figure 3D shows strong staining of intersomitic

vessels in a sagittal section of an E8.0 embryo, whereasCD34 was similarly, although more weakly, stained(Fig. 3E).

Next, we wanted to know whether endomucin is alsoexpressed at sites where endothelial cells and theirprecursors first develop. This development occurs inblood islands of the extraembryonic yolk sac where

Fig. 2. Immunohistochemical staining of embryonic stem (ES) cellderived embryoid bodies ranging from day 3 (d3) to day 12 (d12) ofdifferentiation. ES cells were allowed to differentiate in vitro into embryoidbodies and serial cryostat sections were prepared and stained as de-scribed in Materials and Methods section. Slight morphologic differencesmay occur between embryoid bodies serial sections of the same agebecause of the thickness of sections. Staining for endomucin (mAb

V.5C7) was first detectable at day 6 as demonstrated in the left panels.The middle panels show PECAM-1 immunostaining on serial cryostatsections with specific labeling first detectable at day 5. For negativecontrols, the first antibody was replaced by an irrelevant isotype-matchedreagent as shown in the right panels. Scale bars 5 200 mm in d3,d5,d6,50 mm in d12.


mesenchymal cell aggregates differentiate into endo-thelial cells and erythroid precursor cells. Figure 3Gshows an overview of a transverse section of an E8.0embryo together with the surrounding uterus. Endo-

mucin was clearly stained in blood vessels of the em-bryo as well as in the uterus; however, no staining wasseen in blood islands of the extraembryonic yolk sac, asshown at higher magnifications in Figure 3H,I. These

Fig. 3. Expression of endomucin in embryonic day (E) 7.0, E8.0,E8.5–9.0 mouse embryos. Paraffin sections of mouse embryos at differ-ent stages were analysed by immunohistochemistry. A–C: Transversesections of an E7.0 embryo. Endomucin (A) and CD34 (B) are restrictedto vessels of the uterus, no staining can be observed in the embryo. In C,a negative control with an irrelevant isotype-matched antibody is shown.D–F: Sagittal sections of an E8.0 embryo. Endomucin (D) is stronglystained in intersomitic vessels of the unturned embryo, CD34 (E) wasused as positive control, an irrelevant isotype-matched antibody (F) asnegative control. G–I: Transverse sections of an E8.0 embryo stained for

endomucin. The framed area in (G) is shown at higher magnification in(H), and the framed area in (H) at higher magnification in (I). Note thatblood vessels in the uterus as well as in the embryo are positive, whereasthe blood island in the extraembryonic yolk sac, shown in the framedarea, is negative. K–M: Sagittal sections of the head region of an E8.5–9.0 embryo. Endomucin (K) and CD34 (L) are expressed on endotheliumof all vessels, although anti-CD34 staining was much weaker. M: Nega-tive control. ne, neuroepithelium; mw, myocardial wall of common cham-ber of the heart; ut, uterus; s, somites. Scale bar 5 200 mm in A,H,K(applies to A–C,H,K–M), 50 mm in D,I (applies to D–F,I), 800 mm in G.


results suggest that endomucin is not expressed onendothelial precursors in extraembryonic tissue, incontrast to VE-cadherin that was detected by in situhybridization at such sites (Breier et al., 1996). Thus,

although VE-cadherin and endomucin are induced si-multaneously in differentiating ES cell cultures, onlyVE-cadherin but not endomucin is expressed by endo-thelial precursors in blood islands of the yolk sac. As

Fig. 4. Expression of endomucin in hematopoietic tissues and onputative hematopoietic precursor cells. Immunohistochemistry of paraffinsections of embryonic day (E) 11.5 (A–H), E8.5–9.0 (I) and E15.5(K–M) mouse embryos. Endomucin (monoclonal antibody [mAb] V.5C7)(A–C) and CD34 (D–F) are expressed on endothelium of the aorta as wellas on cell clusters (arrowheads) associated with their endothelium. Eachframed area is shown at higher magnification in the picture on the right.G and H show a section of the subcardinal vein at E11.5 stained forendomucin (mAb V.7C7). H is a detail of the framed area in G at highermagnification. Note that a single cell (denoted by an arrowhead) withinthe tissue in close vicinity to the vessel lumen is positive for endomucin

in addition to small blood vessels and the endothelium of the vein. Idepicts endomucin staining (mAb V.7C7) of a sectioned aorta of anE8.5–9.0 embryo, again with a luminal cell cluster (arrowhead) associ-ated with the vessel wall and positive for endomucin. K–M show a venulewith peripheral blood cells in the abdominal region of an E15.5 embryo,stained with the two mAb V7C7 (K) and V.5C7 (L) against endomucin orwith an isotype matched control antibody (M). All three sections areconsecutive. Note that the same blood cell (marked by an arrow) isstained by each of the two anti-endomucin antibodies. Scale bars 5 200mm in A,D, 50 mm in B,E,G,I, 20 mm in C,F,H,K–M.


analysed on sections of E8.5–9.0 embryos, endomucinwas well expressed on endothelium of capillaries aswell as large blood vessels throughout the embryo(Fig. 3K).

Another interesting site of development of the bloodsystem is the AGM region (Dzierzak et al., 1998) atE10.5 to E11.5, which develops from the paraaortic-splanchnopleura (P-SP) region (Godin et al., 1995;Delassus and Cumano, 1996). Both regions representthe first intraembryonic sites for the development ofhematopoietic progenitors. It was reported that hema-topoietic progenitors express CD34 (Sanchez et al.,1996) and form CD341 clusters that adhere to theventral luminal surface of the dorsal aorta at stageE10.5–11.5 (Garcia-Porrero et al., 1998). As shown inFigure 4A–C, the endothelium of the aorta at stageE11.5 was strongly positive for endomucin, as well as

for CD34 (Fig. 4D–F). At larger magnification, we couldindeed detect cell clusters closely associated with theendothelium that were stained for endomucin and forCD34. In addition, we detected a single cell positive forendomucin within the tissue in close vicinity to thelumen of the subcardinal vein in E11.5 sections (Fig.4G,H). A cluster of endomucin-positive cells inside theaorta, associated to the endothelium, was also detectedin sections of stage E8.5–9.0 (Fig. 4I). Based on thelocation and the embryonal stage, it is conceivable thatthese endomucin-positive, endothelium-associated cellsare potential hematopoietic progenitors. In line withthis interpretation, we detected a single blood cellstained for endomucin with two different mAbs againstendomucin (V.7C7 and V.5C7) in two consecutive sec-tions of a large venule in the abdominal region of anE15.5 embryo (Fig. 4K,L). Whether this finding is a

Fig. 5. Endothelial specificity of endomucin in embryonic day (E) 15.5mouse embryo and expression on embryonal and adult aorta.A–C: Kidney and aorta of E15.5 mouse embryo. Endomucin (A) andCD34 (B) are strongly expressed on endothelium but absent from epi-thelia. C shows negative control with an isotype-matched antibody.D–F: The area of the aorta shown in A–C is shown at higher magnifica-tion. Endomucin (D) shows a focal expression pattern on the endotheliumof the aorta, whereas CD34 is evenly and strongly expressed across the

entire endothelium of the embryonal aorta. F: Negative control.G–I: Transverse sections of the aorta of an adult mouse. G demonstratesfocal expression of endomucin, and H shows staining of the aorta withanti–PECAM-1 antibody as positive control. I: Isotype-matched antibodyas negative control. Arrowheads point to focal endomucin staining ofaorta endothelium. a, aorta; k, kidney. Scale bars 5 200mm in A (appliesto A–C), 50 mm in D,G (applies to D–I).


circulating endothelial precursor or a hematopoieticprogenitor cannot be decided. This finding demon-strates that endomucin-expressing circulating cells arepresent in the embryo at stage E15.5. In combinationwith the stainings shown in Figure 4, our results rep-resent indirect evidence for the expression of endomu-cin on hematopoietic precursor cells.

Expression of endomucin on endothelium of the em-bryonic dorsal aorta at E11.5 and at earlier stages is incontrast to the lack of endomucin staining we detectedearlier when analysing the aorta of adult mice (Morganet al., 1999). To clarify this, we stained sections derivedfrom E15.5 embryos and of adult aorta. As shown in theoverview of Figure 5A, mAb against endomucin specif-ically stained blood vessels of the kidney, whereas theaorta was negative for endomucin, but clearly positivefor CD34 (Fig. 5B). When the aorta was analysed athigher resolution, weak focal endomucin-staining (Fig.5, marked by arrowheads) of some endothelial cells ofthe aorta became detectable (Fig. 5D). Similar weakfocal staining was also detected for endomucin in theadult aorta (Fig. 5G). We conclude that the strongexpression of endomucin on endothelial cells of theaorta at early stages of development vanishes at laterstages and becomes reduced to a weakly detectablelevel of focal expression, whereas expression of endo-mucin on other endothelia of venules and capillariescontinues to be strong. In general, from the first signsof expression at stage E8.0, endomucin was found to bestrongly expressed on capillaries and larger blood ves-sels in all tissues throughout the embryo. No other celltypes such as epithelia, neurons, muscle cells, or fibro-blasts were detected to express endomucin.

CONCLUDING REMARKS

Our results establish endomucin as an endothelial-specific sialomucin throughout embryonal develop-ment. With the exception of hematopoietic progenitors,no other additional cell type expressed endomucin. En-dothelial cells generated in vitro from differentiatingES cells expressed endomucin at a similar stage asreported for VE-cadherin and tie-1 (Vittet et al., 1996).In vivo, endomucin was not detected on extraembry-onic endothelial precursors in blood islands, althoughexpression of VE-cadherin mRNA has been found inthese cells (Breier et al., 1996). Whether intraembry-onic endothelial progenitors generated at a later stageexpress endomucin will be important to analyse in thefuture.

Our results on the expression of endomucin on cellsassociated with the aorta in the AGM region supportthe hypothesis that endomucin might also be expressedon hematopoietic progenitors. This finding would placeendomucin in one group together with the sialomucinsCD164, CD34, and podocalyxin. Future investigationswill tell whether or to what extent these molecules areimportant for the development of hematopoietic cells.Recent results suggest that overexpression of mouseendomucin in human embryonic kidney cells reduces

cell attachment and cell aggregation (Ueno et al. manu-script submitted for publication). Similarly, overex-pression of human endomucin in HEK293T cells wasreported to affect cell attachment to glass surfaces if noextracellular matrix proteins had been precoated (Ki-noshita et al., 2001). It is possible that the regulation ofendomucin expression levels or the binding of not yetidentified endomucin ligands might regulate cell adhe-sive functions. Such effects might be involved in thedetachment of hematopoietic cells from endothelium.

EXPERIMENTAL PROCEDURESStaging of Embryos

Gestational age was initially determined by the dateof formation of the copulation plug and confirmed bycrown-rump length, number of somites, and landmarksof neural development. Mouse embryos at differentembryonic days (E) were embedded in paraffin andused for immunohistochemical analysis (E7.0, E8.0,E8.5–9.0, E11.5, E13.5, E15.5) or used directly forwhole-mount immunolabeling (E9.5).

Embryoid Bodies

Embryoid bodies were generated as described previ-ously (Fassler et al., 1996). Briefly, to initiate ES celldifferentiation and embryoid body formation, droplets(20 ml) containing approximately 600 129/Sv-derivedES cells in Dulbecco’s modified Eagle’s medium with20% fetal calf serum (without LIF) were seeded to aninverted lid of a cell-culture dish. After 2 days, the cellsformed aggregates that were transferred to bacterialdishes and grown in suspension in the same mediumwithout further feeding for up to 12 days. Embryoidbodies were collected at different days of differentia-tion, embedded in tissue freezing medium, and storedat 280°C until processed for immunohistochemicalanalysis.

Serologic Markers

Rat monoclonal antibodies against endomucin weregenerated as described previously (Morgan et al.,1999), and the isotype of the mAb against endomucinwere determined to be IgG1 for V.1A7 and V.5C7 andIgG2a for V.7C7. Antibodies were used as superna-tants. Endothelial specificity was controlled by rat mAbagainst CD34, IgG1, dilution 1:50, and against PE-CAM-1, IgG1, dilution 1:200 (Pharmingen, Heidelberg,Germany) and rat mAb 11D4 against mouse VE-cad-herin (Gotsch et al., 1997).

Immunohistochemical Staining

For paraffin-embedded sections, mouse embryoswere fixed overnight in fresh 4% paraformaldehyde inHEPES buffer, pH 7.4, and 3- to 5-mm consecutivesections were cut on a microtome. After mounting onslides coated with poly-L-lysine (Menzel-Glaser,Nubloch, Germany), dewaxed specimens were im-mersed in sodium citrate buffer (10 mM sodium citrate


monohydrate, pH 6.0) in plastic Coplin jars and incu-bated in an autoclave at 120°C for 10 min. Subse-quently, the slides were allowed to cool down to roomtemperature over a period of 20–30 min, followed byreduction of endogenous peroxidase activity with 0.1%hydrogen peroxide, 0.1 M sodium azide, for 30 min atroom temperature. Nonspecific binding was blockedwith 2% bovine serum albumin in PBS, pH 7.4, for 30min. Tissue sections were incubated for 1 hr with ap-propriate primary antibodies diluted in PBS containing1% bovine serum albumin, followed by incubation withaffinity-purified peroxidase-labeled donkey anti-ratIgG (dilution 1:1,000, Dianova, Hamburg, Germany)and goat anti-rabbit IgG (dilution 1:1,000, Dianova).After the reaction was visualized with 3-amino-9-eth-ylcarbazole, tissue sections were counterstained withMayer’s haematoxylin and mounted. All reactions wereperformed in a humidified chamber at room tempera-ture, and, for control purposes, the first mAb was eitheromitted or replaced by an irrelevant isotype-matchedreagent. These experiments consistently yielded nega-tive results.

For embryoid bodies, 7-mm serial sections were cuton a freezing microtome, mounted on slides coated withpoly-L-lysine, and fixed in acetone for 10 min at 4°C,followed by reduction of endogenous peroxidase activ-ity for 30 min at room temperature. Nonspecific bind-ing was blocked with 2% bovine serum albumin in PBSfor 30 min. Tissue sections were stained and counter-stained as described above.

In Vitro Transcription and Translation

Murine endomucin lacking posttranslational modifi-cations was synthesized by a coupled in vitro transcrip-tion/translation reaction in a reticulocyte lysate by us-ing the “TNT Quick coupled Transcription/TranslationSystem” (Promega, Mannheim, Germany). Based onthe manufacturer’s instructions, each reaction contain-ing 8 ml of TNT Quick Master Mix, 164 kBq 35S-methi-onine (Amersham, Freiburg, Germany) and 0.04-mgvector DNA (either pcDNA3 containing full-lengthmouse endomucin cDNA or vector without insert) wasfilled up to a final volume of 10 ml with DEPC-H2O andincubated for 90 min at 37°C. Subsequently, reactionswere subjected to immunoprecipitations, as previouslydescribed (Weller et al., 1992). As negative or positivestandards, either 10 ml of a reaction containing thevector without insert or 2 ml of a reaction containingthe vector with insert were loaded on the gel, respec-tively.

GST-Fusion Protein

A bacterial fusion protein was generated consistingat its N-terminus of glutathione-S-transferase fused atits C-terminus to the complete extracellular part ofmouse endomucin, ranging from methionine at position1 to the last serine of the extracellular part at position177 followed by amino acids PGSTRAAAS based onsequences of the vector. For construction, the endomu-

cin fragment was synthesized by polymerase chain re-action by using the oligonucleotides CGGGATCCAT-GCGGCTGCTTCAAGCG as upstream and TCCCCCG-GGACTGGAAT AGGAGGGGGTGGT as downstreamprimers and inserted in the bacterial expression vectorpGEX-4T-1 (Pharmacia) by using the restriction sitesBamHI at the 59 end and SmaI at the 39 end of theendomucin fragment. The fusion protein was producedin the protease deficient Escherichia coli strain BL21and isolated as described (Aberle et al., 1994). Gluta-thione-S-transferase was produced from pGEX-4T-1 fornegative controls. Immunoblotting was done as previ-ously described (Morgan et al., 1999). Glutathione-S-transferase was detected by using an affinity-purifiedpolyclonal antiserum (Santa Cruz Biotechnology,Santa Cruz, CA).

ACKNOWLEDGMENTS

The Basel Institute for Immunology was founded andsupported by F. Hoffmann-La Roche Ltd., Basel, Swit-zerland. D.V. received support from the IZKF, Mun-ster; A.K. received support by the Lise-Meitner-Schol-arship; and E.G. received a fellowship from theCraeford Foundation.

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