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E L Krug

Publications and source records attributed to E L Krug.

At least 19 recordsLinked to original sources
PubMed2006

Bidirectional fusion of the heart-forming fields in the developing chick embryo.

It is generally thought that the early pre-tubular chick heart is formed by fusion of the anterior or cephalic limits of the paired cardiogenic fields. However, this study shows that the heart fields initially fuse at their midpoint to form a transitory "butterfly"-shaped, cardiogenic structure. Fusion then progresses bi-directionally along the longitudinal axis in both cranial and caudal directions. Using in vivo labeling, we demonstrate that cells along the ventral fusion line are highly motile, crossing future primitive segments. We found that mesoderm cells migrated cephalically from the unfused tips of the anterior/cephalic wings into the head mesenchyme in the region that has been called the secondary heart field. Perturbing the anterior/cranial fusion results in formation of a bi-conal heart. A theoretical role of the ventral fusion line acting as a "heart organizer" and its role in cardia bifida is discussed.

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PubMed2001

Living morphogenesis of the ventricles and congenital pathology of their component parts.

Living morphogenetic studies show that each definitive ventricle is constructed from different primitive cardiac segments, and each has its specific anatomical features. These ventricular segments are the atrioventricular junction; the primitive inlet segment, part of the primary heart tube, which initially provides the inlets of each ventricle; the primitive outlet segment, which gives rise to both ventricular outlets; and the apical trabeculated regions of the right and left ventricles which grow from the primary heart tube, respectively. In this review, we describe regional pathology based on the relationship of these primitive ventricular components. We propose that the abnormal morphogenesis of one of these segments gives origin to regional ventricular pathology. For example, abnormal embryogenesis of the atrioventricular canal produces malformations of the atrioventricular junctions, such as double inlet ventricle, absence of one atrioventricular connection, and straddling and overriding atrioventricular valves. Similarly, abnormal morphogenesis of the primitive outlet segment gives rise to malformations of the subarterial region of each ventricle, along with the valves guarding these vessels. The principal anatomical features of these malformations of the ventricular inlets and outlets are described, and their possible morphogenesis is discussed. Due to the fact that the apical trabeculated region of each ventricle arises from a separate primitive segment, each ventricle can be identified according to the pattern of its apical trabeculations. This feature is crucial in the elucidation of complex congenital pathology, such as discordant atrioventricular connections.

Animals↗
PubMed1999

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane.

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a "major" and "minor" tectorial membrane (Lim, Hear Res 1986;22:117-146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified. In the present study, a novel monoclonal antibody (TC2) that recognizes a native epitope on glycosaminoglycans enriched in chondroitin-4-sulfate revealed a transient and restricted expression in the developing gerbil TM. The localization patterns suggest that Deiters' and pillar cells secrete a TC2-positive matrix prior to birth that later becomes incorporated into the marginal band and superior layer (cover net) of the TM. The developmental timecourse and patterns of TC2 reactivity suggest that this molecule may play a critical role in the fusion of the minor TM with the major TM.

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PubMed1999

Dynamic expression of a native chondroitin sulfate epitope reveals microheterogeneity of extracellular matrix organization in the embryonic chick heart.

TC2 is a novel monoclonal antibody produced by in vitro immunization of splenocytes with a peanut agglutinin-positive fraction from extracts of prechondrogenic micromass cultures of chick limb mesenchyme. ELISA results demonstrated TC2 reactivity with a native epitope on a glycosaminoglycan (GAG) enriched in chondroitin-4-sulfate and with multiple intact proteoglycans, but not with other GAGs tested. TC2 immunohistochemical reactivity was abolished by pretreatment of sections with chondroitinase AC or preadsorption with chondroitin-4-sulfate GAG. Strong TC2 localization occurred throughout the developing heart at stage 9. As looping ensued, a graded reactivity was observed from lowest in the atrium to highest in the conotruncus that correlated well with versican localization. The superior atrioventricular cushion stained preferentially with TC2 as compared to the inferior cushion at stages 16-18. At these later stages TC2 patterns did not agree completely with anti-versican reactivity. By stage 23 there was a marked reduction in TC2 localization in the heart, however, strong reactivity remained at certain sites, including the conotruncus and in subcompartments of both atrioventricular cushions. A heterogeneous distribution of other native chondroitin sulfate glycosaminoglycan epitopes recognized by monoclonal antibodies d1C4 and CS-56 was observed as well. The distribution of the TC2 epitope usually did not overlap with d1C4 or CS-56 localization at the stages examined. Overall, the spatiotemporal characteristics of TC2 reactivity in the developing chick heart appear to correlate with subdomains of the endocardial cushions as well as with trabecular and atrial septal formation.

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PubMed1998

An autocrine function for transforming growth factor (TGF)-beta3 in the transformation of atrioventricular canal endocardium into mesenchyme during chick heart development.

Transformation of atrioventricular canal endocardium into invasive mesenchyme is a critical antecedent of cardiac septation and valvulogenesis. Previous studies by Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1510-1520, 1991) showed that treatment of atrioventricular canal endocardial and myocardial cocultures with TGFbeta3 antisense oligodeoxynucleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGFbeta3 antisense oligos in this transformation bioassay, and (ii) more clearly define the mechanism of TGFbeta3 function in atrioventricular canal mesenchyme formation. In situ hybridization and immunohistochemistry showed little or no TGFbeta3 mRNA or protein in the atrioventricular canal myocardium or endocardium prior to mesenchyme formation (stage 14; paraformaldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were positive for both TGFbeta3 mRNA and protein. In culture bioassays, atrioventricular canal endocardial monolayers pretreated with antisense phosphorothioate oligodeoxynucleotides to TGFbeta3 did not transform into invasive mesenchyme in response to cardiocyte conditioned medium: the subsequent addition of exogenous TGFbeta3 protein relieved this inhibition. Control cultures without pretreatment or those receiving missense oligos generated similar numbers of invasive mesenchyme in response to cardiocyte conditioned medium. Direct addition of TGFbeta3 protein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associations and stimulated cellular hypertrophy, but did not engender invasive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGFbeta2 protein, either alone or in combination with TGFbeta3. The results of this study indicate that: (i) atrioventricular canal endocardium expresses TGFbeta3 in response to a myocardially derived signal other than TGFbeta3, (ii) atrioventricular canal endocardial TGFbeta3 functions in an autocrine fashion to elicit selected characteristics necessary for cushion tissue formation, and (iii) TGFbeta3 alone or in combination with TGFbeta2 is insufficient to transform atrioventricular canal endocardium into invasive mesenchyme in culture.

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PubMed1997

Temporal and spatial asymmetries in the initial distribution of mesenchyme cells in the atrioventricular canal cushions of the developing chick heart.

BACKGROUND: We investigated potential early asymmetries in the distribution of mesenchymal cells within the inferior and superior AV cushions in the developing chick heart. METHODS: Chick embryos stages 16-20 HH were fixed, embedded in polyacrylamide, and the cell nuclei stained with propidium iodide. Cells counts were determined within the cardiac jelly of the atrioventricular canal (AV) by laser confocal microscopy in coronal planes spanning its entire length. RESULTS: Our data show at the different stages studied, 16-20 HH, that the inferior AV cushion invariably contains more cells than the superior AV cushion. In the inferior cushion, the cell distribution is bimodal, i.e., the proximal and distal regions have more mesenchymal cells than the middle part of the AV canal. In the superior cushion, there is a increasing gradient of mesenchymal cells along the longitudinal axis from the atrium to the ventricle. CONCLUSIONS: Our findings reveal that the temporal and spatial characteristics of mesenchyme formation in the inferior vs. superior AV cushion are different. This asymmetry suggests several potential hypotheses: (1) the distribution of the inducer molecule or its receptor has a distribution similar to that of mesenchymal cells, (2) the extracellular matrix has a differential composition or regionally-specific physical associations, (3) the endocardium is heterogeneous with respect to transformation capacity, or (4) these patterns result from an earlier inductive event. The potential importance of the observed asymmetries in the distribution of AV mesenchyme is discussed relative to localization patterns of molecules critical to successful cardiac morphogenesis and remodeling.

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PubMed1997

Production of a monoclonal antibody by in vitro immunization that recognizes a native chondroitin sulfate epitope in the embryonic chick limb and heart.

We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

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PubMed1996

Origin of the pulmonary venous orifice in the mouse and its relation to the morphogenesis of the sinus venosus, extracardiac mesenchyme (spina vestibuli), and atrium.

BACKGROUND: Human embryology textbooks indicate that the trunks of the pulmonary vein and artery originate from the left atrium and aortic sac, respectively, based on histological analyses of limited human specimens. However, our studies show that the pulmonary venous trunk in the mouse as in other nonhuman vertebrates originates from a vascular "sac" at the venous pole, the sinus venosus. METHODS: Mouse embryos of 9-11 days gestation were obtained and staged according to Theiler's criteria and fixed in Carnoy's solution. Samples were embedded in paraffin and serial sections were prepared. RESULTS: Histological analysis showed that at day 9.5 the pulmonary venous rudiment was initially observed along the left margin in the extracardiac mesenchyme that separated the venous pole of the heart from the lung buds. The endothelium of the pulmonary vein was continuous, with a vascular sac we identified as sinus venosus based on its location immediately posterior to the left sinoatrial fold. The sinus venosus became incorporated into the left atrium (days 10-10.5) to form part of the posterior atrial wall. Similarly, the pulmonary vein and associated extracardiac mesenchyme were "drawn" into the atrium. This extracardiac mesenchyme of the venous pole, also called "spina vestibuli" and containing the pulmonary vein at its left margin, formed a wedge-shaped invagination within the atrium that contributed nonmuscular tissue to the primary atrial septum. CONCLUSIONS: We propose that the orifice of the pulmonary vein establishes a link with the left side of the atrium as a consequence of a venous sac, the sinus venosus, and its associated mesenchyme (in which the root of the pulmonary vein is embedded) being incorporated into the atrium.

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PubMed1995

Transformation of cardiac endothelium into cushion mesenchyme is dependent on ES/130: temporal, spatial, and functional studies in the early chick embryo.

ES/130 is a novel 130-kDa protein that has been linked previously to the transformation of endocardial endothelium into cushion mesenchyme. In the present study we report the localization of protein and mRNA for ES/130 in stages 7-plus through 20 chick embryos and present functional data related to a potential mechanism for ES/130. The temporal and spatial regulation of ES/130 expression suggests that this epithelial-to-mesenchymal transformation is a result of homogenetic induction. Functional studies indicate that myocardially derived ES/130 elicits expression of this protein by target AV endothelial cells, which is linked to a signal transduction cascade. The localization of ES/130 to other sites of inductive interactions (e.g., limb bud ectoderm, gut, and notochord) implies that this protein may have a more widespread importance to embryogenesis beyond its involvement in cardiac cushion tissue formation.

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PubMed1994

Identification of transferrin as one of multiple EDTA-extractable extracellular proteins involved in early chick heart morphogenesis.

It was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial-mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive interactions, e.g., developing heart, limb buds, and neural crest forming region [Krug et al., 1987, Dev Biol 120:348-355; Mjaatvedt et al., 1991, Dev Biol 145:219-230). In the present study, by using an antiserum (ES3) to a similar immunogen, we affinity purified four major EDTA-soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS-PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): 1) amino acid sequencing showed that eight N-terminal residues were identical to those of chicken transferrin, 2) acid hydrolysates of both proteins had nearly identical compositions, 3) the protein co-migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and 4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin demonstrated that anti-transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial-mesenchymal formation. However, both cardiac endothelium and migrating mesenchymal cells became immunoreactive with anti-transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial-mesenchymal transformation.

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PubMed1994

Myocardial regulation of transforming growth factor-beta expression by outflow tract endothelium in the early embryonic chick heart.

We have demonstrated previously that the epithelial-mesenchymal transformation of cardiac endothelium in early chick heart development is induced by EDTA-soluble (ES) extracellular molecules synthesized by the myocardium of specific regions, i.e., the outflow tract (OT) and atrioventricular (AV) canal. Polyclonal antibodies (ES3) prepared against these molecules recognized two major bands, 28 and 46 kDa, in immunoblots and blocked the transformation of OT endothelial cells into mesenchyme in a three-dimensional collagen gel culture system. The studies of Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1516-1520 (1991)) and Potts and Runyan (Dev. Biol. 134, 392-401 (1989)) indicate that transforming growth factor (TGF)-beta expression is necessary for the formation of mesenchyme from cardiac endothelium. In this study, we used ES3 antibodies to test the hypothesis that TGF-beta expression by transforming endothelial cells is regulated by ES antigens. OT and AV endothelial cells treated with embryonic cardiocyte conditioned medium (CCM), which elicits epithelial-mesenchymal transformation, were shown by immunohistochemistry to increase expression of TGF-beta 1-like protein immediately prior to and during their transformation in culture. Endothelium from a nontransforming region of the heart (i.e., ventricle) did not express detectable levels of TGF-beta under similar conditions. The staining pattern for TGF-beta 1-like protein was characterized by a distinct particulate or granular distribution within the Golgi and cytoplasm and at cell surfaces. However, when endothelial transformation was blocked by immunoadsorption of ES proteins from CCM, increased staining for TGF-beta was not observed. These findings suggest an inductive relationship between myocardially derived ES proteins and TGF-beta expression by chick heart endothelial cells which is requisite for their transformation into cushion mesenchyme.

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PubMed1993

Identification of an extracellular 130-kDa protein involved in early cardiac morphogenesis.

Previous studies indicate that the transformation of cardiac endothelium into mesenchyme is dependent upon a developmentally regulated signal expressed by its associated myocardium. This process can be mimicked in culture by substituting a non-cytolytic EDTA extract of embryonic heart tissue for the presence of myocardium. Polyclonal antibodies (ES1) generated against the EDTA-extractable proteins both localized to the cardiac extracellular matrix preceding the transformation of endothelium and blocked this process in culture. Based on these observations, we hypothesized that ES1 antigens participate in the formation of cardiac mesenchyme. The present study was undertaken to prepare cDNA and antibody probes for individual ES1 antigens to better characterize their involvement in this important morphogenetic event. An expression library was constructed in Uni-ZAP using poly(A+) RNA from embryonic cardiocyte cultures that had been shown previously to secrete proteins that engender the formation of cardiac mesenchyme. Screening of this expression library with ES1 antibodies resulted in several clones, one of which ("ES1-2.1a") is described in this report. ES1-2.1a has a 2.6-kilobase pair insert, the sequence of which exhibits no apparent homology to those in data banks. A fragment (852 base pairs) from the 5' region of ES1-2.1a cDNA was subcloned into the expression vector pGEX-2T, and a 20-kDa fragment of the resulting protein used to prepare affinity-purified antibodies. Immunoblotting detected a 130-kDa protein ("ES/130") in two preparations that elicit mesenchyme formation, i.e. EDTA extracts of embryonic hearts and conditioned medium of cardiocyte cultures. Functional studies showed that antibodies to ES/130 inhibited the epithelial-mesenchymal transformation of cardiac endothelium in culture. Immunohistochemistry of cardiocyte cultures localized ES/130 protein to the vacuolar system and secretory granules. By polymerase chain reaction analysis, the message for ES/130 was detected in the developing heart just prior to and during mesenchyme formation. These results are consistent with ES/130 being involved at a critical step in the initiation of the epithelial-mesenchymal transformation of cardiac endothelium.

Amino Acid Sequence↗
PubMed1993

Effects of ectoderm co-culture and conditioned medium on the limb mesoderm in vitro.

Undissociated mesoderm placed onto collagen gels forms three subpopulations of mesenchyme which differentiate along myogenic, chondrogenic and fibrogenic phenotypes. Co-culture with ectoderm appears to inhibit the formation of distinct cartilage elements and myotubes by interfering with the differentiation of chondrogenic and fibrogenic progenitors. Addition of CCM enriched in ES antigens enhances the effects of the ectoderm on chondrogenesis. Culture in the presence of CCM alone retards chondrogenesis and almost completely inhibits myogenesis. These results suggest that the primary effect of ectoderm or CCM in our culture system is on myogenic and chondrogenic differentiation, and ES antigens, if responsible for these effects, appear to have no role in pattern formation.

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PubMed1992

Leg bud mesoderm retains morphogenetic potential to express limb-like characteristics ("limbness") in collagen gel culture.

Recent in situ hybridization studies have correlated expression of potential regulatory genes with pattern formation in limb bud mesoderm (Tabin: Cell 66:199-217, 1991); however, the mechanism(s) controlling their expression in mesoderm and their relevance to the establishment of a limb morphogenetic pattern remain unknown. One likely candidate for regulating patterning events in limb mesoderm is the apical ectodermal ridge, as its removal in ovo results in a graded truncation of limb skeletal elements in the proximal-distal axis dependent upon the time of excision (Rowe and Fallon: J Embryol Exp Morph 68:1-7, 1982). In the present study, we investigate whether the hypothetical imprint of ridge ectoderm is retained in cultured mesoderm. Specifically, we sought to determine if a subpopulation of limb mesoderm that forms in collagen gel culture (Markwald et al: Anat Rec 226:91-107, 1990), retains any expression of "limbness" in the absence of limb ectoderm as characterized by the formation of a predictable number and distribution of limb-like chondrogenic elements in comparison to the temporal and spatial relationships of the in situ proximal, hindlimb skeletal structures. Accordingly, explants of undissociated mesoderm from stage 18-22 chicken leg buds were cultured without ectoderm on collagen gel lattices and the central subpopulation of mesoderm was examined morphologically. We show that this central subset of mesoderm will form chondrogenic cells which were not expressed uniformly throughout the subset, but rather distinct nodules or elements of cartilage were elaborated. Moreover, the number of elements expressed by the central subset increased with the age of the mesoderm at the time of explantation; spatially and temporally, the sequence of elements that formed always proceeded from the proximal, anterior margin of the subset to its distal, posterior border. The shapes of the initial elements (designated I and II) resembled the forms of in situ proximal skeletal structures (girdle and femur-like), whereas more distal elements (III-V) were often fused and without structural similarity to in situ skeletal structures. When cultures were established from the posterior mesoderm of stage 19/20 or 21 mesoblasts, the frequency of element I formation was reduced approximately one-half, whereas formation of more distal elements was unaffected. Conversely, element formation from the central subset established from isolated anterior mesoderm was virtually identical to intact mesoblasts, indicating a capacity to regulate for the loss of mesoderm as occurs in situ (Hampé: Archs Anat Microsc Morph Exp 48:345-378, 1959).(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1992

Multiple glycoproteins localize to a particulate form of extracellular matrix in regions of the embryonic heart where endothelial cells transform into mesenchyme.

Cells derived from an epithelial-mesenchymal transformation within the atrioventricular canal and outflow tract are involved in the partitioning of the early embryonic heart into a four-chambered organ. This transformation process has been shown to proceed from an inductive interaction between the myocardium and competent, target endothelial cells within these regions of the heart. Interestingly, immunohistochemistry revealed the presence of fibronectin-positive particulates within the matrix of mesenchyme-forming regions (Mjaatvedt et al., 1987). This particulate matrix is extractable by EDTA and can elicit the epithelial-mesenchymal transformation in culture (Mjaatvedt and Markwald, 1989). Analysis of EDTA extracts of embryonic heart tissue revealed the presence of fibronectin and about 40 unidentified proteins, 6 of which appeared to be enriched in the biologically active 100,000g pellet fraction (Mjaatvedt and Markwald, 1989). Based on these and other data we have proposed that the particulate matrix is composed of a multicomponent complex of fibronectin and one or more of the low-molecular-weight proteins in this pellet. The purpose of the present study was to begin a biochemical characterization of the nonfibronectin proteins thought to be present in the matrix particulates. Given that many matrix constituents are glycoproteins, lectins were used to initially characterize the particulate constituents. Of the lectins tested, soybean agglutinin (SBA) was found to be specific only for matrix particulates. Histochemical analyses showed that SBA and antibodies against fibronectin colocalized regionally and temporally to the same matrix particulates in embryonic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

An antiserum (ES1) against a particulate form of extracellular matrix blocks the transition of cardiac endothelium into mesenchyme in culture.

The epithelial-mesenchymal transition of cardiac endothelium is a critical developmental event in the formation of valvular and septal anlagen. We have demonstrated previously that this event can be mimicked in culture by treating atrioventricular canal (AV) endothelium with EDTA-soluble proteins extracted from embryonic heart tissue. This activity was fractionated by ultracentrifugation of the EDTA extract, indicating that the critical proteins existed as a multicomponent complex. Based on these results we propose that: (1) the in vitro particulates in EDTA extracts correspond to an observed particulate form of extracellular matrix within the myocardial basement membrane (MBM) of mesenchyme-forming regions and (2) one or more of the proteins in the MBM particulates function to elicit the epithelial-mesenchymal transition. To test these hypotheses we utilized an antiserum, termed ES1, prepared against EDTA-extractable particulates from embryonic chick hearts. Both ES1 and an anti-fibronectin monoclonal antibody (M3H) co-localized in situ to particles within the MBM; however, no ES1 reactivity towards fibronectin could be detected by ELISA or immunoblot analysis. The ES1-positive MBM particulates were removed by extraction with EDTA, but not with PBS, indicating a divalent cation-mediated association of the constituent proteins. ES1 antibodies recognized two major (28 and 46 kDa) and three minor (93, 109, and 180 kDa) proteins on immunoblots of EDTA-extractable proteins. When tested in culture, ES1 antiserum inhibited the formation of mesenchyme from AV endothelium in a dose-dependent manner, while M3H did not. These results are consistent with an active role for one or more of the ES1 antigens in initiating the formation of AV mesenchyme. The localization of ES1 antigens to the extracellular matrix at other dynamic interfaces, e.g., ectoderm/neural tube and limb bud ectoderm/mesoderm, point to a potentially general importance of ES1 antigens in mediating similar developmental interactions.

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