Embryonic development of the heart involves multiple morphogenetic processes. Toward the end of heart looping stages, the tubular heart begins to undergo chamber specification. In the outflow tract and the endocardial cushion (or atrioventricular canal), a subpopulation of endocardial endothelial cells undergoes transition to a mesenchymal phenotype, delaminates from the endocardial surface, and migrates into regional swellings of extracellular matrix (ECM) by means of a process called epithelial–mesenchymal transformation (see Markwald, 1995; Eisenberg and Markwald, 1995; Baldwin, 1996). Several ECM molecules are known to be associated with this process, including laminin, fibronectin, vitronectin, JB-3, tenascin, fibulin-1, and fibulin-2 (Davis et al., 1989; Loeber and Runyan, 1990; Spence et al., 1992; Zhang et al., 1993, 1995; Bouchey et al., 1996), but their specific roles are not fully understood. In the outflow tract, the cardiac neural crest cells penetrate into the subendothelial tissue of the truncus arteriosus and conal cushion tissue (Jiang et al., 2000) and later give rise to the septum between the two great vessels and two ventricular outflow tracts (see Kirby and Waldo, 1995). These transformed mesenchymal cells are thought to be responsible for producing structural connective tissue of heart valves and atrioventricular septa in the later stages of heart development (Manasek et al., 1986; Markwald et al., 1990; Borg et al., 1990).
The embryonic development of aortic arch vessels also involves migration of the transformed mesenchymal cells onto the ECM of the vessels. Aortic arch vessels develop in accordance with migration of the neural crest cells to the basement membrane of the truncus arteriosus and the pharyngeal arch arteries. Neural crest cells, upon settling at the basement membrane of the aortic arch vessels, proliferate to form multilayer vascular smooth muscle cells and produce new ECM organization. Eventually, aortic arch vessels compose a unique structure, called tunica media, of alternating smooth muscle cell and elastin layers, which occupies the majority of the vessel wall (Wolinsky and Glagov, 1967; Davis, 1993a). Several extracellular matrix molecules form this elastin lamina (Clark and Glagov, 1985; Hungerford et al., 1996), but the mechanism of development of this unique structure of aorta is not fully understood.
In the development of coronary vessels, epithelial–mesenchymal transformation is also known to be a critical step for vasculogenesis and angiogenesis. Coronary vascular cells are derived from epicardial cells, which migrate over the looped heart to form a single-layer epithelium and later undergo epithelial–mesenchymal transformation to give rise to vascular tissue (Vrancken Peeters et al., 1999). These mesenchymal cells interact with subepicardial extracellular matrix, proliferate, and differentiate into three distinct cell lineages: coronary smooth muscle cells, perivascular fibroblasts, and intermyocardial fibroblasts (Mikawa and Gourdie, 1996; Dettman et al., 1998). These cells subsequently migrate cranially to envelope the heart from the sinus venosus to the outflow tract. This morphogenetic transition is crucially important in optimal myocardial proliferation and differentiation. The mechanisms that regulate the cell transformation, cell migration, cell differentiation, extracellular matrix remodeling, and connective tissue production, however, are still unclear.
The fibulins are an emerging family of extracellular matrix proteins characterized by tandem arrays of calcium-binding epidermal growth factor (EGF)-like modules and a common C-terminal globular domain. Five members have been identified to date and all, except fibulin-3, have been implicated to play a role in cardiovascular development (Argraves et al., 1990; Pan et al., 1993a,b; Tran et al., 1997; Giltay et al., 1999; Nakamura et al., 1999; Kowal et al., 1999). The first two members, fibulin-1 and -2, have been studied in more detail in the context of heart development. In the early avian embryo, fibulin-1 is associated with epithelial–mesenchymal transformation in the endocardial cushion, developing myotomes and neural crest (Spence et al., 1992). In the developing avian heart, fibulin-1 is expressed throughout the cardiac jelly and is up-regulated where epithelial cells transform into migratory mesenchymal cells. In mouse embryos, both fibulin-1 and -2 are expressed at sites of epithelial–mesenchymal transformation and cell migration during cardiac valvuloseptal and great vessel development (Zhang et al., 1993, 1995), but the expression at early stages of heart development have not been critically examined. Both proteins remain as prominent components of adult valves (Zhang et al., 1995; Miosge et al., 1998), suggesting a crucial role in maintaining tensile integrity of the valve tissue. However, there are distinct temporal and spatial differences between fibulin-1 and -2 expression pattern. In particular, fibulin-2 expression is more restricted to developing cardiovascular system whereas fibulin-1 is expressed in a more unrestricted manner (Zhang et al., 1996). Fibulin-2 is prominent in the epicardium of day 11 embryo, whereas little fibulin-1 expression is noted (Zhang et al., 1995). In addition, only fibulin-2 is expressed at the coronary artery wall in the adult mouse heart (Zhang et al., 1995). Despite these differences, both proteins are present in the elastic fibers of the blood vessel walls where they colocalize with and bind to endostatin, the angiogenesis inhibitor (Sasaki et al., 1998; Miosge et al., 1999). These findings suggest that both fibulin-1 and -2 are involved in epithelial–mesenchymal transformation and vascular remodeling, but their specific roles seem to be quite different.
To further assess possible biological roles of fibulin-2 during cardiovascular development, we have used detailed immunohistochemistry and in situ hybridization studies to identify the localization of protein and transcripts in the mouse from early embryonic to adult stages. We have found that fibulin-2 expression is up-regulated by the epithelial–mesenchymal transformation not only in the cardiac valves and the media of aortic arch vessels, but also in the coronary vessels. However, the origin of fibulin-2 is different in these three anatomic sites. In the cardiac valves, fibulin-2 is produced by the endocardial-derived mesenchymal cells. In the media of aortic arch vessels, fibulin-2 is produced abundantly by the smooth muscle precursor cells originated from the cardiac neural crest cells. In the coronary vessels, fibulin-2 is predominantly produced by the epicardial cells and epicardial-derived vascular endothelial cells. Fibulin-2, thus, serves as an excellent marker for epithelial–mesenchymal transformation and vascular remodeling throughout mouse cardiovascular development.
Fibulin-2 Expression Is Abruptly Increased at E9.5 Upon Epithelial–Mesenchymal Transformation in the Outflow Tract and the Endocardial Cushion (E8.5 to 9.5)
In the embryonic day 8.5 (E8.5: day of vaginal plug = E0) mouse embryo, fibulin-2 protein is faintly expressed at the basement membrane of neural tube, skin (presumptive epidermis), dorsal aorta, and foregut (Fig. 1A,B). It is only seen barely in the ECM of the tubular heart, especially along the basal side of the myocardial surface. At E9.0, fibulin-2 continues to be weakly expressed at the basement membrane of the various organs, including ECM of the tubular heart (Fig. 1C), but it is not expressed in association with the migrating neural crest cells. At E9.5, however, fibulin-2 expression becomes suddenly increased in accordance with the endocardial cells and the transformed mesenchymal cells in ECM of the cardiac outflow tract and the endocardial cushion (Fig. 1D–F). Fibulin-2 forms a specific fibrillar organization in association with endocardial cells and the transformed mesenchymal cells.
Increase of Fibulin-2 Expression Is Associated With the Proliferating Mesenchymal Cells in the Cardiac Jelly and the Epicardial Cells (E10.5 to 11)
At E10.5, abundant expression of fibulin-2 protein is concentrated around the migrating mesenchymal cells and endocardial cells to form a fibrillar network (Fig. 2A,B). Fibulin-2 is also seen along the subepicardial surface (Fig. 2C). In situ hybridization with 35S labeling of the E11 mouse embryo shows that fibulin-2 transcript is predominantly localized in the vessel wall of the aortic arch arteries, the proliferating mesenchymal cells in the outflow tract and the endocardial cushion, and the epicardial cells (Fig. 2D–F). Fibulin-2 transcript is exclusively expressed by the mesenchymal cells in the ECM, but not by the endocardial cells or myocardial cells (Fig. 2F). Localization of fibulin-2 transcript is well correlated with that of the protein expression.
Fibulin-2 Expression Becomes More Restricted to the Heart Valves, Aortic Arch Vessels, and Epicardium (E13 to E15)
At E13, fibulin-2 protein is expressed predominantly within the future cardiac valve region but also is localized within the subendocardium (Fig. 3A,B). In situ hybridization image at E13.5 reveals that fibulin-2 transcript is specifically expressed in the mesenchymal cells in the semilunar and atrioventricular valves, the wall of great vessels (aorta and pulmonary artery), the interatrial septum, and the epicardium (Fig. 3C–F). In the tricuspid valve area, the fibulin-2 transcript is specifically localized in the mesenchymal cells close to the endocardial surface, suggesting a specific cell population of the mesenchymal cells in the valve tissue, which is responsible for producing fibulin-2 (Fig. 3E). In the aorta, fibulin-2 transcript is abundantly expressed at the mesenchymal cell layers (Fig. 3C,D). Fibulin-2 transcript continues to be expressed in the epicardial cells (Fig. 3D,F).
Fibulin-2 Expression Is Developmentally Regulated Throughout the Maturation of the Aortic Arch Vessels (E13.5 to Postnatal Day 13)
At E11, there is a significant increase in fibulin-2 transcript expression in the wall of the aortic arch vessels (Fig. 2D). Although the vessel wall remains a relatively thin structure, some populations of the migrated neural crest cells begin to express smooth muscle protein (α-smooth muscle actin, see Fig. 4A). At E13, fibulin-2 transcript remains to be highly expressed in the proliferating mesenchymal cells, which are differentiating into vascular smooth muscle cells (Fig. 3C). Fibulin-2 protein is abundantly expressed in a pericellular manner around each proliferating medial smooth muscle precursor cell (Fig. 4B,C). In the later stages when the smooth muscle precursor cells are increasingly expressing α-smooth muscle actin (from E15 to E18), fibulin-2 protein remains abundantly expressed in the ECM of the media (see Fig. 4D–F). At postnatal 13 days, although fibulin-2 transcript expression is remarkably down-regulated (Fig. 4I), the fibulin-2 protein remains expressed at the basement membrane of the endothelial cells, the surface of elastin lamina, the pericellular region of the smooth muscle cells, and in the adventitia (Fig. 4G,H). At this stage, elastin has formed a continuous layer and become a major component of elastin lamina (Fig. 4G,H; also see Davis, 1995) and smooth muscle cells have reached a relatively mature stage.
Fibulin-2 Is Expressed Throughout Coronary Vascular Development: Epicardial Cells to Coronary Vessels (E13.5 to Adult)
Fibulin-2 begins to be localized at the subepicardial region at E10.5 upon formation of epicardial cell layer (Fig. 2C). At E13.5, the coronary vessels begin to develop from the epicardial cells with strong expression of fibulin-2 and the transformed vascular cells also express fibulin-2 (Fig. 5A,B). Fibulin-2 transcript is expressed by the epicardial cells (see Fig. 3D,F), which gives rise to the abundant protein expression in the subepicardial ECM. Some of these vascular cells begin to express smooth muscle protein in addition to fibulin-2 (Fig. 5B).
In the 13-day-old postnatal mouse heart, in situ hybridization of fibulin-2 reveals that fibulin-2 transcript is predominantly expressed by the epicardial cells and the coronary vascular endothelial cells (Fig. 5C,D). Fibulin-2 protein is expressed around the cell surface of the vascular endothelial cells and also gives rise to a fibrillar structure between vascular smooth muscle cells and adjacent myocardial cells (Fig. 5E,H). At this stage, the coronary vasculature consists of three morphologically distinct subpopulations. The majority of the coronary vessels show a two-layer structure with endothelial cells in the inner layer (fibulin-2 is localized along the cell surface) and smooth muscle cells in an outer layer (Fig. 5H). The second type of coronary vessels does not have an obvious smooth muscle layer but has fibulin-2 localization at the basement membrane of the vascular endothelium (Fig. 5G,I). In these vessels, the endothelial cells are flat-shaped as opposed to compact and condensed unilayer structure seen in the predominant type of coronary arteries (compare Fig. 5G,I with E,F,H). This type of coronary vessels may represent coronary veins. The third type of coronary vasculature are intermyocardial capillary vessels, which are made of PECAM-1– positive endothelial cells without fibulin-2 protein localization. These capillary vessels are prevalently seen throughout the myocardium (see yellow arrows in Fig. 5E and arrowheads in Fig. 5I).
At an adult stage (10 weeks of age), fibulin-2 protein remains to be expressed in association with the coronary arterial and venous endothelial cells, but no fibulin-2 protein expression is detectable in the capillary endothelium (data not shown).
Fibulin-2 Begins to Be Synthesized by the Transformed Mesenchymal Cells Upon Their Exposure to the Preexisting Cardiac ECM
In the endocardial cushion (atrioventricular canal), mesenchymal cells are induced through a transformation of endocardial cells by means of reciprocal signaling between the endocardial and myocardial cell layers, mediated in part by TGF-β family members (Eisenberg and Markwald, 1995; see Fishman and Chien, 1997). A previous study revealed that fibulin-2 is synthesized by the transformed mesenchymal cells in the embryonic endocardial cushion tissue in the mouse embryo on E11 and E13 (Zhang et al., 1995), but expression in the earlier stages before the mesenchymal cell migration was not investigated. Our current sequential analyses of the temporal expression have suggested that the epithelial–mesenchymal transformation induces sudden up-regulation of fibulin-2. Before the advent of mesenchymal cells within the cardiac ECM (E8.5 to 9.0), fibulin-2 expression is barely detected, unlike other extracellular matrix molecules such as fibronectin, flectin (Tsuda et al., 1998), and fibulin-1 (Tsuda et al., unpublished observation). It is upon the presence of transformed mesenchymal cells underneath the endocardium that fibulin-2 protein expression is suddenly increased (see Fig. 1D,E), and the protein localization persists thereafter.
The protein localization pattern suggests a close interaction between mesenchymal cells and endocardial cells (see Figs. 2A,B, 3A,C,E). It is possible that the formation of fibulin-2 fibrillar network in the ECM serves to facilitate optimal migration, proliferation, and differentiation of the mesenchymal cells, but further experiments are required to test this hypothesis.
Fibulin-2 and Aortic Arch Vessel Development
In aortic development, fibulin-2 specifically marks the transformed mesenchymal cells after their settlement in the basement membrane of the endothelial tube. Compared with other middle-sized arteries, aortic arch vessels are known to have specific architecture consisting of alternating layers of smooth muscle and elastin lamina. Fibulin-2 expression is rapidly increased when the neural crest cells reach the basement membrane of the arch vessels at E11 (Fig. 2D). This sudden increase of fibulin-2 synthesis seems to be triggered by the mesenchymal cell migration onto the existing ECM, which follows exactly the same mechanism observed in the endocardial cushion and outflow tract regions.
Previous immunohistochemical studies showed that α-smooth muscle actin induction begins immediately after the mesenchymal cells reach the basement membrane of endothelial tube of aortic arch vessels, as early as E10.5 (Takahashi et al., 1996). The coexpression of fibulin-2 transcript and α-smooth muscle actin at E11 (see Figs. 2D, 3C, 4A) suggests that fibulin-2 may be a good marker for precursor smooth muscle cells. The role of fibulin-2 after this sudden increase may be closely related to proliferation and differentiation of the mesenchymal cells. Although the smooth muscle precursor cells become more differentiated into mature smooth muscle cells with the establishment of an alternating structure of elastin lamina, fibulin-2 expression remains relatively the same (Fig. 4E–H). It is speculated that the first task facing vascular smooth muscle precursor cells is to produce extracellular matrix to assemble the specific scaffolding in which to undergo optimal proliferation before becoming differentiated into smooth muscle cells (see Drake et al., 1998). Fibulin-2 is actively produced by the smooth muscle precursor cells of the aortic arch vessels during embryonic development, but transcript production becomes down-regulated once a basic frame of alternating ECM layers is established. This finding suggests its role in organizing the initial steps of the process resulting in aortic arch vessel wall assembly.
The formation of an alternating structure of ECM layers and smooth muscle layers in the aortic media is a complex process that is not fully understood. A model of embryonic development of aortic arch vessels is proposed here from the standpoint of fibulin-2 expression (Fig. 6). Upon the migration of neural crest cells onto the basement membrane, there is a sudden production of fibulin-2 by the settled mesenchymal cells (Settlement) and simultaneously they begin to produce α-smooth muscle actin to become smooth muscle precursor cells (Commitment) (E11.0). In accordance with increased fibulin-2 production in the ECM, smooth muscle precursor cells proliferate to form a multilayered structure (Proliferation) (E13). Fibulin-2 mRNA synthesis begins to slow down, although fibulin-2 protein expression remains to be expressed abundantly in the ECM layers (Differentiation) (E18). As early as E15, small patchy deposits of elastin begin to appear within this circumferential ECM layer and its deposition becomes progressively thicker and continuous with further development (Davis, 1995). Postnatally, when vascular smooth muscle cells complete terminal differentiation, elastin has become a predominant component of the ECM layer (Elastin lamina formation). Previous studies revealed that fibulin-2 binds fibrillin-1 and elastin and is localized at the interphase of amorphous elastin cores and fibrillin-1 microfibrils (Reinhardt et al., 1996; Sasaki et al., 1999). Electron microscopic studies indicated that the smooth muscle cells become linked with the elastic laminae by bundles of microfibrils early in development. Subsequently, these microfibrils become progressively infiltrated with elastin so as to form extensions of elastin from the elastin laminae in the adult media. (Davis, 1993a). Fibulin-2, along with other matrix molecules such as fibulin-1 and fibrillin-1, may have similar roles in aortic vessel development.
A physical attachment of endothelial cells to the subendothelial matrix must exist to maintain the structural integrity of the aortic intima. In the 5-day postnatal aorta by electron microscopy, extensive filament bundles extend along the subendothelial matrix connecting the endothelial cells to the underlying elastin lamina (Davis, 1993b). The temporal and spatial expression of these anchoring filaments in the early postnatal life in the aortic intima well correlates with fibulin-2 localization. This finding indicates that fibulin-2 may participate in anchoring filaments in the aortic intima to support physical integrity of the developing aorta.
Fibulin-2 and Coronary Vascular Development
At the late looping stage (E9.5), epicardial cells migrate from proepicardium, derivative of the developing liver, and form an epithelial sheet over the naked myocardium (Viragh et al., 1993). The epicardium is the site of initial cardiac neovascularization and formation of coronary circulatory system, and certain extracellular matrix proteins are thought to have some roles in blood vessel formation (Mikawa and Fischman, 1992). The critical importance of epicardium during coronary vascular development was demonstrated by inactivation of the genes encoding integrin α4 (Yang et al., 1995) and vascular cell adhesion molecule-1 (or VCAM-1) (Kwee et al., 1995) where formation of epicardium is deficient and coronary vasculature fails to develop. It is the epicardial-derived cell population that will later penetrate into the myocardial wall and will give rise to coronary vascular smooth muscle, fibroblasts, and vascular endothelial cells (Poelmann et al., 1993, Mikawa and Gourdie, 1996, Dettman et al., 1998).
Previously, fibulin-2 was reported to be expressed in the epicardium around E13 and in the wall of coronary vessels in adult mice, but their specific developmental role was not clearly addressed (Zhang et al., 1995, 1996). Fibulin-2 begins to be produced by the epicardial cells before formation of the coronary vessels (Fig. 3D), and coronary endothelium continues to express fibulin-2 through adulthood. Fibulin-2 mRNA is also exclusively seen within the coronary endothelial cells and epicardial cells (Fig. 5C,D), suggesting its unique involvement in coronary vascular development. The protein localization along the cell borders and basement membrane of the vascular endothelial cells suggests its specific role in cell–cell and cell–matrix interaction, respectively (Fig. 5), probably participating in maintaining structural integrity of the endothelial cell layer during vessel formation and later quiescent stage. These findings suggest that fibulin-2 may be an important structural component of cell–cell interaction among the endothelial cells as well as one of key signaling molecules that facilitates epithelial–mesenchymal transformation and subsequent differentiation of the epicardial cells.
Endothelial expression of fibulin-2 has never been described in the past. Unlike coronary arteries and veins, however, capillary endothelium does not express fibulin-2. This difference may indicate different lineages of endothelial cells in the coronary vascular network. Fibulin-2 serves as an excellent marker for coronary vascular endothelium other than capillary vessels, and it will be of great interest to study how it is expressed during revascularization after myocardial ischemia.
Possible Role of Fibulin-2 and Other ECM Proteins in Epithelial–Mesenchymal Transformation
Compared with other ECM proteins, fibulin-2 is unique as its expression is relatively restricted to the developing cardiovascular system in the mouse embryo and is closely related to epithelial–mesenchymal transformation, including coronary vascular development. In the endocardial cushion matrix and the basement membrane of the aortic arch vessels, abrupt increase of fibulin-2 synthesis appears initiated by the exposure of migrating mesenchymal cells into the existing ECM. On the other hand, coronary vasculogenesis, another form of epithelial–mesenchymal transformation, takes place in association with the increased fibulin-2 expression in the subepicardial region, but the involvement of fibulin-2 in the coronary vascular development appears to be different from that in the endocardial cushion matrix and the aortic arch vessels. During the formation of epicardium, epicardial cells migrate over the myocardial surface and several ECM molecules, including fibulin-2, are produced thereafter.
Why does this difference take place? It is probably due to the specific interactions among ECM molecules in different tissues and in different developmental stages. Previous in vitro studies indicated that fibulin-2 has multifunctional binding properties and, thus, has several alternative routes for its integration into basement membranes and other extracellular structures (Sasaki et al., 1995). Extracellular matrix molecules in the basement membrane and blood vessel wall that interact with fibulin-2 include laminin, nidogen-1, fibronectin, fibrillin-1, elastin, perlecan, and endostatin (type XVIII collagen) (Sasaki et al., 1995, 1999; Reinhardt et al., 1996; Brown et al., 1997). These ECM molecules may participate in combination with fibulin-2 in regulating epithelial–mesenchymal transformation during tissue differentiation. Understanding possible interactions among the ECM proteins may produce some answers to numerous questions regarding tissue morphogenesis in the cardiovascular development.
C57Bl/6J mice (Jackson Laboratories) were used in this study. The morning of the vaginal plug was designated as embryonic day 0 (E0). The dissected embryos were first fixed in 4% paraformaldehyde for overnight and then transferred to 100% methanol through ascending concentrations. The stored embryos were either used for whole-mount immunohistochemistry or cryosectioned at 7–8 μm for immunostaining and in situ hybridization. Adult heart tissue was immediately frozen with liquid nitrogen upon dissection and was fixed with acetone after cryosectioning.
Antibodies and Immunofluorescent Staining
Rabbit anti-mouse fibulin-2 antiserum (1028+: Pan et al., 1993b) was used at a dilution of 1:1,000. Mouse anti–α-smooth muscle actin (Sigma, St. Louis, MO) and rat anti-CD 31 (PECAM-1; BD PharMingen, San Diego, CA) were used at l:400 dilution and 1:30, respectively. Cy-3 conjugated goat anti rabbit IgG (Jackson ImmunoResearch Laboratory, West Grove, PA), FITC-conjugated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA), and Cy-2 conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratory) were used as secondary antibodies. Some slides (Figs. 4, 5) were also treated with DAPI nuclear staining.
In general, we applied whole-mount immunostaining with subsequent plastic embedment for embryos at younger stages (E8.5 to 9.5 as whole embryos; E10.5 to 13 as isolated embryonic hearts). The detailed protocol for whole-mount immunohistochemistry with plastic sectioning was described previously (Linask and Tsuda, 2000). Excellent antibody penetration has been obtained in whole-mount specimen through this protocol (Tsuda et al., 1998). Briefly, after the immunoreaction, the specimen was dehydrated through ascending concentrations of methanol and was embedded in araldite (Ted Peller, Redding, CA). The plastic-embedded specimen was sectioned at 1.5 μm throughout the heart region. For embryos older than E11, immunostaining was performed with 7- to 8-μm cryosections. Microscopic examination was performed with Zeiss (Oberkochen, Germany) Axioskop microscope equipped with epifluorescence attachments. The image was obtained with Toshiba 3CCD camera and was digitally analyzed and stored in Image-Pro Plus (Media Cybernetics, MD).
In Situ Hybridization of Fibulin-2
In situ hybridization was performed with 35S-labeled RNA probe following the protocol described previously (Wawersik and Epstein, 2000). A 578-bp fragment of mouse fibulin-2 cDNA (nucleotide position 1-578, Pan et al., 1993b) was subcloned into PCR II vector (Invitrogen) and used as a template for producing sense and antisense riboprobes with α-[35S]-UTP (ICN) and T7 or Sp6 polymerase (Roche Molecular Biochemicals). Sense probe was used as a negative control.
We thank Dr. Kersti Linask for critical reading of the manuscript, Dr. Franscois-Xavier Sicot for subcloning the fibulin-2 cDNA probe, and Dr. Grace Loredo for advice on in situ hybridization.