Temporally Compartmentalized Expression of Ephrin-B2 during Renal Glomerular Development
Takamune Takahashi*,
Keiko Takahashi*,
Sebastian Gerety,
Hai Wang,
David J. Anderson and
Thomas O. Daniel*
*Nephrology Division and Center for Vascular Biology, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Biology, California Institute of Technology, Pasadena, California.
Correspondence to Dr. Thomas O. Daniel, Immunex Corporation, 51 University Street, Seattle, WA 98101. Phone: 206-470-4875; Fax: 206-933-3733; E-mail:danielt{at}immunex.com
ABSTRACT. Glomerular development proceeds through the spatiallyordered and sequential recruitment, proliferation, assembly,and differentiation of endothelial, mesangial, and epithelialprogenitors. The molecular determinants of cell-cell recognitionand targeting in this process have yet to be defined. The Eph/ephrinfamily of membrane receptors and counter-receptors are criticalparticipants of developmental vascular assembly in extrarenalsites. Renal expression patterns of ephrin-B2 and EphB4 wereinvestigated using mice expressing ß-galactosidaseunder control of ephrin-B2 or EphB4 promoters. The earliestglomerular expression of ephrin-B2 was identified in a subsetof differentiating comma-stage glomerular epithelial cells (podocyteprogenitors) adjacent to the vascular cleft where endothelialprogenitors are subsequently recruited. Epithelial ephrin-B2expression was accompanied by expression in endothelial andmesangial cells as capillary assembly progressed. At or nearcompletion of glomerular maturation, epithelial ephrin-B2 expressionwas extinguished, with persistence in glomerular endothelialcells. Throughout development, one of several ephrin-B2 receptors,EphB4, was persistently and exclusively expressed in endothelialcells of venous structures. The findings show sequential ephrin-B2expression across glomerular lineages, first in a distinct subsetof podocyte progenitors and subsequently in endothelial cellsof the developing glomerulus. Given targeting functions forEph/ephrin family proteins, the findings suggest that ephrin-B2expression marks podocyte progenitors at the site of vascularcleft formation, where expression may establish an "address"to which endothelial and mesangial progenitors are recruited.Thus, the present results suggest that ephrin-B2 and EphB interactionsplay an important role in glomerular microvascular assembly.
Developmental assembly of the renal glomerular microcirculationis a precise and coordinated process that forms a highly specializedvascular filtering apparatus (1,2). Glomerular endothelial cellsare initially dispersed throughout the metanephric mesenchymaltissue (3,4) and are subsequently recruited to the clefts ofcomma-shaped glomeruli, where they proliferate and assembleinto glomerular capillaries adjacent to mesangial and glomerularepithelial cells. A distinct population of endothelial progenitors,recruited from the extrarenal para-aortic region, assemblesa basket-like network on the superficial surface of the developingkidney, where they interconnect to integrate large vessel andglomerular microvascular circulations (5). Available data implicateboth vasculogenic and angiogenic processes in this integratedassembly.
Several growth factors and their receptors mediate criticalsteps in glomerular capillary development. Vascular endothelialgrowth factor is expressed in developing podocytes and seemsto promote differentiation and recruitment of endothelial progenitorsexpressing the vascular endothelial growth factor receptor flk-1from the adjacent metanephric mesenchyme (6,7). Endothelialproduction of platelet-derived growth factor-BB (PDGF-BB) andmesangial progenitor expression of platelet-derived growth factorreceptor-ß (PDGFR-ß) are required for thecoordinated recruitment, proliferation, and assembly of mesangialcells (8). The endothelial tie-2 receptor and its angiopoietinligands, which mediate endothelial-pericyte interactions andmicrovascular maturation (9–11), have been detected inendothelial and mesangial cells of developing glomeruli (12,13).Transforming growth factor-ß1 actions are implicatedat several steps during glomerulogenesis, most prominently inthe maturation phase required to stabilize vascular structure(14).
In addition to these examples of paracrine intercellular communication,cell-cell contact is implicated in correct targeting and assemblyof the glomerulus, yet little is known about the molecular regulationof targeting and assembly. The Eph receptor tyrosine kinasesare transmembrane proteins that are engaged by contact withtheir counter-receptors, ephrins, upon cell-cell contact (15,16).Both Eph and ephrin proteins contribute to developmental patterningand neuronal targeting (17). Among ephrins, the ephrin-A subclassproteins (ephrin-A1 to -A5) are membrane attached through glycosyl-phosphatidyl-inositollinkages, whereas the ephrin-B subclasses (ephrin-B1 to -B3)are transmembrane proteins with highly conserved cytoplasmicdomains. Ephrin-As interact with the A subset of Eph receptors(EphA1 to A8) and Ephrin-B ligands with the B subset of Ephreceptors (EphB1 to EphB6). Mice homozygous for ephrin-B2 orEphB4 allele mutation display early embryonic vascularizationdefects at the developmental stage when interconnection of ephrin-B2expressing arterial limb vascular networks normally links withEphB4 expressing venous limb networks (18,19). Lethal vasculardefects are also detected in EphB2/EphB3 double mutants (20).EphB2 expression in mesenchymal cells adjacent to vessels hassuggested that Ephrin-B/EphB signaling may participate in intercellularinteractions between endothelial cells and adjacent mesenchymalcells (20).
As for renal expression of ephrin-B and EphB, early studieshave shown that glomerular endothelial progenitors display EphB1immunoreactivity both before and during recruitment to comma-and S-shaped glomeruli (21). Moreover, specific oligomerizedforms of ephrin-B1 stimulate in vitro assembly of human renalmicrovascular endothelial cells, expressing EphB1, into capillary-likestructures (21,22), and endothelial cells derived from differentvascular beds display striking differences in capillary assemblyresponses to specific members of the ephrin-A and ephrin-B families(21). Furthermore, more recent studies have demonstrated a highlevel of ephrin-B2 expression in adult glomeruli (23,24). Thesefindings suggested an important role of ephrin-B and EphB engagementin glomerular microvascular assembly.
In the present study, we explored the spatial and temporal patternof ephrin-B2 and EphB4 expression to define their potentialroles in renal glomerular development. The data demonstratea sequential, compartmentalized pattern of ephrin-B2 expressionthrough distinct cell lineages and suggest important roles ofephrin-B2 and EphB interactions during glomerular microvascularassembly.
Animals
The ephrin-B2tlacZ/+ or EphB4tlacZ/+ mice were generated asdescribed previously (18,19). Disruption of the ephrin-B2 orEphB4 gene by Tau-ß-galactosidase insertion drivesexpression under control of the endogenous ephrin-B2 or EphB4promoter. Animals were genotyped at weaning by PCR detectingß-galactosidase sequences or targeted exon as describedpreviously (19). Flk1tm1Jrt mice and wild-type C57Bl6/J micewere purchased from the Jackson Laboratory (Bar Harbor, ME)and genotyped by PCR amplifying ß-galactosidase orNeo-resistant gene sequences.
ß-Galactosidase Development Procedures
Timed pregnant mice were killed at the indicated gestationalday. Kidney tissues were removed from dissected embryos andfixed with 4% paraformaldehyde in phosphate-buffered saline(PBS) for 5 to 60 min at 4°C. The tissues were rinsed twicewith cold PBS and permeabilized with PBS containing 0.02% NP-40,0.01% sodium deoxycholate, and 2 mM MgCl2 for 15 min at 4°C.Color development was carried out for at least 6 h at room temperaturein solution containing 0.02% NP-40, 5 mM potassium ferricyanide,5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-D-galactopyranoside(X-gal; Sigma, St. Louis, MO) in PBS. Tissues were postfixedwith 4% paraformaldehyde in PBS for 60 min at 4°C, washedin PBS, dehydrated through graded ethanol series and isopropanolor Histo-Clear (National Diagnostics, Atlanta, GA), and embeddedin paraffin. Serial sections, 5 µm thick, were cut, dehydrated,mounted, and examined by light microscopy using Nomarski optics(Zeiss Axiophot; Carl Zeiss, Thornwood, NY).
Combined Immunostaining with ß-Galactosidase Histochemistry
Immunohistochemistry with anti- smooth muscle actin (SMA) andanti-Tamm Horsfall protein and histochemistry with the lectinfrom Dolichos biflorus agglutinin (DBA) were superimposed onß-galactosidase histochemistry. The kidneys subjectedto ß-galactosidase development were embedded in paraffin,and the paraffin sections were dewaxed, dehydrated, and blockedwith 5% donkey serum/PBS or 0.1% bovine serum albumin/PBS for30 min at room temperature. Then, the sections were incubatedwith the following: (1) horseradish peroxidase (HRP)-labeledanti-SMA monoclonal (1:200, clone1A4; DAKO, Carpinteria, CA),(2) anti-Tamm Horsfall protein goat antiserum (1:400; ICN BiomedicalsInc., Costa Mesa, CA), and (3) HRP-conjugated DBA (5 µg/ml,Sigma) for 60 min at room temperature. Tamm Horsfall proteinimmunostaining was then conducted by reaction with HRP-conjugatedanti-goat IgG (3.2 µg/ml, Jackson Immunoresearch Laboratories,West Grove, PA) for 60 min at room temperature. Thereafter,the slides were rinsed three times with PBS, and color reactionwas developed using diaminobenzidine chromogen solution (LiquidDAB, DAKO), dehydrated, mounted, and examined by light microscopy(Zeiss Axiophot).
Immunohistochemistry Procedures
Kidney tissues were snap-frozen in a dry ice-acetone bath. Cryostatsections (5 µm) were fixed in acetone for 10 min at -20°C,washed three times with cold PBS, and blocked with 5% normaldonkey serum or with 5% normal donkey serum plus 5% mouse orrat serum for 20 min. The sections were incubated with the following:(1) rabbit anti-ß-galactosidase (2.1 µg/ml;5 Prime Inc., Boulder, CO), (2) phycoerythrin-conjugated ratanti-CD31 monoclonal (10 µg/ml, clone MEC13.3; Pharmingen,Sanprego, CA), (3) Cy3-conjugated mouse anti-SMA monoclonal(1:200, clone1A4; Sigma), or (4) sheep anti-laminin (20 µg/ml,provided by Dale Abrahamson, University of Kansas Medical Center,Kansas City, KS) in the combinations indicated in the figurelegends. Tissue sections were incubated for 60 min at room temperature,washed with cold PBS, and, when necessary, incubated with FITC-or rhodamine-conjugated donkey anti-rabbit IgG (6.5 µg/ml;Jackson Immunoresearch Laboratories) or rhodamine-conjugatedanti-sheep IgG (6.5 µg/ml Jackson Immunoresearch Laboratories)for 30 min at room temperature. Washed sections were mounted(Vectashield; Vector Laboratories, Burlingame, CA) and analyzedby confocal microscopy (Zeiss LSM410). For the double-labelingstudy with anti-LacZ and anti-thiazide-sensitive Na-Cl cotransporter(TSC-1), anti-LacZ directly labeled with FITC was prepared asdescribed previously (25). The frozen section was first incubatedwith an affinity-purified anti-rat TSC1 rabbit polyclonal (providedfrom Steven Hebert, Yale University, New Haven, CT) (26) ata dilution of 1:1000 for 60 min at room temperature and thenwith rhodamine-conjugated donkey anti-rabbit IgG (15 µg/ml,Jackson Immunoresearch Laboratories) for 30 min. Subsequently,the section was incubated with rabbit IgG (20 µg/ml) for30 min. After rinsing in PBS, the section was reacted with FITC-conjugatedanti-LacZ (0.2 mg/ml) for 60 min.
The pattern of ephrin-B2 expression in adult and developingmouse kidney was evaluated in heterozygous mice carrying a homologousrecombinant ephrin-B2 allele expressing a Tau-ß-galactosidasefusion protein under control of the endogenous ephrin-B2 promoter(18). Tau sequences promote association of the expressed ß-galactosidasewith microtubules (27), permitting clear definition of the cellularpatterns of ephrin-B2 promoter activity. At low-power resolutionin Figure 1, ß-galactosidase expression is prominentin vascular structures, including the branch of renal artery,interlobular artery (Figure 1, A and B), arterioles and glomeruli(Figure 1A), peritubular capillaries (Figure 1A), and subsetsof medullary vascular bundles (Figure 1, C, G, and I) of adultmouse kidney. The ephrin-B2 promoter-driven ß-galactosidaseactivity was prominent in arterial but not venous endothelialcells, as previously reported (18).
Figure 1. Ephrin-B2 expression in adult mouse kidney. (A through E) Renal tissues from ephrin-B2 heterozygous mice were stained by ß-galactosidase histochemistry. The staining marks ephrin-B2 promoter activity in adult kidney vasculature, including the branch of renal artery (B), interlobular arteries (A), afferent and efferent arterioles (aa, ea), glomerulus (Glom), peritubular capillaries (PCap), vascular bundles of the outer medulla (arrows), and tubular components including connecting tubules (CT) and collecting ducts (CD). Staining is not detected in the sections from wild-type mice (D). It is noteworthy that ephrin-B2 promoter is active in vascular smooth muscle cells (VSMC) of large muscular artery (B) and interlobular artery (E-a and E-b). This pattern apparently differs from the endothelial-restricted staining of flk-1 heterozygous mice at postnatal day 7 (E-c). The outer surface of the vessels are indicated by hatched lines and circles. (F) Cryosections of ephrin-B2 heterozygous mice were immunostained with anti-ß-galactosidase. In renal arteries, ephrin-B2 expression is detected in endothelial cells (ECs) and VSMC. Endothelial expression of ephrin-B2 is higher in afferent arteriole and glomerular capillaries than in the branch of renal artery. Ephrin-B2 expression of glomerular mesangial cells (MCs) is obscure. (G and H) The paraffin sections of adult kidney, subjected to ß-galactosidase development, were stained with Dolichos biflorus agglutinin (DBA) peroxidase conjugate (brown). The staining defines ephrin-B2 expression in collecting ducts (CD), adjacent connecting tubules (CT), and vascular bundles (arrows). The double-labeling study for anti-LacZ (green) and anti-TSC1 (red) demonstrates lack of ephrin-B2 expression in distal convoluted tubules (DCT) (H, insert). (I) The immunostaining with anti-Tamm Horsfall protein (brown) was combined with ß-galactosidase histochemistry. Ephrin-B2 expression is not observed in ascending limb (AL) of distal tubules. It is noteworthy that vascular bundles are composed of ephrin-B2-positive (arrowheads) and -negative (arrows) vessels (insert). The thin limbs of Henle (TL) are negative for ephrin-B2 expression. Magnifications: x250 in A through E; x400 in F (right); x600 in F (middle and left); x80 in G; x200 in H; x100 in I.
On closer inspection, we determined that ephrin-B2 expressionwas not limited to endothelial cells but extended to vascularsmooth muscle cells (VSMC) in muscular arteries, including therenal artery, interlobular artery, and afferent arterioles (Figure 1, E and F).The staining pattern was distinct from the endothelial-restrictedß-galactosidase expression driven by the endogenousflk-1 promoter (Figure 1E). Ephrin-B2 promoter activity wasdetected also in medullary collecting ducts labeled by DBA andadjacent distal tubules (Figure 1, C and G) but not in ascendinglimb of Henle labeled by anti-Tamm Horsfall protein (Figure 1I)and distal convoluted tubules identified by TSC1 expression(Figure 1H), consistent with medullary collecting duct derivationfrom the ureteric bud epithelium that stains at early developmentalstages.
Motivated by the prominent vascular and particularly glomerularexpression of ephrin-B2, we set out to define the temporal andspatial pattern of ephrin-B2 promoter activity and to ascertainits potential roles in glomerular vascular assembly. We examinedephrin-B2 expression from embryonic day 12.5, the earliest developmentalstage at which capillaries develop in metanephric stroma, topostnatal day 7, when the overall functional pattern of mousekidney has been established. Shown in the E12.5 metanephrictissue of Figure 2A, ß-galactosidase activity wasdetected in arterial endothelial cells sprouting from dorsalaorta and in the ureteric bud branches extending into the metanephricmesenchyme. Weak ß-galactosidase activity was alsodetected in undifferentiated mesenchymal cells surrounding metanephricducts. At E13.5, glomerular epithelial cells of comma-shapedglomeruli as well as branching arteries and ureteric buds showedprominent ß-galactosidase activity (Figure 2, B and C).At E16.5, high-level ß-galactosidase activitywas detected in developing vessels, including those in musculararteries, arterioles, glomerular capillaries, and both collectingducts and adjacent connecting tubules (Figure 2, D through F).This vascular and distal tubular expression pattern persistsfrom renal development to maturity.
Figure 2. Ephrin-B2 expression in developing mouse kidney. The ephrin-B2 promoter activity of developing kidney was visualized by ß-galactosidase histochemistry. (A) At embryonic day 12.5 (E12.5), ephrin-B2 expression is apparent in the ureteric bud epithelium (UB), in the early arterial vessels (Art) entering into metanephros, and in the vascular networks (arrowhead) surrounding the metanephric blastema and assembled around the ureteric bud. (B through F) At E13.5 (B) and E14.5 (C), staining is evident in the developing glomeruli, including comma-shaped (C), S-shaped (S), and capillary loop stage (CL) glomeruli. Similar expression pattern of ephrin-B2 is observed in E16.5 (D), E18.5 (E), and postnatal day 7 (F) kidneys. The combined DBA/ß-galactosidase histochemistry demonstrates the ephrin-B2 expression in the connecting tubules (CT) to be continuous with collecting ducts (CD) (E, insert). Magnification, x150.
The high level of ephrin-B2 promoter activity in the developingglomeruli led us to analyze further the identity of the expressingcell types. Shown in Figure 3A-a, ephrin-B2 surrogate ß-galactosidaseactivity was restricted to the epithelial cells from which visceralepithelial cells are derived in comma-shaped developing glomeruli.Weak ephrin-B2 expression was seen in parietal glomerular epithelialsites with some extension to proximal tubules (not shown). Thisexpression pattern is distinct from that of heterozygous miceexpressing ß-galactosidase under control of the flk-1promoter (Figure 3A-e). Flk-1 has been used to define the originand pattern of assembly of glomerular endothelial progenitorsin previous studies (5). Flk-1 promoter-driven ß-galactosidaseactivity was detected in endothelial precursors that are discontinuouslydistributed in metanephric stroma. These cells are thought tomigrate to vascular clefts of comma-shaped glomeruli (Figure 3A-e).Low level and less frequent expression of ephrin-B2 wasdetected in these endothelial precursors (Figure 3A-a). As glomerulardevelopment progressed to the S-shaped stage (Figure 3A-b) andcapillary loop stage (Figure 3A-c), prominent ephrin-B2-drivenß-galactosidase expression was detected in glomerularvascular cells, including not only visceral epithelial cellsbut also endothelial and mesangial cells. Emphasizing the temporaldistinctions in the pattern of ephrin-B2 expression during glomerulardevelopment, the cellular expression pattern changed markedlyby completion of glomerular maturation. Ephrin-B2 expressionin glomerular epithelial cells was reduced by postnatal day7 (not shown), and no ß-galactosidase activity wasdetected in glomerular epithelial cells of adult mouse kidney(Figure 3A-d).
Figure 3. Progression of glomerular ephrin-B2 expression in distinct lineages. (A) Embryonic and adult kidneys from ephrin-B2 (a through d) or flk-1 (e through h) heterozygous mice were examined by ß-galactosidase histochemistry (a and e, E14.5; b, c, f, and g, E16.5; d and h, adult mice). Ephrin-B2 expression is initially detected in precursors of the visceral glomerular epithelial cells (VE) adjacent to the vascular cleft (VC) of comma-shaped glomeruli (a) and subsequently in glomerular capillary tufts of S-shaped (b) and capillary loop stage glomeruli (c). Ephrin-B2 and flk-1 expression is seen in endothelial progenitors (arrowheads) distributed around comma-shaped glomeruli (a and e). In the glomeruli of adult animals (d), glomerular epithelial ephrin-B2 expression is markedly attenuated. In contrast, flk-1 expression is initially observed in endothelial progenitors migrating into the vascular cleft (VC) (e) and remains restricted to glomerular endothelial cells during glomerulogenesis (f and g). In adult kidney, flk-1 is continuously expressed in glomeruli, whereas arterial (not shown) and arteriolar flk-1 are markedly diminished (h). (B) Frozen tissue sections of neonatal (a through c) and adult (d) kidneys were double-immunolabeled with anti-ßgalactosidase (ephrin-B2, green) and anti-laminin (red). In comma- and S-shaped glomeruli (a), ephrin-B2 is predominantly expressed in glomerular epithelium (Epi). Subsequently, definitive expression of ephrin-B2 can be seen in glomerular endothelial (En) and mesangial (Mes) cells as well as in glomerular epithelial cells (Epi) of capillary stage glomeruli (b and c). Finally, ephrin-B2 remains to be expressed in glomerular endothelial cells of adult kidney (d). Magnifications: x400 in A; x800 in B-a; x1000 in B-b; x1000 in B-c; x1000 in B-d.
The shifting pattern of expression led us to evaluate more closelythe relationship of ß-galactosidase activity to theglomerular basement membrane as a means of discriminating expressionin visceral epithelial from expression in endothelial cells.Our targeting strategy with Tau-LacZ protein enabled us to analyzethe lineage expression using anti-ß-galactosidaseimmunohistochemistry. In double-labeling experiments that evaluatedß-galactosidase and laminin immunoreactivities inthe same sections by confocal microscopy (Figure 3B), anti-ß-galactosidaseinitially labeled visceral glomerular epithelial cells (Figure 3B-a).At the next stages of glomerular development, ephrin-B2expression was evident in all glomerular cell components, includingepithelial, endothelial, and mesangial cells. (Figure 3, B-b and B-c).Subsequently, at full maturity, ephrin-B2 expressionwas restricted to glomerular endothelial cells (Figure 3B-d).
This progression of expression through distinct cell lineagesled us to evaluate ephrin-B2 expression in vascular cells oflarger arteries as well as of the glomerulus. For this purpose,we conducted simultaneous detection of ß-galactosidaseimmunoreactivity and SMA immunoreactivity. In E14.5 kidneys,ephrin-B2-driven ß-galactosidase was detected onlyin sprouting and branching arterial endothelial cells but notin surrounding VSMC labeled by anti-SMA (Figure 4A). As arteriogenesisprogressed to the neonatal stage, ephrin-B2 expression was apparentlydetected in VSMC of the renal arterial circulation (Figure 4B).In adulthood, VSMC dominantly express ephrin-B2, with some attenuationin smooth muscle cells of afferent arteriole and glomerularcapillaries (Figure 4C).
Figure 4. Lineage progression of ephrin-B2 expression in arteries. Immunofluorescence stainings were conducted on frozen kidney tissue sections with anti-ß-galactosidase (green) and anti-CD31 (red) or anti- smooth muscle actin (SMA; red) in combinations indicated in each panel. (A) In E14.5 stage kidney, ephrin-B2 is expressed in endothelial cells (ECs) of developing arterial vessels (overlapping staining for ß-galactosidase and CD31) but not in VSMC labeled by anti-SMA. (B) In neonatal kidney, ephrin-B2 expression is detected in VSMC as well as in endothelial cells. High-level expression of endothelial ephrin-B2 is revealed by overlapping staining (yellow) for ß-galactosidase and CD31 (right). (C) In adult arteries, ephrin-B2 is dominantly expressed in VSMC, and endothelial ephrin-B2 is reduced. Magnification, x600
To explore the distribution of cells expressing receptors withwhich ephrin-B2 may interact, we examined patterns of EphB4expression. EphB4 was shown previously to be expressed in venouslimb endothelial cells during vascular development in extrarenalsites, and mice genetically deficient in EphB4 were shown tohave a phenotype complementary to that of ephrin-B2 null mice(19). Using recombinant mice expressing tau-ß-galactosidaseunder control of the EphB4 promoter (19), we detected ß-galactosidaseactivity in endothelial cells of the renal vein extending tometanephric tissue at E14.5 (Figure 5A). Definitive EphB4 promoteractivity was not observed in developing glomeruli at comma-shaped,S-shaped, or later stages of development (Figure 5, A, B, D, and E).Renal venous endothelial EphB4 expression was dramaticallyreduced after postnatal day 7 stage (not shown) and was notdetectable in adult kidney using optimized conditions (Figure 5C).Low levels of expression were identified in the endocapillaryspace of a small subset (<10%) of glomeruli, as shown inFigure 5C. The unfavorable signal-to-noise ratio and the infrequentglomerular expression made further definition of the responsiblecell population impossible. It is noteworthy that the consistentand stable EphB4 expression in venous endothelium contrastssharply with the progression of ephrin-B2 expression throughmultiple cell lineages.
Figure 5. Venous expression of EphB4 in developing mouse kidney. The renal tissues from EphB4 heterozygous mice were stained by ß-galactosidase histochemistry. (A through C) At E14.5 (A), the staining visualizes EphB4 promoter activity in renal veins (V) extending to metanephros. No apparent signals are detected in developing glomeruli, including S-shaped (S) and capillary loop stage (CL) glomeruli. In neonate kidney (B), EphB4 expression is detected in venous endothelial cells (V) constructing postglomerular circulations. EphB4 expression is dramatically reduced in adult kidney (C). It is noteworthy that a few significant EphB4 signals are observed in mature glomeruli (arrows) and in some blood cells (arrowhead). (D and E) Anti-SMA immunohistochemistry (brown) was combined with ß-galactosidase histochemistry to define venous endothelial expression of EphB4. EphB4 expression is exclusively observed in venous endothelial cells in E14.5 (D) and neonate (E) kidneys. No stainings can be seen in arterial endothelial cells. Magnifications: x100 in A through C; x200 in D; x150 in E.
A highly organized glomerular structure is formed through orchestratedintercellular connections between three distinct cell populations(1). However, little is known about cell targeting receptorsthat assemble glomerular cells to appropriate positions uponcell-cell contact. Recently, it was shown that ephrin-B andits EphB receptor participate in developmental vascular cellassembly. Here we show the sequential, compartmentalized expressionof ephrin-B2 through different cell lineages during glomerulardevelopment.
The initial studies of ephrin-B2 demonstrated that its expressionmarks arterial endothelial cells, whereas its binding partner,EphB4, is restricted in expression to venous endothelial cellsin early embryonic angiogenesis (18,19). This spatial segregation,coupled with the developmental timing of vascularization failurein both ephrin-B2-and EphB4-deficient mice, suggests a rolefor ephrin-B2/EphB4 engagement in the coordinated targetingand interconnection of venous and arterial limb structures.
In formation of the glomerulus, endothelial progenitors seemto develop from metanephric mesenchyme, migrate to the developingglomerulus, and assemble into glomerular capillaries througha vasculogenic process (3,4). Ultimately they are integratedto arterial and venous circulations through afferent and efferentarterioles. On the basis of developmental restriction of ephrin-B2expression to arterial and EphB4 to venous endothelial cells,we sought to define the boundary of ephrin-B2/EphB4 interaction.We demonstrated that arterial and glomerular endothelial cellsexpress ephrin-B2, whereas EphB4 is indeed expressed in venouslimb vessels branched from renal vein.
These findings raise two points. First, early endothelial progenitors,identified by recruitment to the vascular cleft and expressionof Flk-1, express ephrin-B2, similar to arterial endothelialcells. This arterial "programming" of glomerular endotheliumis maintained even in the efferent end of glomerular endothelialcapillaries and efferent arteriole (Figure 1A). Second, thedistinct site at which ephrin-B2-expressing efferent arteriolesinterconnect with EphB4-expressing peritubular capillaries remainsill-defined at present. It is noteworthy that the medullaryvascular bundle contains two distinct populations of capillaries,including ephrin-B2-expressing and -nonexpressing endothelium.Although a rare cell expresses EphB4 in mature glomeruli (Figure 5, B and C),this seems most likely to represent hematopoietic-derivedcells (28–30).
We were surprised to find that during renal development, ephrin-B2expression is first prominent in glomerular epithelial, notendothelial, cells. This expression is restricted to a subpopulationof epithelial cells adjacent to the vascular cleft, representingpodocyte progenitor cells. Developmental studies in Xenopusdemonstrated that ephrin-B1 and ephrin-B2 are expressed in thesomites, whereas EphB4 is present on the posterior cardinalvein and intersomitic vein (31). A recent study demonstratedthat either ectopic expression of ephrin-B1 or disruption ofEphB4 signaling leads to aberrant projection of intersomiticvessels (32). Consistent with these findings, ephrin-B2 nullmice develop aberrant sprouting of intersomitic vessels (33).These findings suggest that sprouting intersomitic vessels areguided through migration routes by somitic ephrin-B and endothelialEphB engagement (32). By analogy, we speculate that glomerularepithelial ephrin-B2 may guide endothelial progenitors expressingEphB1 to glomerular sites (21).
Ephrin-B2 expression was identified in developing glomerularmesangial cells, as defined by SMA expression (data not shown).Mesangial expression persisted through the early perinatal periodbut was extinguished in mature glomeruli (Table 1). This stage-restrictedexpression of mesangial ephrin-B2 appears analogous to its expressionin periendothelial mesenchymal cells in developing intersomiticvessels at E9.5. In early extrarenal embryonic angiogenesis,arterial limb endothelial cells express ephrin-B1, ephrin-B2,and EphB3, whereas mesenchymal cells that surround these vesselsexpress ephrin-B2 and EphB2 (20). Some (30%) of EphB2/EphB3double knockout mice experience embryonic vascularization failure,implicating other EphB receptors in cell-cell interactions necessaryfor proper vascular assembly. By extrapolation, one may expectthat mesangial EphB3 (34) and/or other EphB proteins engagethe endothelial ephrin-B2 to direct or modulate endothelial-mesangialcell assembly.
Table 1. Temporal and spatial expression of ephrin-B2 in renal vascular developmenta
We noted prominent expression of ephrin-B2 in VSMC of maturelarge arteries, in addition to its endothelial expression (Table 1).This vascular smooth muscle expression is not restrictedto specific developmental stages and continues to maturity.On the basis of evidence that ephrin-B2 and EphB6 engagementarrests tumor cell growth (35), ephrin-B2 may participate instabilizing and maintaining the vascular smooth muscle layer.Similarly, persistent ephrin-B2 expression in glomerular andarterial endothelial cells may be involved in the maintenanceof endothelial integrity.
Expression of ephrin-B2 in medullary and cortical collectingducts and connecting tubules of developing and adult kidneys(Figure 1, C and G) suggests possible roles in developmentaltubular/endothelial interactions. By analogy to the glomerularepithelial/endothelial interactions, tubule epithelial ephrin-B2may direct the spatial organization of peritubular capillariesat these sites in cortex and medulla. Although earlier studiesfocused on functions for ephrin-B2 and EphB4 in embryonic arterial-venousvascular assembly, spatial- and timing-restricted expressionof ephrin-B2 and EphB4 during glomerular development implicatesthese proteins in the integration of glomerular cell assembly.
Acknowledgments
This work was supported by National Institutes of Health GrantR01-DK-47078 (T.O.D.). Analysis was performed in part throughuse of the VUMC Cell Imaging Resource supported by CA68485 andDK20593. The authors thank Raymond Harris and Matthew Breyerfor support and advice, Dale Abrahamson for providing the anti-lamininantibody, Steven Hebert for providing the anti-rTSC1 antibody,and John Schlueter and Amy Hsu for technical assistance.
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Received for publication October 27, 2000.
Accepted for publication July 11, 2001.
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Abstract
Introduction
smooth muscle actin (
