J Am Soc Nephrol 12: 2673–2682, 2001 Temporally Compartmentalized Expression of -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; and †Department of Biology, California Institute of Technology, Pasadena, California.

Abstract. Glomerular development proceeds through the spa- progressed. At or near completion of glomerular maturation, tially ordered and sequential recruitment, proliferation, assem- epithelial ephrin-B2 expression was extinguished, with persis- bly, and differentiation of endothelial, mesangial, and epithe- tence in glomerular endothelial cells. Throughout develop- lial progenitors. The molecular determinants of cell-cell ment, one of several ephrin-B2 receptors, EphB4, was persis- recognition and targeting in this process have yet to be defined. tently and exclusively expressed in endothelial cells of venous The Eph/ephrin family of membrane receptors and counter- structures. The findings show sequential ephrin-B2 expression receptors are critical participants of developmental vascular across glomerular lineages, first in a distinct subset of podocyte assembly in extrarenal sites. Renal expression patterns of eph- progenitors and subsequently in endothelial cells of the devel- rin-B2 and EphB4 were investigated using mice expressing oping glomerulus. Given targeting functions for Eph/ephrin ␤-galactosidase under control of ephrin-B2 or EphB4 promot- family , the findings suggest that ephrin-B2 expression ers. The earliest glomerular expression of ephrin-B2 was iden- marks podocyte progenitors at the site of vascular cleft forma- tified in a subset of differentiating comma-stage glomerular tion, where expression may establish an “address” to which epithelial cells (podocyte progenitors) adjacent to the vascular endothelial and mesangial progenitors are recruited. Thus, the cleft where endothelial progenitors are subsequently recruited. present results suggest that ephrin-B2 and EphB interactions Epithelial ephrin-B2 expression was accompanied by expres- play an important role in glomerular microvascular assembly. sion in endothelial and mesangial cells as capillary assembly

Developmental assembly of the renal glomerular microcircu- receptor flk-1 from the adjacent metanephric mesenchyme lation is a precise and coordinated process that forms a highly (6,7). Endothelial production of platelet-derived growth fac- specialized vascular filtering apparatus (1,2). Glomerular en- tor–BB (PDGF-BB) and mesangial progenitor expression of dothelial cells are initially dispersed throughout the metaneph- platelet-derived receptor–␤ (PDGFR-␤) are re- ric mesenchymal tissue (3,4) and are subsequently recruited to quired for the coordinated recruitment, proliferation, and as- the clefts of comma-shaped glomeruli, where they proliferate sembly of mesangial cells (8). The endothelial tie-2 receptor and assemble into glomerular capillaries adjacent to mesangial and its ligands, which mediate endothelial-peri- and glomerular epithelial cells. A distinct population of endo- cyte interactions and microvascular maturation (9–11), have thelial progenitors, recruited from the extrarenal para-aortic been detected in endothelial and mesangial cells of developing region, assembles a basket-like network on the superficial glomeruli (12,13). Transforming growth factor-␤1 actions are surface of the developing kidney, where they interconnect to implicated at several steps during glomerulogenesis, most integrate large vessel and glomerular microvascular circula- prominently in the maturation phase required to stabilize vas- tions (5). Available data implicate both vasculogenic and an- cular structure (14). giogenic processes in this integrated assembly. In addition to these examples of paracrine intercellular Several growth factors and their receptors mediate critical communication, cell-cell contact is implicated in correct steps in glomerular capillary development. Vascular endothe- targeting and assembly of the glomerulus, yet little is known lial growth factor is expressed in developing podocytes and about the molecular regulation of targeting and assembly. seems to promote differentiation and recruitment of endothelial The Eph receptor tyrosine kinases are transmembrane pro- progenitors expressing the vascular endothelial growth factor teins that are engaged by contact with their counter-recep- tors, , upon cell-cell contact (15,16). Both Eph and ephrin proteins contribute to developmental patterning and Received October 27, 2000. Accepted July 11, 2001. neuronal targeting (17). Among ephrins, the ephrin-A sub- Correspondence to Dr. Thomas O. Daniel, Immunex Corporation, 51 Univer- class proteins (ephrin-A1 to -A5) are membrane attached sity Street, Seattle, WA 98101. Phone: 206-470-4875; Fax: 206-933-3733; E-mail: [email protected] through glycosyl-phosphatidyl-inositol linkages, whereas 1046-6673/1212-2673 the ephrin-B subclasses (ephrin-B1 to -B3) are transmem- Journal of the American Society of Nephrology brane proteins with highly conserved cytoplasmic domains. Copyright © 2001 by the American Society of Nephrology Ephrin-As interact with the A subset of Eph receptors 2674 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2673–2682, 2001

(EphA1 to A8) and Ephrin-B ligands with the B subset of washed in PBS, dehydrated through graded ethanol series and isopro- Eph receptors (EphB1 to EphB6). Mice homozygous for panol or Histo-Clear (National Diagnostics, Atlanta, GA), and em- ephrin-B2 or EphB4 allele mutation display early embryonic bedded in paraffin. Serial sections, 5 ␮m thick, were cut, dehydrated, vascularization defects at the developmental stage when mounted, and examined by light microscopy using Nomarski optics interconnection of ephrin-B2 expressing arterial limb vas- (Zeiss Axiophot; Carl Zeiss, Thornwood, NY). cular networks normally links with EphB4 expressing ve- nous limb networks (18,19). Lethal vascular defects are also Combined Immunostaining with detected in EphB2/EphB3 double mutants (20). EphB2 ex- ␤ pression in mesenchymal cells adjacent to vessels has sug- -Galactosidase Histochemistry ␣ ␣ gested that Ephrin-B/EphB signaling may participate in Immunohistochemistry with anti– smooth muscle actin ( SMA) and anti–Tamm Horsfall and histochemistry with the lectin intercellular interactions between endothelial cells and ad- from Dolichos biflorus agglutinin (DBA) were superimposed on ␤-ga- jacent mesenchymal cells (20). lactosidase histochemistry. The kidneys subjected to ␤-galactosidase As for renal expression of ephrin-B and EphB, early studies development were embedded in paraffin, and the paraffin sections have shown that glomerular endothelial progenitors display were dewaxed, dehydrated, and blocked with 5% donkey serum/PBS EphB1 immunoreactivity both before and during recruitment to or 0.1% bovine serum albumin/PBS for 30 min at room temperature. comma- and S-shaped glomeruli (21). Moreover, specific oli- Then, the sections were incubated with the following: (1) horseradish gomerized forms of ephrin-B1 stimulate in vitro assembly of peroxidase (HRP)-labeled anti-␣SMA monoclonal (1:200, clone1A4; human renal microvascular endothelial cells, expressing DAKO, Carpinteria, CA), (2) anti–Tamm Horsfall protein goat anti- EphB1, into capillary-like structures (21,22), and endothelial serum (1:400; ICN Biomedicals Inc., Costa Mesa, CA), and (3) cells derived from different vascular beds display striking HRP-conjugated DBA (5 ␮g/ml, Sigma) for 60 min at room temper- differences in capillary assembly responses to specific mem- ature. Tamm Horsfall protein immunostaining was then conducted by ␮ bers of the ephrin-A and ephrin-B families (21). Furthermore, reaction with HRP-conjugated anti-goat IgG (3.2 g/ml, Jackson Immunoresearch Laboratories, West Grove, PA) for 60 min at room more recent studies have demonstrated a high level of eph- temperature. Thereafter, the slides were rinsed three times with PBS, rin-B2 expression in adult glomeruli (23,24). These findings and color reaction was developed using diaminobenzidine chromogen suggested an important role of ephrin-B and EphB engagement solution (Liquid DAB, DAKO), dehydrated, mounted, and examined in glomerular microvascular assembly. by light microscopy (Zeiss Axiophot). In the present study, we explored the spatial and temporal pattern of ephrin-B2 and EphB4 expression to define their potential roles in renal glomerular development. The data Immunohistochemistry Procedures demonstrate a sequential, compartmentalized pattern of eph- Kidney tissues were snap-frozen in a dry ice–acetone bath. Cryostat rin-B2 expression through distinct cell lineages and suggest sections (5 ␮m) were fixed in acetone for 10 min at Ϫ20°C, washed important roles of ephrin-B2 and EphB interactions during three times with cold PBS, and blocked with 5% normal donkey glomerular microvascular assembly. serum or with 5% normal donkey serum plus 5% mouse or rat serum for 20 min. The sections were incubated with the following: (1) rabbit anti–␤-galactosidase (2.1 ␮g/ml; 5 Prime Inc., Boulder, CO), (2) Materials and Methods ␮ Animals phycoerythrin-conjugated rat anti-CD31 monoclonal (10 g/ml, clone MEC13.3; Pharmingen, Sanprego, CA), (3) Cy3-conjugated mouse The ephrin-B2tlacZ/ϩ or EphB4tlacZ/ϩ mice were generated as anti-␣SMA monoclonal (1:200, clone1A4; Sigma), or (4) sheep anti- described previously (18,19). Disruption of the ephrin-B2 or EphB4 laminin (20 ␮g/ml, provided by Dale Abrahamson, University of gene by Tau-␤-galactosidase insertion drives expression under control Kansas Medical Center, Kansas City, KS) in the combinations indi- of the endogenous ephrin-B2 or EphB4 promoter. Animals were cated in the figure legends. Tissue sections were incubated for 60 min genotyped at weaning by PCR detecting ␤-galactosidase sequences or at room temperature, washed with cold PBS, and, when necessary, targeted exon as described previously (19). Flk1tm1Jrt mice and wild- incubated with FITC- or rhodamine-conjugated donkey anti-rabbit type C57Bl6/J mice were purchased from the Jackson Laboratory (Bar IgG (6.5 ␮g/ml; Jackson Immunoresearch Laboratories) or rhodam- Harbor, ME) and genotyped by PCR amplifying ␤-galactosidase or ine-conjugated anti-sheep IgG (6.5 ␮g/ml Jackson Immunoresearch Neo-resistant gene sequences. Laboratories) for 30 min at room temperature. Washed sections were mounted (Vectashield; Vector Laboratories, Burlingame, CA) and ␤-Galactosidase Development Procedures analyzed by confocal microscopy (Zeiss LSM410). For the double- Timed pregnant mice were killed at the indicated gestational day. labeling study with anti-LacZ and anti–thiazide-sensitive Na-Cl co- Kidney tissues were removed from dissected embryos and fixed with transporter (TSC-1), anti-LacZ directly labeled with FITC was pre- 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 to 60 pared as described previously (25). The frozen section was first min at 4°C. The tissues were rinsed twice with cold PBS and perme- incubated with an affinity-purified anti-rat TSC1 rabbit polyclonal abilized with PBS containing 0.02% NP-40, 0.01% sodium deoxy- (provided from Steven Hebert, Yale University, New Haven, CT) (26)

cholate, and 2 mM MgCl2 for 15 min at 4°C. Color development was at a dilution of 1:1000 for 60 min at room temperature and then with carried out for at least6hatroom temperature in solution containing rhodamine-conjugated donkey anti-rabbit IgG (15 ␮g/ml, Jackson 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferro- Immunoresearch Laboratories) for 30 min. Subsequently, the section ␮ cyanide, 2 mM MgCl2, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-D- was incubated with rabbit IgG (20 g/ml) for 30 min. After rinsing in galactopyranoside (X-gal; Sigma, St. Louis, MO) in PBS. Tissues PBS, the section was reacted with FITC-conjugated anti-LacZ (0.2 were postfixed with 4% paraformaldehyde in PBS for 60 min at 4°C, mg/ml) for 60 min. J Am Soc Nephrol 12: 2673–2682, 2001 Ephrin-B2 in Renal Glomerular Development 2675

Results rogate ␤-galactosidase activity was restricted to the epithelial The pattern of ephrin-B2 expression in adult and developing cells from which visceral epithelial cells are derived in comma- mouse kidney was evaluated in heterozygous mice carrying a shaped developing glomeruli. Weak ephrin-B2 expression was homologous recombinant ephrin-B2 allele expressing a Tau- seen in parietal glomerular epithelial sites with some extension ␤-galactosidase fusion protein under control of the endogenous to proximal tubules (not shown). This expression pattern is ephrin-B2 promoter (18). Tau sequences promote association distinct from that of heterozygous mice expressing ␤-galacto- of the expressed ␤-galactosidase with microtubules (27), per- sidase under control of the flk-1 promoter (Figure 3A-e). Flk-1 mitting clear definition of the cellular patterns of ephrin-B2 has been used to define the origin and pattern of assembly of promoter activity. At low-power resolution in Figure 1, ␤-ga- glomerular endothelial progenitors in previous studies (5). ␤ lactosidase expression is prominent in vascular structures, in- Flk-1 promoter-driven -galactosidase activity was detected in cluding the branch of renal artery, interlobular artery (Figure 1, endothelial precursors that are discontinuously distributed in A and B), arterioles and glomeruli (Figure 1A), peritubular metanephric stroma. These cells are thought to migrate to capillaries (Figure 1A), and subsets of medullary vascular vascular clefts of comma-shaped glomeruli (Figure 3A-e). Low bundles (Figure 1, C, G, and I) of adult mouse kidney. The level and less frequent expression of ephrin-B2 was detected in ephrin-B2 promoter-driven ␤-galactosidase activity was prom- these endothelial precursors (Figure 3A-a). As glomerular de- inent in arterial but not venous endothelial cells, as previously velopment progressed to the S-shaped stage (Figure 3A-b) and reported (18). capillary loop stage (Figure 3A-c), prominent ephrin-B2– ␤ On closer inspection, we determined that ephrin-B2 expres- driven -galactosidase expression was detected in glomerular sion was not limited to endothelial cells but extended to vas- vascular cells, including not only visceral epithelial cells but cular smooth muscle cells (VSMC) in muscular arteries, in- also endothelial and mesangial cells. Emphasizing the temporal distinctions in the pattern of ephrin-B2 expression during glo- cluding the renal artery, interlobular artery, and afferent merular development, the cellular expression pattern changed arterioles (Figure 1, E and F). The staining pattern was distinct markedly by completion of glomerular maturation. Ephrin-B2 from the endothelial-restricted ␤-galactosidase expression expression in glomerular epithelial cells was reduced by post- driven by the endogenous flk-1 promoter (Figure 1E). Eph- natal day 7 (not shown), and no ␤-galactosidase activity was rin-B2 promoter activity was detected also in medullary col- detected in glomerular epithelial cells of adult mouse kidney lecting ducts labeled by DBA and adjacent distal tubules (Fig- (Figure 3A-d). ure 1, C and G) but not in ascending limb of Henle labeled by The shifting pattern of expression led us to evaluate more anti–Tamm Horsfall protein (Figure 1I) and distal convoluted closely the relationship of ␤-galactosidase activity to the glo- tubules identified by TSC1 expression (Figure 1H), consistent merular basement membrane as a means of discriminating with medullary collecting duct derivation from the ureteric bud expression in visceral epithelial from expression in endothelial epithelium that stains at early developmental stages. cells. Our targeting strategy with Tau-LacZ protein enabled us Motivated by the prominent vascular and particularly glo- to analyze the lineage expression using anti–␤-galactosidase merular expression of ephrin-B2, we set out to define the immunohistochemistry. In double-labeling experiments that temporal and spatial pattern of ephrin-B2 promoter activity and evaluated ␤-galactosidase and laminin immunoreactivities in to ascertain its potential roles in glomerular vascular assembly. the same sections by confocal microscopy (Figure 3B), anti– We examined ephrin-B2 expression from embryonic day 12.5, ␤-galactosidase initially labeled visceral glomerular epithelial the earliest developmental stage at which capillaries develop in cells (Figure 3B-a). At the next stages of glomerular develop- metanephric stroma, to postnatal day 7, when the overall func- ment, ephrin-B2 expression was evident in all glomerular cell tional pattern of mouse kidney has been established. Shown in components, including epithelial, endothelial, and mesangial ␤ the E12.5 metanephric tissue of Figure 2A, -galactosidase cells. (Figure 3, B-b and B-c). Subsequently, at full maturity, activity was detected in arterial endothelial cells sprouting ephrin-B2 expression was restricted to glomerular endothelial from dorsal aorta and in the ureteric bud branches extending cells (Figure 3B-d). into the metanephric mesenchyme. Weak ␤-galactosidase ac- This progression of expression through distinct cell lineages tivity was also detected in undifferentiated mesenchymal cells led us to evaluate ephrin-B2 expression in vascular cells of surrounding metanephric ducts. At E13.5, glomerular epithelial larger arteries as well as of the glomerulus. For this purpose, cells of comma-shaped glomeruli as well as branching arteries we conducted simultaneous detection of ␤-galactosidase im- ␤ ␣ and ureteric buds showed prominent -galactosidase activity munoreactivity and SMA immunoreactivity. In E14.5 kidneys, ␤ (Figure 2, B and C). At E16.5, high-level -galactosidase ac- ephrin-B2–driven ␤-galactosidase was detected only in sprout- tivity was detected in developing vessels, including those in ing and branching arterial endothelial cells but not in surround- muscular arteries, arterioles, glomerular capillaries, and both ing VSMC labeled by anti-␣SMA (Figure 4A). As arteriogen- collecting ducts and adjacent connecting tubules (Figure 2, D esis progressed to the neonatal stage, ephrin-B2 expression was through F). This vascular and distal tubular expression pattern apparently detected in VSMC of the renal arterial circulation persists from renal development to maturity. (Figure 4B). In adulthood, VSMC dominantly express ephrin- The high level of ephrin-B2 promoter activity in the devel- B2, with some attenuation in smooth muscle cells of afferent oping glomeruli led us to analyze further the identity of the arteriole and glomerular capillaries (Figure 4C). expressing cell types. Shown in Figure 3A-a, ephrin-B2 sur- To explore the distribution of cells expressing receptors with 2676 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2673–2682, 2001

Figure 1. Ephrin-B2 expression in adult mouse kidney. (A through E) Renal tissues from ephrin-B2 heterozygous mice were stained by ␤-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 J Am Soc Nephrol 12: 2673–2682, 2001 Ephrin-B2 in Renal Glomerular Development 2677

Figure 2. Ephrin-B2 expression in developing mouse kidney. The ephrin-B2 promoter activity of developing kidney was visualized by ␤ -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/ -galactosidase histochemistry demonstrates the ephrin-B2 expression in the connecting tubules (CT) to be continuous with collecting ducts (CD) (E, insert). Magnification, ϫ150.

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–␤-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 ␤-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 ␤-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: ϫ250 in A through E; ϫ400 in F (right); ϫ600 in F (middle and left); ϫ80 in G; ϫ200 in H; ϫ100 in I. 2678 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2673–2682, 2001

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 -galactosidase histochemistry (a and e, E14.5;b,c,f,andg,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–␤galactosidase (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: ϫ400 in A; ϫ800 in B-a; ϫ1000 in B-b; ϫ1000 in B-c; ϫ1000 in B-d. J Am Soc Nephrol 12: 2673–2682, 2001 Ephrin-B2 in Renal Glomerular Development 2679

Figure 4. Lineage progression of ephrin-B2 expression in arteries. Immunofluorescence stainings were conducted on frozen kidney tissue sections with anti–␤-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 ␤-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 ␤-galactosidase and CD31 (right). (C) In adult arteries, ephrin-B2 is dominantly expressed in VSMC, and endothelial ephrin-B2 is reduced. Magnification, ϫ600 which ephrin-B2 may interact, we examined patterns of EphB4 (Figure 5, A, B, D, and E). Renal venous endothelial EphB4 expression. EphB4 was shown previously to be expressed in expression was dramatically reduced after postnatal day 7 stage venous limb endothelial cells during vascular development in (not shown) and was not detectable in adult kidney using opti- extrarenal sites, and mice genetically deficient in EphB4 were mized conditions (Figure 5C). Low levels of expression were shown to have a phenotype complementary to that of ephrin-B2 identified in the endocapillary space of a small subset (Ͻ10%) of null mice (19). Using recombinant mice expressing tau-␤-galac- glomeruli, as shown in Figure 5C. The unfavorable signal-to- tosidase under control of the EphB4 promoter (19), we detected noise ratio and the infrequent glomerular expression made further ␤-galactosidase activity in endothelial cells of the renal vein definition of the responsible cell population impossible. It is extending to metanephric tissue at E14.5 (Figure 5A). Definitive noteworthy that the consistent and stable EphB4 expression in EphB4 promoter activity was not observed in developing glomer- venous endothelium contrasts sharply with the progression of uli at comma-shaped, S-shaped, or later stages of development ephrin-B2 expression through multiple cell lineages. 2680 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2673–2682, 2001

Figure 5. Venous expression of EphB4 in developing mouse kidney. The renal tissues from EphB4 heterozygous mice were stained by ␤ -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 ␤-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: ϫ100 in A through C; ϫ200 in D; ϫ150 in E.

Discussion dinated targeting and interconnection of venous and arterial A highly organized glomerular structure is formed through limb structures. orchestrated intercellular connections between three distinct In formation of the glomerulus, endothelial progenitors seem cell populations (1). However, little is known about cell tar- to develop from metanephric mesenchyme, migrate to the geting receptors that assemble glomerular cells to appropriate developing glomerulus, and assemble into glomerular capillar- positions upon cell-cell contact. Recently, it was shown that ies through a vasculogenic process (3,4). Ultimately they are ephrin-B and its EphB receptor participate in developmental integrated to arterial and venous circulations through afferent vascular cell assembly. Here we show the sequential, compart- and efferent arterioles. On the basis of developmental restric- mentalized expression of ephrin-B2 through different cell lin- tion of ephrin-B2 expression to arterial and EphB4 to venous eages during glomerular development. endothelial cells, we sought to define the boundary of ephrin- The initial studies of ephrin-B2 demonstrated that its expres- B2/EphB4 interaction. We demonstrated that arterial and glo- sion marks arterial endothelial cells, whereas its binding part- merular endothelial cells express ephrin-B2, whereas EphB4 is ner, EphB4, is restricted in expression to venous endothelial indeed expressed in venous limb vessels branched from renal cells in early embryonic (18,19). This spatial vein. segregation, coupled with the developmental timing of vascu- These findings raise two points. First, early endothelial larization failure in both ephrin-B2–and EphB4-deficient mice, progenitors, identified by recruitment to the vascular cleft and suggests a role for ephrin-B2/EphB4 engagement in the coor- expression of Flk-1, express ephrin-B2, similar to arterial en- J Am Soc Nephrol 12: 2673–2682, 2001 Ephrin-B2 in Renal Glomerular Development 2681 dothelial cells. This arterial “programming” of glomerular en- appears analogous to its expression in periendothelial mesen- dothelium is maintained even in the efferent end of glomerular chymal cells in developing intersomitic vessels at E9.5. In early endothelial capillaries and efferent arteriole (Figure 1A). Sec- extrarenal embryonic angiogenesis, arterial limb endothelial ond, the distinct site at which ephrin-B2–expressing efferent cells express ephrin-B1, ephrin-B2, and EphB3, whereas mes- arterioles interconnect with EphB4-expressing peritubular cap- enchymal cells that surround these vessels express ephrin-B2 illaries remains ill-defined at present. It is noteworthy that the and EphB2 (20). Some (30%) of EphB2/EphB3 double knock- medullary vascular bundle contains two distinct populations of out mice experience embryonic vascularization failure, impli- capillaries, including ephrin-B2–expressing and –nonexpress- cating other EphB receptors in cell-cell interactions necessary ing endothelium. Although a rare cell expresses EphB4 in for proper vascular assembly. By extrapolation, one may ex- mature glomeruli (Figure 5, B and C), this seems most likely to pect that mesangial EphB3 (34) and/or other EphB proteins represent hematopoietic-derived cells (28–30). engage the endothelial ephrin-B2 to direct or modulate endo- We were surprised to find that during renal development, thelial-mesangial cell assembly. ephrin-B2 expression is first prominent in glomerular epithe- We noted prominent expression of ephrin-B2 in VSMC of lial, not endothelial, cells. This expression is restricted to a mature large arteries, in addition to its endothelial expression subpopulation of epithelial cells adjacent to the vascular cleft, (Table 1). This vascular smooth muscle expression is not representing podocyte progenitor cells. Developmental studies restricted to specific developmental stages and continues to in Xenopus demonstrated that ephrin-B1 and ephrin-B2 are maturity. On the basis of evidence that ephrin-B2 and EphB6 expressed in the somites, whereas EphB4 is present on the engagement arrests tumor cell growth (35), ephrin-B2 may posterior cardinal vein and intersomitic vein (31). A recent participate in stabilizing and maintaining the vascular smooth study demonstrated that either ectopic expression of ephrin-B1 muscle layer. Similarly, persistent ephrin-B2 expression in or disruption of EphB4 signaling leads to aberrant projection of glomerular and arterial endothelial cells may be involved in the intersomitic vessels (32). Consistent with these findings, eph- maintenance of endothelial integrity. rin-B2 null mice develop aberrant sprouting of intersomitic Expression of ephrin-B2 in medullary and cortical collecting vessels (33). These findings suggest that sprouting intersomitic ducts and connecting tubules of developing and adult kidneys vessels are guided through migration routes by somitic eph- (Figure 1, C and G) suggests possible roles in developmental rin-B and endothelial EphB engagement (32). By analogy, we tubular/endothelial interactions. By analogy to the glomerular speculate that glomerular epithelial ephrin-B2 may guide en- epithelial/endothelial interactions, tubule epithelial ephrin-B2 dothelial progenitors expressing EphB1 to glomerular sites may direct the spatial organization of peritubular capillaries at (21). these sites in cortex and medulla. Although earlier studies Ephrin-B2 expression was identified in developing glomer- focused on functions for ephrin-B2 and EphB4 in embryonic ular mesangial cells, as defined by ␣SMA expression (data not arterial-venous vascular assembly, spatial- and timing-re- shown). Mesangial expression persisted through the early peri- stricted expression of ephrin-B2 and EphB4 during glomerular natal period but was extinguished in mature glomeruli (Table development implicates these proteins in the integration of 1). This stage-restricted expression of mesangial ephrin-B2 glomerular cell assembly.

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