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Defective Glomerulogenesis in the Absence of Laminin α5 Demonstrates a Developmental Role for the Kidney Glomerular Basement Membrane
Developmental Biology 217, 278 –289 (2000) doi:10.1006/dbio.1999.9546, available online at http://www.idealibrary.com on
Defective Glomerulogenesis in the Absence of Laminin a5 Demonstrates a Developmental Role for the Kidney Glomerular Basement Membrane Jeffrey H. Miner and Cong Li Renal Division, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110
Laminins are major components of all basement membranes. They are a diverse group of a/b/g heterotrimers formed from five a, three b, and three g chains. Laminin a5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, a5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin a chain deposition in which a5 replaces a1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin a5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin a5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells. © 2000 Academic Press Key Words: laminin; basement membrane; development; kidney glomerulus; urogenital abnormalities.
INTRODUCTION Development of the definitive kidney (the metanephros) involves branching morphogenesis and a conversion of mesenchyme to epithelium. In the mouse, metanephric development begins on embryonic day (E) 11 when the ureteric bud, an epithelial outgrowth of the Wolffian duct, invades the metanephric blastema, a collection of mesenchymal cells determined to form renal tissue. Reciprocal signaling between the ureteric bud and the metanephric mesenchyme (reviewed in Bard et al., 1994; Patterson and Dressler, 1994; Sariola and Sainio, 1997; Vainio and Muller, 1997) results in branching and growth of the ureteric bud and condensation of a subset of the mesenchyme into an epithelial ball (Saxen, 1987). This epithelium, referred to as a renal vesicle, undergoes a series of morphological changes that result in the formation of a nephron, a filtration unit consisting of a glomerulus and tubule. The nature of these morphological changes allows a natural subdivision of nephrogenesis into several distinct stages: the commashape stage, the S-shape stage, the capillary loop or cup-
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shape stage, and the maturing glomerulus stage (Saxen, 1987). Along with these changes in epithelial structure, the developing glomerulus becomes vascularized by endothelial cells which first invade the vascular cleft at the S-shape stage (Sorokin and Ekblom, 1992). In addition, mesangial cells, which are smooth muscle-like cells, join the endothelial cells inside the glomerulus and maintain the looped configuration of the glomerular capillaries (Leveen et al., 1994; Soriano, 1994). A major structural and functional component of the glomerular capillaries is the glomerular basement membrane (GBM). Basement membranes are, in general, sheets of extracellular matrix that abut many cell types, including epithelia, endothelia, muscle, fat, and peripheral nerve. They play roles in cell adhesion, proliferation, differentiation, and migration. All basement membranes are composed of laminin, collagen IV, entactin/nidogen, and sulfated proteoglycans (reviewed in Paulsson, 1992; Timpl, 1989, 1996; Timpl and Brown, 1996). The GBM contributes to the glomerular filtration apparatus and contains an atypical assortment of basement membrane protein iso0012-1606/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Kidney Defects in Laminin a5 Mutant Mice
forms (reviewed in Miner, 1998, 1999). The importance of the GBM’s atypical composition is illustrated by the dramatic effects of mutations in GBM component genes: mutation in any one of the collagen a3–a5(IV) chain genes causes Alport syndrome (hereditary nephritis) in humans (Barker et al., 1990; Lemmink et al., 1994; Mochizuki et al., 1994) and a similar disease in dogs (Zheng et al., 1994) and knockout mice (Cosgrove et al., 1996; Miner and Sanes, 1996), and a targeted mutation in the mouse laminin b2 chain gene results in severe proteinuria and death at 3–5 weeks of age (Noakes et al., 1995). In the glomerular capillary loops, two cell types are in intimate contact with the GBM. Endothelial cells lie on the inner face of the GBM and line the capillary lumina; podocytes, also called glomerular or visceral epithelial cells, lie on the opposite face of the GBM within the urinary space. During glomerulogenesis, first the epithelial and then the invading endothelial cells produce their own basement membranes, and these subsequently fuse to form the definitive GBM (Abrahamson, 1985; Sariola et al., 1984). As part of this process, there are transitions in the basement membrane component isoforms that are deposited in the developing GBM (reviewed in Miner, 1998), and these transitions are especially dramatic for laminin. Laminin is a heterotrimeric glycoprotein composed of a, b, and g chains; there are five a, three b, and three g chains that can associate to form at least 12 heterotrimers (Aumailley and Smyth, 1998; Engvall and Wewer, 1996; Iivanainen et al., 1999; Koch et al., 1999; Timpl, 1996). During glomerulogenesis, the nascent GBM initially contains laminins-1 (a1b1g1) and -8 (a4b1g1), and laminin-10 (a5b1g1) joins them at the S-shape stage. By the capillary loop stage, laminin-1 is eliminated from the GBM, and then laminins-9 (a4b2g1) and -11 (a5b2g1) begin to accumulate. At maturity, only components of laminin-11 are detected in the GBM (Miner, 1998, 1999). Whether there is any functional significance to these complex transitions, in terms of a role in glomerular development, has been only partially tested. In knockout mice lacking laminin b2, laminins-9 and -11 are absent, yet a morphologically normal (albeit functionally inadequate) glomerulus forms (Noakes et al., 1995). Previously, we reported lethality at E14 –E17 and defects in neural tube closure, digit septation, and placentation in embryos with a targeted mutation in the laminin a5 chain gene Lama5 (Miner et al., 1998). Here, we have studied the effects of the mutation and the resulting absence of laminins-10 and -11 in the kidney. We found that glomerulogenesis failed in Lama5 2/2 mutants due to disruption of the GBM and aberrant behavior of the associated cells. In addition, a minority of embryos lacked one or both kidneys, indicating a dual role for laminin a5 in kidney development.
MATERIALS AND METHODS Breeding of mice. Lama5 2/2 mice were produced as described (Miner et al., 1998) and maintained on a mixed 129/
279 C57BL/6J background. Timed matings were arranged between heterozygous mice to produce homozygous and control embryos of the desired ages. Noon on the day a vaginal plug was detected was considered E0.5. Homozygotes were identified by the 100% penetrant syndactyly phenotype first evident at E12.5 (Miner et al., 1998). Heterozygotes have never displayed any differences from wild-type mice, so both were used as controls. To obtain E10.75 normal embryos, timed matings were arranged between outbred ICR mice (Harlan, Indianapolis, IN). Metanephric organ culture. Kidneys were removed from E13.5 mutant and littermate control embryos and cultured on a Nuclepore filter in serum-free medium for 6 days as previously described (Rogers et al., 1991). Antibody and lectin reagents. Rat mAbs 8B3 to laminin a1 and 5A2 to laminin b1 (Abrahamson et al., 1989) were gifts from Dale Abrahamson (University of Kansas Medical Center, Kansas City, KS). Rabbit antiserum to laminin a2 (Cheng et al., 1997) was a gift from Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ). Rat mAb 1914 to laminin g1 was purchased from Chemicon International (Temecula, CA). Rabbit antisera to laminins a4 and a5 have been previously described (Miner et al., 1997). Rabbit antibody to mouse entactin was a gift from Albert Chung (University of Pittsburgh, Pittsburgh, PA). Rat mAb MEC 13.3 to platelet endothelial cell adhesion molecule (PECAM) was purchased from Pharmingen (San Diego, CA). Rabbit antiserum sc-192 to Wilms’ tumor protein (WT1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse mAb D33 to desmin was purchased from Dako Corporation (Carpinteria, CA). Cy3- and FITC-conjugated second antibodies were purchased from ICN/ Cappel (Costa Mesa, CA). Horseradish peroxidase-conjugated Dolichos biflorus agglutinin (DBA) lectin was purchased from Sigma Chemical Co. (St. Louis, MO). Histology. For immunohistochemistry, whole embryos were immersed in OCT, frozen in dry ice/ethanol-cooled isopentane, and sectioned frontally at 7 mm on a cryostat. Sections were then sometimes fixed in acetone, ethanol, or 2% paraformaldehyde in PBS. Primary and secondary antibodies were diluted in PBS/1% BSA and applied for 1 h. A denaturation protocol was employed for laminin a4 staining (Miner et al., 1997). Sections were mounted in 90% glycerol/0.13 PBS/1 mg/ml p-phenylenediamine. For conventional histology, embryos were fixed in 10% buffered formalin and embedded in paraffin by standard methods. Four-micrometer sections were cut and stained with hematoxylin and eosin. For lectin histochemistry, cultured metanephroi were fixed in 10% buffered formalin and embedded in paraffin. Four-micrometer sections were cut, deparaffinized, rehydrated, and stained with horseradish peroxidase-conjugated DBA lectin as previously described (Rogers et al., 1993). For semithin and thin sectioning, embryonic kidneys were fixed in 4% paraformaldehyde, 4% glutaraldehyde in 0.1 M cacodylate buffer and processed as described (Noakes et al., 1995). Twomicrometer sections were cut with a glass knife and stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife and stained with lead citrate plus uranyl acetate for transmission electron microscopy. Fluorescent and light microscopic images were captured off a Nikon Eclipse 800 microscope with a Spot 2 cooled color digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) using Spot Software Version 2.1. Images were imported into Adobe PhotoShop 5.0 for final processing and layout.
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RESULTS Expression of Laminin a5 during Metanephrogenesis We are interested in finding roles for laminin a5 in kidney development. Toward this end, we have determined the expression pattern of laminin a5 during metanephrogenesis. Previously, we showed that in postinduction fetal and newborn kidneys, laminin a5 is a component of the ureteric bud basement membrane and is deposited in the developing GBM and tubular basement membrane (TBM) at the S-shape stage of nephrogenesis (Miner et al., 1997). As nephrogenesis proceeds, a5 becomes the exclusive GBM a chain and is found throughout the length of the TBM (Miner et al., 1997; Sorokin et al., 1997). Here, we have immunostained for a5 and for other laminin chains in the developing urogenital region at a much earlier stage, E10.75. This is a point in development when the ureteric bud has only recently branched off the Wolffian duct toward the metanephric mesenchyme, and we wanted to determine which laminins were present in its basement membrane. Antibodies to the Wilms’ tumor protein (WT1) were used to identify the metanephric mesenchyme (Fig. 1A). Laminin a5 was present in basement membranes associated with the Wolffian duct and the recently branched ureteric bud (Fig. 1C). Importantly, both these epithelia are involved in preinduction events that are required for successful metanephrogenesis (Davies and Bard, 1998; Vainio and Muller, 1997). Laminins a1, b1, and g1 colocalized with a5 (Fig. 1B and data not shown), indicating that both laminins-1 and -10 are likely the trimers normally present in these basement membranes.
Sporadic Renal Agenesis in Lama5 2/2 Embryos The presence of laminin a5 in the Wolffian duct and nascent ureteric bud basement membranes suggested that it could play a role in early kidney development. Indeed, dissection or sectioning of Lama5 2/2 embryos at E13.5 to E16.5 revealed an absence of either one or both kidneys in ;20% of cases (Fig. 2). In some cases of renal agenesis the ureter was also absent, but in others the ureter was present (Fig. 2, arrows). Renal agenesis was never observed in control littermates. Most mutant embryos had two kidneys that were smaller than those of controls (Fig. 2). However, occasionally one mutant kidney was significantly smaller than the other (not shown). Together, these results suggest that in the absence of laminin a5 there is a variable inhibition of or defect in metanephric development, with heterogeneous outcomes. This is somewhat reminiscent of the variably penetrant exencephaly in Lama5 2/2 embryos we described previously (Miner et al., 1998) and is consistent with the variable phenotypes observed for other mutations which affect kidney development (Davies and Bard, 1998; Vainio and Muller, 1997). It is worth noting here that the presence or absence of kidneys did not correlate with the occurrence of exencephaly. Based on its expression
FIG. 1. Expression of laminins in Wolffian duct and nascent ureteric bud. Sections through the urogenital region at E10.75 were stained with antibodies to the Wilms’ tumor protein WT1 to identify metanephric mesenchyme (A). The section in A was doubly labeled with an antibody to laminin a1 (B), which stains both the ureteric bud (u) and the Wolffian duct (w). An adjacent section was labeled with an antibody to laminin a5 (C), which also labels the ureteric bud and the Wolffian duct but not the metanephric mesenchyme. Bar, 50 mm.
pattern (Fig. 1), laminin a5 may have a role in branching of the ureter, either off the Wolffian duct or within the metanephric mesenchyme. In addition, laminin-1 may have
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Kidney Defects in Laminin a5 Mutant Mice
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an important role in compensating—in most cases successfully—for the absence of laminin-10.
Defective Ureteric Bud Branching in Cultured Lama5 2/2 Metanephroi The possibility existed that the small kidneys of Lama5 2/2 embryos were the result of extrinsic defects, such as those previously documented in the placenta (Miner et al., 1998). Therefore, we removed metanephroi from mutant and control embryos at E13.5 and cultured them for 6 days in vitro. After being cultured they were fixed, embedded in paraffin, sectioned, and stained with D. biflorus agglutinin, a lectin which specifically binds the ureteric bud and its derivatives. Control metanephroi showed extensive ureteric bud branching and nephrogenesis, while mutants exhibited greatly attenuated branching of the ureteric bud and reduced nephrogenesis (Fig. 3). Thus, Lama5 2/2 kidneys have an intrinsic ureteric bud branching defect. Interestingly, the defect appears to be more severe in vitro than in vivo. This may be related to the harsher, suboptimal conditions of growth in culture versus growth in the natural embryonic environment.
Defective Glomerulogenesis in Lama5 2/2 Kidneys We showed previously that in the developing nephron, a5 is first detectable in the peripheral basement membrane of the comma-shaped figure, and then at the S-shape stage it appears in the basement membranes destined to become part of the GBM and distal TBMs (Miner et al., 1997). Thus, the possibility existed that in the absence of a5, the comma- and S-shaped structures might not form properly. However, examination of kidney sections at E13.5 revealed that primitive nephron structures were present and were indistinguishable from controls (Figs. 4A and 4B). This indicates that a5 is required neither for ureteric bud induction of the metanephric mesenchyme nor for the morphological changes that characterize the early period of nephrogenesis. To study later stage kidneys at high resolution, semithin plastic sections of E16.5 control and mutant kidneys were cut and stained with toluidine blue. Capillary loop stage glomeruli were present in both controls and mutants (Figs. 4C and 4D). However, while control kidneys also contained maturing, vascularized glomeruli (Fig. 4E), such glomeruli
were never observed in mutant kidneys. Instead, mutant kidneys contained aberrant structures not found in normal kidneys (Fig. 4H). Examination of many sections from several different mutant kidneys revealed that the aberrant structures resulted from the failure to complete glomerulogenesis. After the capillary loop stage, the glomerulus normally constricts at the vascular pole to encase the endothelial and mesangial cells that will compose and pattern the glomerular capillaries. In the mutant kidneys, constriction began on schedule, but a group of cells was improperly extruded out of the interior of the glomerulus (Figs. 4F and 4G). These extruded cells remained clustered juxtaposed to the avascular glomerulus, and together these constitute the observed aberrant structures (Fig. 4H). To further investigate this defect, we used cell-typespecific antibodies to identify the cells in the aberrant structures, with the expectation that this would aid in the determination of their origins and the cause of the defect. Frozen sections of E15.5–E16.5 control and mutant kidneys were stained with antibodies to WT1, PECAM, and desmin to label podocytes, endothelial cells, and mesangial cells, respectively. In both control and mutant constricting glomeruli, WT1 was detected in the presumptive podocytes, while the PECAM antibody identified the endothelial cells adjacent to the concave aspect of the “cup-shaped” glomerular epithelium (Figs. 5A and 5B). In later stage control glomeruli, podocytes were observed in a narrow band at the periphery, and endothelial cells lined the interior (Fig. 5C). However, in later stage mutant glomeruli, constriction resulted in extrusion of endothelial cells, leaving an avascular cluster of podocytes (Fig. 5D; see also Fig. 4H). Mesangial cells, which provide structural support for the looped glomerular capillaries, were also displaced. These cells, which can be identified with a desmin antibody, are normally found associated with endothelial cells in the interior of the glomerulus (Fig. 5E). However, in the mutant, they were localized just outside of the constricting glomerulus (Fig. 5F), most likely in association with extruded endothelial cells. Thus, Lama5 2/2 kidneys exhibit a defect in glomerulogenesis leading to avascular glomeruli and displaced endothelial and mesangial cells. To understand more about this defect and how it relates to the absence of laminin a5, we used immunohistochemistry to characterize the basement membranes associated with the developing nephrons. Frozen sections of E15.5–
FIG. 2. Sporadic renal agenesis in Lama5 2/2 embryos. Urogenital organs were dissected out of formaldehyde-fixed control and mutant embryos at the indicated gestational ages. Mutant kidneys that do form are smaller than those of control littermates. Note bilateral (C) and unilateral (F) renal agenesis in mutants. Arrows indicate the presence of ureters despite the absence of kidneys; however, only one ureter is present in C. a, adrenal gland; k, kidney; o, ovary; b, bladder; t, testis. Bar, 1 mm. FIG. 3. Defective ureteric bud branching in cultured Lama5 2/2 metanephroi. Control (A, B) and mutant (C, D) metanephroi were removed from E13.5 embryos and cultured for 6 days on Nuclepore filters. Sections of fixed, paraffin-embedded metanephroi were stained with horseradish peroxidase-labeled DBA lectin, which specifically labels ureteric bud derivatives (arrows). Note the reduced branching of the ureter in the two mutant metanephroi, which were obtained from two different embryos. Bar, 200 mm.
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Kidney Defects in Laminin a5 Mutant Mice
FIG. 4. Defective glomerulogenesis in Lama5 2/2 kidneys. (A and B) Low-power views of control (A) and mutant (B) E13.5 kidneys reveal that nephrogenesis, as indicated by the presence of epithelial structures, is occurring in both. (C–H) High-power views of developing glomeruli in control (C, E) and mutant (D, F, G, H) E16.5 kidneys. Sections in C and D show capillary loop stage glomeruli; no significant differences between control and mutant are apparent. Section in E shows a maturing control glomerulus; such glomeruli were never observed in the mutant. Sections in F and G show consecutive stages of glomerular constriction with
283 E16.5 kidneys were stained with polyclonal antibodies to laminin-1, entactin/nidogen, and type IV collagen (Figs. 5 and 6 and data not shown). Other than the glomerular defects described above, no differences between control and mutant basement membranes were detected with these antibodies. Mutations in basement membrane component genes, whether naturally occurring or experimentally induced, have in many cases been shown to lead to ectopic deposition of related components (Cosgrove et al., 1996; Kashtan and Kim, 1992; Miner and Sanes, 1996; Noakes et al., 1995; Patton et al., 1997, 1999). Such ectopic deposition, sometimes referred to as compensation, may serve to furnish some of the functions of the missing or mutated component. Alternatively, compensation may lead to abnormal physiological responses. To determine the molecular nature of the laminin deposited in mutant basement membranes, we stained control and Lama5 2/2 E15.5–E16.5 kidney sections with chain-specific antibodies to the laminin a1, a2, a4, a5, b1, and g1 chains (Fig. 6 and data not shown). Notable findings in the mutant include: (1) proper disappearance of laminin a1 from the developing GBM at the capillary loop stage (Figs. 6A and 6C); (2) proper appearance of a5 in control GBM, but complete absence of laminin a5 immunoreactivity in the mutant (Figs. 6B and 6D); (3) ectopic accumulation of laminin a2 in medullary collecting duct basement membranes (Fig. 6G); and (4) presence of laminins a4, b1, and g1 in both control and mutant capillary loop stage GBM (Figs. 6I and 6J and data not shown), indicating that laminin-8 may be the major laminin remaining in mutant GBM. That laminin a4 is the only a chain detected in the mutant GBM is highly significant, because a4 is a truncated chain which lacks a short arm. The short arms of laminin have been shown to be involved in polymerization of the laminin network (Cheng et al., 1997), which is presumed to be necessary for formation of a basement membrane (Smyth et al., 1999). Thus, the GBM might not form properly without a laminin containing a full-length a chain. To further investigate the structure of mutant GBM, we used transmission electron microscopy to visualize developing GBMs in E16.5 control and mutant kidneys. In the S-shaped figure, the control and mutant presumptive GBM appeared very similar: the basement membrane underlay the podocytes, and the invading endothelial cells made sporadic contact with it (Figs. 7A and 7B). At this stage of glomerulogenesis, the developing GBM is normally undergoing a developmental switch in laminin a chain deposition—laminin a1 begins to disappear, and laminin a5
evidence of cells being extruded out of the developing glomerulus rather than being encased within. (H) The aberrant structures that are the end products of failed glomerulogenesis. The asterisks mark the clusters of cells extruded from within developing glomeruli. Bars, (B) 100 mm, (H) 25 mm.
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glomerular capillaries (Figs. 7C and 7D). The most straightforward interpretation is that the absence of laminin a5, coupled with the programmed disappearance of a1, results in the absence of a definitive GBM by the late capillary loop stage. Low-power observations with the electron microscope revealed another defect relating to the absence of laminin a5. In constricting glomeruli, the podocytes normally form what is, for the most part, a single-cell layer lying atop the GBM (Fig. 7E). However, the podocytes in mutant glomeruli did not maintain this arrangement, but rather formed a double layer (Fig. 7F). This suggests that podocyte interactions with laminin a5 and/or the GBM may be necessary for maintaining their columnar arrangement within the developing glomerulus. The failure to maintain this arrangement and the inability of the invading endothelial cells to interact with a normal basement membrane are, together, likely responsible for the observed aberrant glomerulogenesis.
DISCUSSION
FIG. 5. Identification of the cells in the aberrant glomerular structures. (A–D) E16.5 control and Lama5 2/2 mutant kidney sections were doubly labeled with antibodies to WT1 to label podocytes (red) and to PECAM to label endothelial cells (green). In constricting glomeruli (A, B), endothelial cells line the interior adjacent to the podocytes. At a later stage (C, D), in the control the podocytes are arranged in a single-cell-layer epithelium adjacent to the glomerular capillaries formed by the endothelial cells. In the mutant, the podocytes are in disarray, and the endothelial cells are in the process of being extruded from within the constricting glomerulus. (E and F) E16.5 control and mutant kidney sections were doubly labeled with antibodies to laminin-1 to label basement membranes (red) and to desmin to label mesangial cells (green). In the control, mesangial cells are scattered in the interior of the constricting glomerulus (cg), but in the mutant they are clustered outside (asterisk). Bar, 50 mm.
accumulates and becomes predominant by the capillary loop stage. As shown above, a1 did disappear in the mutant, but a5 could not replace it (Figs. 6C and 6D). At the ultrastructural level, this was reflected by the absence of a discernible GBM at the late capillary loop stage in the mutant, while the control exhibited a clear, albeit somewhat irregular, GBM separating the podocytes from the
That basement membranes are critical for the function of mature kidneys is a fundamental of renal physiology. However, no previous studies have shown that basement membranes play any role in kidney development in vivo. Kidney development involves branching morphogenesis, mesenchyme to epithelium transitions, complex morphological changes in epithelial structure, and vascularization. All of these processes are accompanied by molecular and structural alterations of basement membranes. Here, we have studied these processes in a mouse mutant lacking the laminin a5 chain, a chain whose pattern of expression predicted that it might have an important role in metanephric development. Indeed, we found two defects which stem from the absence of laminin a5: renal agenesis and aberrant glomerulogenesis. The renal agenesis observed in ;20% of mutant embryos suggests a defect in branching or outgrowth of the ureteric bud from the Wolffian duct. Although we could not determine the mechanism leading to this defect, we showed that both the Wolffian duct and the ureteric bud are normally surrounded by basement membranes which contain a5, most likely as part of laminin-10 (Fig. 1 and data not shown). There are several hypotheses that could explain renal agenesis. Outgrowth of the ureteric bud is initiated by a signal emanating from the metanephric mesenchyme (Davies and Bard, 1998; Vainio and Muller, 1997). The absence of a5 from the mutant Wolffian duct basement membrane may somehow interfere with reception or slow the transduction of this signal. Indeed, laminin-1 and -2 have recently been shown to influence the localization of transmembrane receptors and the underlying cytoskeleton in cultured myotubes (Colognato et al., 1999). Laminin-10 may exhibit a similar activity in vivo, both in the Wolffian duct and in the ureteric bud. Alternatively, the budding and/or outgrowth process may
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Kidney Defects in Laminin a5 Mutant Mice
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FIG. 6. Deposition of laminin chains and entactin in developing control and Lama5 2/2 mutant kidneys. (A–H) Sections from E16.5 kidneys were doubly labeled with antibodies to laminins a1 1 a5 or to laminins a2 1 b1, as indicated. Laminin a1 has been properly eliminated from both control and mutant capillary loop stage GBM (arrows in A, C) and has been replaced by a5 in the control (B) but not in the mutant (D). In the medulla, laminin a2 is normally present only in tubular and not in collecting duct (cd) basement membranes, which all contain laminin b1 (E, F). In the mutant, a2 accumulates abundantly in the collecting duct basement membranes (G, H). This is likely due to the absence of a5, the major collecting duct laminin a chain. (I–L) Sections from E16.5 kidneys were labeled with antibodies to laminin a4 and entactin (En), as indicated. a4 is detected in both control and mutant GBM, suggesting that a4-containing laminins predominate in mutant GBM. Entactin is present in mutant GBM and is also associated with the extruded endothelial and mesangial cells (asterisk). g, developing glomerulus. Bar, 50 mm.
rely on cell/matrix interactions that are impaired by the absence of a5. The ureteric bud must extend some 200 –300 mm to reach the metanephric mesenchyme (Davies and Bard, 1998), and if outgrowth is slowed, this distance may not be covered quickly enough for successful induction of metanephrogenesis. In support of this, mutation of integrin a8 also results in the absence of kidneys, and this was correlated with slowed ureteric bud outgrowth (Muller et al., 1997), suggesting that cell/matrix interactions are critical for proper ureteric bud outgrowth.
On the other hand, the fact that an apparently normal ureter could develop in the absence of a kidney in Lama5 mutants (Fig. 2) suggests a defect in differentiation of the metanephric mesenchyme. However, if it exists, such a defect is most likely secondary to aberrant signaling by the ureteric bud, since the metanephric mesenchyme does not express detectable levels of a5 (Fig. 1C). The presence of small kidneys in the mutant also suggests a branching defect within the developing kidney. This defect was mirrored in metanephric organ culture experi-
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FIG. 7. Ultrastructural analysis of the developing GBM in control and mutant kidneys. (A and B) S-shape stage of nephrogenesis. A somewhat discontinuous basement membrane (arrowheads) underlies the podocytes (p) in both control and mutant. Note endothelial cell (e) processes making contact with the basement membrane in both cases. (C and D) Capillary loop stage glomeruli. The control maintains a basement membrane (arrowheads) underlying the podocytes, but the mutant does not have a structurally sound basement membrane, though some disorganized matrix material is evident (arrow). (E and F) Low-power view of constricting glomeruli. Podocytes (some marked by asterisks) are arranged in an essentially single-cell layer in the control but are in disarray in the mutant. Bars, (D) 1 mm, (F) 10 mm.
ments. Cultured mutant metanephroi routinely exhibited greatly reduced ureteric bud branching compared to control littermate metanephroi (Fig. 3). This suggests that the branching defect is intrinsic to the kidney and indicates a role for laminin a5 and the ureteric bud basement membrane in branching morphogenesis. In a perhaps related phenomenon, chemical and genetic interference with production of proteoglycans, which are found in all basement
membranes, also inhibits branching of the ureteric bud in vitro (Davies et al., 1995; Kispert et al., 1996) and in vivo (Bullock et al., 1998). We did not find defects in the conversion of metanephric mesenchyme to epithelium (in that nephrons were induced and did develop). This is consistent with the fact that laminin a5 is not normally found in the basement membrane initially laid down by the nascent epithelium (Miner
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Kidney Defects in Laminin a5 Mutant Mice
et al., 1997; Sorokin et al., 1997). In contrast, the nascent epithelial basement membrane does contain laminin a1, and antibodies to laminin a1 inhibit the mesenchyme to epithelium transition in vitro (Klein et al., 1988; Sorokin et al., 1992). As far as the nephron is concerned, only at the S-shape stage is there significant accumulation of a5, in both the presumptive glomerular and the tubular basement membranes (Miner et al., 1997). No defects were found in mutant S-shape bodies, but at the capillary loop and later stages, several defects became obvious. First, there was no discernible GBM lying between the podocytes and the endothelial cells. Although with antibodies we could detect components of basement membranes, such as laminin-8 (a4b1g1), type IV collagen, and entactin (Fig. 6 and data not shown), there was no ultrastructurally sound GBM (Fig. 7). This could be related to the fact that there was no fulllength laminin a chain present, and laminin-8 may not, on its own, be able to polymerize into a laminin network capable of contributing to basement membrane formation. Second, in constricting glomeruli, the podocytes were not properly configured (Figs. 5 and 7). Normally at this stage, the podocytes form what is, for the most part, a one cell layer thick epithelium lying atop the GBM (Fig. 7E). However, in the mutant, the podocytes exhibited what appears to be a stratified or pseudostratified epithelial configuration (Fig. 7F). The most straightforward interpretation is that podocytes normally maintain themselves in a single cell layer by adhering to the GBM, with which they are closely associated (Fig. 7C). Because the GBM was disrupted in the mutant (Fig. 7D), the podocytes failed to maintain their columnar epithelial arrangement. The most striking defect in the Lama5 2/2 kidney was the aborted glomerulogenesis. As glomerular constriction progressed, cells were forced out of the interior, leaving a cluster of podocytes adjacent to a group of endothelial and mesangial cells (Figs. 4 and 5). The absence of the GBM may have prevented the endothelial cells from maintaining their position within the glomerulus, due to lack of adhesive interactions, or perhaps the disorder of the podocytes altered the strength or nature of the mechanical forces generated by constriction. In either case, the results suggest that the GBM is crucial for proper vascularization and thus proper formation of glomeruli. Thus, the GBM not only is intimately involved in glomerular filtration, but also has a crucial, unique role in glomerulogenesis. The timing of the disruption of the mutant GBM correlates with the elimination of laminin a1 and the failed accumulation of laminin a5. As discussed above, we were able to detect type IV collagen, entactin/nidogen, and the components of laminin-8 (a4b1g1) in mutant constricting glomeruli (Fig. 6). However, these did not assemble into a proper basement membrane. Normally, laminin-10 (a5b1g1) is present in the GBM to fulfill the presumed requirement of a full-length a chain for laminin network self-assembly (Cheng et al., 1997). It is possible that preventing the elimination of laminin a1 (and thus laminin-1)
287 might rescue the disruption of the GBM. However, that a1 was eliminated on schedule even in the absence of a5 deposition suggests that there is an active process of basement membrane remodeling during this critical stage of glomerular development which is responsible, at least in part, for the a1 to a5 developmental switch. Of all the mutations that have been found to affect mouse kidney development, only a few result in overt defects in glomerulogenesis. These include platelet-derived growth factor-B, platelet-derived growth factor receptor b, and integrin a3 mutations. In both the Pdgfb and the Pdgfrb knockouts, there is a striking absence of mesangial cells in glomeruli, resulting in a single, distended, but otherwise normal capillary loop (Leveen et al., 1994; Soriano, 1994). In the Itga3 knockout, the glomerular capillaries are dilated and less branched, the GBM is disorganized, and podocytes fail to form foot processes (Kreidberg et al., 1996). Importantly, integrin a3 is expressed basally on podocytes at S-shape and later stages (Korhonen et al., 1990; Kreidberg et al., 1996), and it has been shown to be a receptor for laminin-10 and/or -11 (Kikkawa et al., 1998; Tani et al., 1999), both of which contain the a5 chain. That both Itga3 and Lama5 mutations affect the structure of the GBM is consistent with their gene products interacting during glomerulogenesis to (1) organize the GBM, (2) maintain the columnar arrangement of the podocyte epithelium, and (3) induce foot process formation. Another mutation which, like Lama5, affects vascularization of glomeruli, has been described in zebrafish. In cloche zebrafish mutants, endothelial cells throughout the embryo are absent (Stainier et al., 1995). Nevertheless, cloche pronephric glomeruli develop, and the podocytes lay down a GBM and form foot processes (Majumdar and Drummond, 1999). This is analogous to what occurs in mouse metanephric organ culture in vitro, in which essentially normal but avascular glomeruli form (Bernstein et al., 1981). However, it is distinctly different from the aberrant glomerulogenesis we observed in Lama5 2/2 kidneys in vivo. In conclusion, our results allow us to assign novel functions to the GBM: it is necessary for proper configuration of podocytes, for their subsequent differentiation, and for vascularization of glomeruli. Thus, the GBM is not only a major component of the glomerular filtration apparatus, but also a crucial organizer of glomerulogenesis.
ACKNOWLEDGMENTS We thank D. Abrahamson, P. Yurchenco, and A. Chung for gifts of antibodies; S. Rogers for metanephric organ culture and histology; J. Cunningham for help with electron microscopy; M. Nichol, J. Gross, C. Borgmeyer, and S. Weng for care of mice; J. Mudd for technical assistance; and M. Rauchman for comments on the manuscript. This work was supported by a George M. O’Brien Kidney Research Center grant from the NIH (P50 DK45181) to J. H. Miner.
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Koch, M., Olson, P. F., Albus, A., Jin, W., Hunter, D. D., Brunken, W. J., Burgeson, R. E., and Champliaud, M. F. (1999). Characterization and expression of the laminin g3 chain: A novel, nonbasement membrane-associated, laminin chain. J. Cell Biol. 145, 605– 618. Korhonen, M., Ylanne, J., Laitinen, L., and Virtanen, I. (1990). The a1-a6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J. Cell Biol. 111, 1245–1254. Kreidberg, J. A., Donovan, M. J., Goldstein, S. L., Rennke, H., Shepherd, K., Jones, R. C., and Jaenisch, R. (1996). Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development 122, 3537–3547. Lemmink, H. H., Mochizuki, T., van den Heuvel, P. W. J., Schroder, C. H., Barrientos, A., Monnens, L. A. H., van Oost, B. A., Brunner, H. G., Reeders, S. T., and Smeets, H. J. M. (1994). Mutations in the type IV collagen a3 (COL4A3) gene in autosomal recessive Alport syndrome. Hum. Mol. Genet. 3, 1269 –1273. Leveen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E., and Betsholtz, C. (1994). Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 8, 1875–1887. Majumdar, A., and Drummond, I. A. (1999). Podocyte differentiation in the absence of endothelial cells as revealed in the zebrafish avascular mutant, cloche. Dev. Genet. 24, 220 –229. Miner, J. H. (1998). Developmental biology of glomerular basement membrane components. Curr. Opin. Nephrol. Hypertens. 7, 13–19. Miner, J. H. (1999). Renal basement membrane components. Kidney Int. 56, 2016 –2024. Miner, J. H., Cunningham, J., and Sanes, J. R. (1998). Roles for laminin in embryogenesis: Exencephaly, syndactyly, and placentopathy in mice lacking the laminin a5 chain. J. Cell Biol. 143, 1713–1723. Miner, J. H., Patton, B. L., Lentz, S. I., Gilbert, D. J., Snider, W. D., Jenkins, N. A., Copeland, N. G., and Sanes, J. R. (1997). The laminin a chains: Expression, developmental transitions, and chromosomal locations of a1–5, identification of heterotrimeric laminins 8 –11, and cloning of a novel a3 isoform. J. Cell Biol. 137, 685–701. Miner, J. H., and Sanes, J. R. (1996). Molecular and functional defects in kidneys of mice lacking collagen a3(IV): Implications for Alport syndrome. J. Cell Biol. 135, 1403–1413. Mochizuki, T., Lemmink, H. H., Mariyama, M., Antignac, C., Gubler, M.-C., Pirson, Y., Verellen-Dumoulin, C., Chan, B., Schroder, C. H., Smeets, H. J., and Reeders, S. T. (1994). Identification of mutations in the a3(IV) and a4(IV) collagen genes in autosomal recessive Alport syndrome. Nat. Genet. 8, 77– 82. Muller, U., Wang, D., Denda, S., Meneses, J. J., Pedersen, R. A., and Reichardt, L. F. (1997). Integrin a8b1 is critically important for epithelial–mesenchymal interactions during kidney morphogenesis. Cell 88, 603– 613. Noakes, P. G., Miner, J. H., Gautam, M., Cunningham, J. M., Sanes, J. R., and Merlie, J. P. (1995). The renal glomerulus of mice lacking s-laminin/laminin b2: Nephrosis despite molecular compensation by laminin b1. Nat. Genet. 10, 400 – 406. Patterson, L. T., and Dressler, G. R. (1994). The regulation of kidney development: New insights from an old model. Curr. Opin. Genet. Dev. 4, 696 –702. Patton, B. L., Connolly, A. M., Martin, P. T., Cunningham, J. M., Mehta, S., Pestronk, A., Miner, J. H., and Sanes, J. R. (1999).
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Distribution of ten laminin chains in dystrophic, regenerating, and denervated muscles. Neuromuscul. Disord. 9, 423– 433. Patton, B. L., Miner, J. H., Chiu, A. Y., and Sanes, J. R. (1997). Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice. J. Cell Biol. 139, 1507–1521. Paulsson, M. (1992). Basement membrane proteins: Structure, assembly, and cellular interactions. Crit. Rev. Biochem. Mol. Biol. 27, 93–127. Rogers, S. A., Ryan, G., and Hammerman, M. R. (1991). Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro. J. Cell Biol. 113, 1447–1453. Rogers, S. A., Ryan, G., Purchio, A. F., and Hammerman, M. R. (1993). Metanephric transforming growth factor-b1 regulates nephrogenesis in vitro. Am. J. Physiol. 264, F996 –F1002. Sariola, H., and Sainio, K. (1997). The tip-top branching ureter. Curr. Opin. Cell Biol. 9, 877– 884. Sariola, H., Timpl, R., von der Mark, K., Mayne, R., Fitch, J. M., Linsenmayer, T. F., and Ekblom, P. (1984). Dual origin of glomerular basement membrane. Dev. Biol. 101, 86 –96. Saxen, L. (1987). “Organogenesis of the Kidney.” Cambridge Univ. Press, Cambridge, UK. Smyth, N., Vatansever, H. S., Murray, P., Meyer, M., Frie, C., Paulsson, M., and Edgar, D. (1999). Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation. J. Cell Biol. 144, 151–160. Soriano, P. (1994). Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 8, 1888 –1896. Sorokin, L., and Ekblom, P. (1992). Development of tubular and glomerular cells of the kidney. Kidney Int. 41, 657– 664.
289 Sorokin, L. M., Conzelmann, S., Ekblom, P., Battaglia, C., Aumailley, M., and Timpl, R. (1992). Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development. Exp. Cell Res. 201, 137–144. Sorokin, L. M., Pausch, F., Durbeej, M., and Ekblom, P. (1997). Differential expression of five laminin a (1–5) chains in developing and adult mouse kidney. Dev. Dyn. 210, 446 – 462. Stainier, D. Y., Weinstein, B. M., Detrich, H. W., 3rd, Zon, L. I., and Fishman, M. C. (1995). Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development 121, 3141–3150. Tani, T., Lehto, V. P., and Virtanen, I. (1999). Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion. Exp. Cell Res. 248, 115–21. Timpl, R. (1989). Structure and biological activity of basement membrane proteins. Eur. J. Biochem. 180, 487–502. Timpl, R. (1996). Macromolecular organization of basement membranes. Curr. Opin. Cell Biol. 8, 618 – 624. Timpl, R., and Brown, J. C. (1996). Supramolecular assembly of basement membranes. BioEssays 18, 123–132. Vainio, S., and Muller, U. (1997). Inductive tissue interactions, cell signaling, and the control of kidney organogenesis. Cell 90, 975–978. Zheng, K., Thorner, P. S., Marrano, P., Baumal, R., and McInnes, R. R. (1994). Canine X chromosome-linked hereditary nephritis: A genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the a5 chain of collagen type IV. Proc. Natl. Acad. Sci. USA 91, 3989 –3993. Received for publication September 24, 1999 Revised October 26, 1999 Accepted October 26, 1999
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Defective Glomerulogenesis in the Absence of Laminin a5 Demonstrates a Developmental Role for the Kidney Glomerular Basement Membrane Jeffrey H. Miner and Cong Li Renal Division, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110
Laminins are major components of all basement membranes. They are a diverse group of a/b/g heterotrimers formed from five a, three b, and three g chains. Laminin a5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, a5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin a chain deposition in which a5 replaces a1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin a5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin a5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells. © 2000 Academic Press Key Words: laminin; basement membrane; development; kidney glomerulus; urogenital abnormalities.
INTRODUCTION Development of the definitive kidney (the metanephros) involves branching morphogenesis and a conversion of mesenchyme to epithelium. In the mouse, metanephric development begins on embryonic day (E) 11 when the ureteric bud, an epithelial outgrowth of the Wolffian duct, invades the metanephric blastema, a collection of mesenchymal cells determined to form renal tissue. Reciprocal signaling between the ureteric bud and the metanephric mesenchyme (reviewed in Bard et al., 1994; Patterson and Dressler, 1994; Sariola and Sainio, 1997; Vainio and Muller, 1997) results in branching and growth of the ureteric bud and condensation of a subset of the mesenchyme into an epithelial ball (Saxen, 1987). This epithelium, referred to as a renal vesicle, undergoes a series of morphological changes that result in the formation of a nephron, a filtration unit consisting of a glomerulus and tubule. The nature of these morphological changes allows a natural subdivision of nephrogenesis into several distinct stages: the commashape stage, the S-shape stage, the capillary loop or cup-
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shape stage, and the maturing glomerulus stage (Saxen, 1987). Along with these changes in epithelial structure, the developing glomerulus becomes vascularized by endothelial cells which first invade the vascular cleft at the S-shape stage (Sorokin and Ekblom, 1992). In addition, mesangial cells, which are smooth muscle-like cells, join the endothelial cells inside the glomerulus and maintain the looped configuration of the glomerular capillaries (Leveen et al., 1994; Soriano, 1994). A major structural and functional component of the glomerular capillaries is the glomerular basement membrane (GBM). Basement membranes are, in general, sheets of extracellular matrix that abut many cell types, including epithelia, endothelia, muscle, fat, and peripheral nerve. They play roles in cell adhesion, proliferation, differentiation, and migration. All basement membranes are composed of laminin, collagen IV, entactin/nidogen, and sulfated proteoglycans (reviewed in Paulsson, 1992; Timpl, 1989, 1996; Timpl and Brown, 1996). The GBM contributes to the glomerular filtration apparatus and contains an atypical assortment of basement membrane protein iso0012-1606/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Kidney Defects in Laminin a5 Mutant Mice
forms (reviewed in Miner, 1998, 1999). The importance of the GBM’s atypical composition is illustrated by the dramatic effects of mutations in GBM component genes: mutation in any one of the collagen a3–a5(IV) chain genes causes Alport syndrome (hereditary nephritis) in humans (Barker et al., 1990; Lemmink et al., 1994; Mochizuki et al., 1994) and a similar disease in dogs (Zheng et al., 1994) and knockout mice (Cosgrove et al., 1996; Miner and Sanes, 1996), and a targeted mutation in the mouse laminin b2 chain gene results in severe proteinuria and death at 3–5 weeks of age (Noakes et al., 1995). In the glomerular capillary loops, two cell types are in intimate contact with the GBM. Endothelial cells lie on the inner face of the GBM and line the capillary lumina; podocytes, also called glomerular or visceral epithelial cells, lie on the opposite face of the GBM within the urinary space. During glomerulogenesis, first the epithelial and then the invading endothelial cells produce their own basement membranes, and these subsequently fuse to form the definitive GBM (Abrahamson, 1985; Sariola et al., 1984). As part of this process, there are transitions in the basement membrane component isoforms that are deposited in the developing GBM (reviewed in Miner, 1998), and these transitions are especially dramatic for laminin. Laminin is a heterotrimeric glycoprotein composed of a, b, and g chains; there are five a, three b, and three g chains that can associate to form at least 12 heterotrimers (Aumailley and Smyth, 1998; Engvall and Wewer, 1996; Iivanainen et al., 1999; Koch et al., 1999; Timpl, 1996). During glomerulogenesis, the nascent GBM initially contains laminins-1 (a1b1g1) and -8 (a4b1g1), and laminin-10 (a5b1g1) joins them at the S-shape stage. By the capillary loop stage, laminin-1 is eliminated from the GBM, and then laminins-9 (a4b2g1) and -11 (a5b2g1) begin to accumulate. At maturity, only components of laminin-11 are detected in the GBM (Miner, 1998, 1999). Whether there is any functional significance to these complex transitions, in terms of a role in glomerular development, has been only partially tested. In knockout mice lacking laminin b2, laminins-9 and -11 are absent, yet a morphologically normal (albeit functionally inadequate) glomerulus forms (Noakes et al., 1995). Previously, we reported lethality at E14 –E17 and defects in neural tube closure, digit septation, and placentation in embryos with a targeted mutation in the laminin a5 chain gene Lama5 (Miner et al., 1998). Here, we have studied the effects of the mutation and the resulting absence of laminins-10 and -11 in the kidney. We found that glomerulogenesis failed in Lama5 2/2 mutants due to disruption of the GBM and aberrant behavior of the associated cells. In addition, a minority of embryos lacked one or both kidneys, indicating a dual role for laminin a5 in kidney development.
MATERIALS AND METHODS Breeding of mice. Lama5 2/2 mice were produced as described (Miner et al., 1998) and maintained on a mixed 129/
279 C57BL/6J background. Timed matings were arranged between heterozygous mice to produce homozygous and control embryos of the desired ages. Noon on the day a vaginal plug was detected was considered E0.5. Homozygotes were identified by the 100% penetrant syndactyly phenotype first evident at E12.5 (Miner et al., 1998). Heterozygotes have never displayed any differences from wild-type mice, so both were used as controls. To obtain E10.75 normal embryos, timed matings were arranged between outbred ICR mice (Harlan, Indianapolis, IN). Metanephric organ culture. Kidneys were removed from E13.5 mutant and littermate control embryos and cultured on a Nuclepore filter in serum-free medium for 6 days as previously described (Rogers et al., 1991). Antibody and lectin reagents. Rat mAbs 8B3 to laminin a1 and 5A2 to laminin b1 (Abrahamson et al., 1989) were gifts from Dale Abrahamson (University of Kansas Medical Center, Kansas City, KS). Rabbit antiserum to laminin a2 (Cheng et al., 1997) was a gift from Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ). Rat mAb 1914 to laminin g1 was purchased from Chemicon International (Temecula, CA). Rabbit antisera to laminins a4 and a5 have been previously described (Miner et al., 1997). Rabbit antibody to mouse entactin was a gift from Albert Chung (University of Pittsburgh, Pittsburgh, PA). Rat mAb MEC 13.3 to platelet endothelial cell adhesion molecule (PECAM) was purchased from Pharmingen (San Diego, CA). Rabbit antiserum sc-192 to Wilms’ tumor protein (WT1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse mAb D33 to desmin was purchased from Dako Corporation (Carpinteria, CA). Cy3- and FITC-conjugated second antibodies were purchased from ICN/ Cappel (Costa Mesa, CA). Horseradish peroxidase-conjugated Dolichos biflorus agglutinin (DBA) lectin was purchased from Sigma Chemical Co. (St. Louis, MO). Histology. For immunohistochemistry, whole embryos were immersed in OCT, frozen in dry ice/ethanol-cooled isopentane, and sectioned frontally at 7 mm on a cryostat. Sections were then sometimes fixed in acetone, ethanol, or 2% paraformaldehyde in PBS. Primary and secondary antibodies were diluted in PBS/1% BSA and applied for 1 h. A denaturation protocol was employed for laminin a4 staining (Miner et al., 1997). Sections were mounted in 90% glycerol/0.13 PBS/1 mg/ml p-phenylenediamine. For conventional histology, embryos were fixed in 10% buffered formalin and embedded in paraffin by standard methods. Four-micrometer sections were cut and stained with hematoxylin and eosin. For lectin histochemistry, cultured metanephroi were fixed in 10% buffered formalin and embedded in paraffin. Four-micrometer sections were cut, deparaffinized, rehydrated, and stained with horseradish peroxidase-conjugated DBA lectin as previously described (Rogers et al., 1993). For semithin and thin sectioning, embryonic kidneys were fixed in 4% paraformaldehyde, 4% glutaraldehyde in 0.1 M cacodylate buffer and processed as described (Noakes et al., 1995). Twomicrometer sections were cut with a glass knife and stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife and stained with lead citrate plus uranyl acetate for transmission electron microscopy. Fluorescent and light microscopic images were captured off a Nikon Eclipse 800 microscope with a Spot 2 cooled color digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) using Spot Software Version 2.1. Images were imported into Adobe PhotoShop 5.0 for final processing and layout.
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RESULTS Expression of Laminin a5 during Metanephrogenesis We are interested in finding roles for laminin a5 in kidney development. Toward this end, we have determined the expression pattern of laminin a5 during metanephrogenesis. Previously, we showed that in postinduction fetal and newborn kidneys, laminin a5 is a component of the ureteric bud basement membrane and is deposited in the developing GBM and tubular basement membrane (TBM) at the S-shape stage of nephrogenesis (Miner et al., 1997). As nephrogenesis proceeds, a5 becomes the exclusive GBM a chain and is found throughout the length of the TBM (Miner et al., 1997; Sorokin et al., 1997). Here, we have immunostained for a5 and for other laminin chains in the developing urogenital region at a much earlier stage, E10.75. This is a point in development when the ureteric bud has only recently branched off the Wolffian duct toward the metanephric mesenchyme, and we wanted to determine which laminins were present in its basement membrane. Antibodies to the Wilms’ tumor protein (WT1) were used to identify the metanephric mesenchyme (Fig. 1A). Laminin a5 was present in basement membranes associated with the Wolffian duct and the recently branched ureteric bud (Fig. 1C). Importantly, both these epithelia are involved in preinduction events that are required for successful metanephrogenesis (Davies and Bard, 1998; Vainio and Muller, 1997). Laminins a1, b1, and g1 colocalized with a5 (Fig. 1B and data not shown), indicating that both laminins-1 and -10 are likely the trimers normally present in these basement membranes.
Sporadic Renal Agenesis in Lama5 2/2 Embryos The presence of laminin a5 in the Wolffian duct and nascent ureteric bud basement membranes suggested that it could play a role in early kidney development. Indeed, dissection or sectioning of Lama5 2/2 embryos at E13.5 to E16.5 revealed an absence of either one or both kidneys in ;20% of cases (Fig. 2). In some cases of renal agenesis the ureter was also absent, but in others the ureter was present (Fig. 2, arrows). Renal agenesis was never observed in control littermates. Most mutant embryos had two kidneys that were smaller than those of controls (Fig. 2). However, occasionally one mutant kidney was significantly smaller than the other (not shown). Together, these results suggest that in the absence of laminin a5 there is a variable inhibition of or defect in metanephric development, with heterogeneous outcomes. This is somewhat reminiscent of the variably penetrant exencephaly in Lama5 2/2 embryos we described previously (Miner et al., 1998) and is consistent with the variable phenotypes observed for other mutations which affect kidney development (Davies and Bard, 1998; Vainio and Muller, 1997). It is worth noting here that the presence or absence of kidneys did not correlate with the occurrence of exencephaly. Based on its expression
FIG. 1. Expression of laminins in Wolffian duct and nascent ureteric bud. Sections through the urogenital region at E10.75 were stained with antibodies to the Wilms’ tumor protein WT1 to identify metanephric mesenchyme (A). The section in A was doubly labeled with an antibody to laminin a1 (B), which stains both the ureteric bud (u) and the Wolffian duct (w). An adjacent section was labeled with an antibody to laminin a5 (C), which also labels the ureteric bud and the Wolffian duct but not the metanephric mesenchyme. Bar, 50 mm.
pattern (Fig. 1), laminin a5 may have a role in branching of the ureter, either off the Wolffian duct or within the metanephric mesenchyme. In addition, laminin-1 may have
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Kidney Defects in Laminin a5 Mutant Mice
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an important role in compensating—in most cases successfully—for the absence of laminin-10.
Defective Ureteric Bud Branching in Cultured Lama5 2/2 Metanephroi The possibility existed that the small kidneys of Lama5 2/2 embryos were the result of extrinsic defects, such as those previously documented in the placenta (Miner et al., 1998). Therefore, we removed metanephroi from mutant and control embryos at E13.5 and cultured them for 6 days in vitro. After being cultured they were fixed, embedded in paraffin, sectioned, and stained with D. biflorus agglutinin, a lectin which specifically binds the ureteric bud and its derivatives. Control metanephroi showed extensive ureteric bud branching and nephrogenesis, while mutants exhibited greatly attenuated branching of the ureteric bud and reduced nephrogenesis (Fig. 3). Thus, Lama5 2/2 kidneys have an intrinsic ureteric bud branching defect. Interestingly, the defect appears to be more severe in vitro than in vivo. This may be related to the harsher, suboptimal conditions of growth in culture versus growth in the natural embryonic environment.
Defective Glomerulogenesis in Lama5 2/2 Kidneys We showed previously that in the developing nephron, a5 is first detectable in the peripheral basement membrane of the comma-shaped figure, and then at the S-shape stage it appears in the basement membranes destined to become part of the GBM and distal TBMs (Miner et al., 1997). Thus, the possibility existed that in the absence of a5, the comma- and S-shaped structures might not form properly. However, examination of kidney sections at E13.5 revealed that primitive nephron structures were present and were indistinguishable from controls (Figs. 4A and 4B). This indicates that a5 is required neither for ureteric bud induction of the metanephric mesenchyme nor for the morphological changes that characterize the early period of nephrogenesis. To study later stage kidneys at high resolution, semithin plastic sections of E16.5 control and mutant kidneys were cut and stained with toluidine blue. Capillary loop stage glomeruli were present in both controls and mutants (Figs. 4C and 4D). However, while control kidneys also contained maturing, vascularized glomeruli (Fig. 4E), such glomeruli
were never observed in mutant kidneys. Instead, mutant kidneys contained aberrant structures not found in normal kidneys (Fig. 4H). Examination of many sections from several different mutant kidneys revealed that the aberrant structures resulted from the failure to complete glomerulogenesis. After the capillary loop stage, the glomerulus normally constricts at the vascular pole to encase the endothelial and mesangial cells that will compose and pattern the glomerular capillaries. In the mutant kidneys, constriction began on schedule, but a group of cells was improperly extruded out of the interior of the glomerulus (Figs. 4F and 4G). These extruded cells remained clustered juxtaposed to the avascular glomerulus, and together these constitute the observed aberrant structures (Fig. 4H). To further investigate this defect, we used cell-typespecific antibodies to identify the cells in the aberrant structures, with the expectation that this would aid in the determination of their origins and the cause of the defect. Frozen sections of E15.5–E16.5 control and mutant kidneys were stained with antibodies to WT1, PECAM, and desmin to label podocytes, endothelial cells, and mesangial cells, respectively. In both control and mutant constricting glomeruli, WT1 was detected in the presumptive podocytes, while the PECAM antibody identified the endothelial cells adjacent to the concave aspect of the “cup-shaped” glomerular epithelium (Figs. 5A and 5B). In later stage control glomeruli, podocytes were observed in a narrow band at the periphery, and endothelial cells lined the interior (Fig. 5C). However, in later stage mutant glomeruli, constriction resulted in extrusion of endothelial cells, leaving an avascular cluster of podocytes (Fig. 5D; see also Fig. 4H). Mesangial cells, which provide structural support for the looped glomerular capillaries, were also displaced. These cells, which can be identified with a desmin antibody, are normally found associated with endothelial cells in the interior of the glomerulus (Fig. 5E). However, in the mutant, they were localized just outside of the constricting glomerulus (Fig. 5F), most likely in association with extruded endothelial cells. Thus, Lama5 2/2 kidneys exhibit a defect in glomerulogenesis leading to avascular glomeruli and displaced endothelial and mesangial cells. To understand more about this defect and how it relates to the absence of laminin a5, we used immunohistochemistry to characterize the basement membranes associated with the developing nephrons. Frozen sections of E15.5–
FIG. 2. Sporadic renal agenesis in Lama5 2/2 embryos. Urogenital organs were dissected out of formaldehyde-fixed control and mutant embryos at the indicated gestational ages. Mutant kidneys that do form are smaller than those of control littermates. Note bilateral (C) and unilateral (F) renal agenesis in mutants. Arrows indicate the presence of ureters despite the absence of kidneys; however, only one ureter is present in C. a, adrenal gland; k, kidney; o, ovary; b, bladder; t, testis. Bar, 1 mm. FIG. 3. Defective ureteric bud branching in cultured Lama5 2/2 metanephroi. Control (A, B) and mutant (C, D) metanephroi were removed from E13.5 embryos and cultured for 6 days on Nuclepore filters. Sections of fixed, paraffin-embedded metanephroi were stained with horseradish peroxidase-labeled DBA lectin, which specifically labels ureteric bud derivatives (arrows). Note the reduced branching of the ureter in the two mutant metanephroi, which were obtained from two different embryos. Bar, 200 mm.
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Kidney Defects in Laminin a5 Mutant Mice
FIG. 4. Defective glomerulogenesis in Lama5 2/2 kidneys. (A and B) Low-power views of control (A) and mutant (B) E13.5 kidneys reveal that nephrogenesis, as indicated by the presence of epithelial structures, is occurring in both. (C–H) High-power views of developing glomeruli in control (C, E) and mutant (D, F, G, H) E16.5 kidneys. Sections in C and D show capillary loop stage glomeruli; no significant differences between control and mutant are apparent. Section in E shows a maturing control glomerulus; such glomeruli were never observed in the mutant. Sections in F and G show consecutive stages of glomerular constriction with
283 E16.5 kidneys were stained with polyclonal antibodies to laminin-1, entactin/nidogen, and type IV collagen (Figs. 5 and 6 and data not shown). Other than the glomerular defects described above, no differences between control and mutant basement membranes were detected with these antibodies. Mutations in basement membrane component genes, whether naturally occurring or experimentally induced, have in many cases been shown to lead to ectopic deposition of related components (Cosgrove et al., 1996; Kashtan and Kim, 1992; Miner and Sanes, 1996; Noakes et al., 1995; Patton et al., 1997, 1999). Such ectopic deposition, sometimes referred to as compensation, may serve to furnish some of the functions of the missing or mutated component. Alternatively, compensation may lead to abnormal physiological responses. To determine the molecular nature of the laminin deposited in mutant basement membranes, we stained control and Lama5 2/2 E15.5–E16.5 kidney sections with chain-specific antibodies to the laminin a1, a2, a4, a5, b1, and g1 chains (Fig. 6 and data not shown). Notable findings in the mutant include: (1) proper disappearance of laminin a1 from the developing GBM at the capillary loop stage (Figs. 6A and 6C); (2) proper appearance of a5 in control GBM, but complete absence of laminin a5 immunoreactivity in the mutant (Figs. 6B and 6D); (3) ectopic accumulation of laminin a2 in medullary collecting duct basement membranes (Fig. 6G); and (4) presence of laminins a4, b1, and g1 in both control and mutant capillary loop stage GBM (Figs. 6I and 6J and data not shown), indicating that laminin-8 may be the major laminin remaining in mutant GBM. That laminin a4 is the only a chain detected in the mutant GBM is highly significant, because a4 is a truncated chain which lacks a short arm. The short arms of laminin have been shown to be involved in polymerization of the laminin network (Cheng et al., 1997), which is presumed to be necessary for formation of a basement membrane (Smyth et al., 1999). Thus, the GBM might not form properly without a laminin containing a full-length a chain. To further investigate the structure of mutant GBM, we used transmission electron microscopy to visualize developing GBMs in E16.5 control and mutant kidneys. In the S-shaped figure, the control and mutant presumptive GBM appeared very similar: the basement membrane underlay the podocytes, and the invading endothelial cells made sporadic contact with it (Figs. 7A and 7B). At this stage of glomerulogenesis, the developing GBM is normally undergoing a developmental switch in laminin a chain deposition—laminin a1 begins to disappear, and laminin a5
evidence of cells being extruded out of the developing glomerulus rather than being encased within. (H) The aberrant structures that are the end products of failed glomerulogenesis. The asterisks mark the clusters of cells extruded from within developing glomeruli. Bars, (B) 100 mm, (H) 25 mm.
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glomerular capillaries (Figs. 7C and 7D). The most straightforward interpretation is that the absence of laminin a5, coupled with the programmed disappearance of a1, results in the absence of a definitive GBM by the late capillary loop stage. Low-power observations with the electron microscope revealed another defect relating to the absence of laminin a5. In constricting glomeruli, the podocytes normally form what is, for the most part, a single-cell layer lying atop the GBM (Fig. 7E). However, the podocytes in mutant glomeruli did not maintain this arrangement, but rather formed a double layer (Fig. 7F). This suggests that podocyte interactions with laminin a5 and/or the GBM may be necessary for maintaining their columnar arrangement within the developing glomerulus. The failure to maintain this arrangement and the inability of the invading endothelial cells to interact with a normal basement membrane are, together, likely responsible for the observed aberrant glomerulogenesis.
DISCUSSION
FIG. 5. Identification of the cells in the aberrant glomerular structures. (A–D) E16.5 control and Lama5 2/2 mutant kidney sections were doubly labeled with antibodies to WT1 to label podocytes (red) and to PECAM to label endothelial cells (green). In constricting glomeruli (A, B), endothelial cells line the interior adjacent to the podocytes. At a later stage (C, D), in the control the podocytes are arranged in a single-cell-layer epithelium adjacent to the glomerular capillaries formed by the endothelial cells. In the mutant, the podocytes are in disarray, and the endothelial cells are in the process of being extruded from within the constricting glomerulus. (E and F) E16.5 control and mutant kidney sections were doubly labeled with antibodies to laminin-1 to label basement membranes (red) and to desmin to label mesangial cells (green). In the control, mesangial cells are scattered in the interior of the constricting glomerulus (cg), but in the mutant they are clustered outside (asterisk). Bar, 50 mm.
accumulates and becomes predominant by the capillary loop stage. As shown above, a1 did disappear in the mutant, but a5 could not replace it (Figs. 6C and 6D). At the ultrastructural level, this was reflected by the absence of a discernible GBM at the late capillary loop stage in the mutant, while the control exhibited a clear, albeit somewhat irregular, GBM separating the podocytes from the
That basement membranes are critical for the function of mature kidneys is a fundamental of renal physiology. However, no previous studies have shown that basement membranes play any role in kidney development in vivo. Kidney development involves branching morphogenesis, mesenchyme to epithelium transitions, complex morphological changes in epithelial structure, and vascularization. All of these processes are accompanied by molecular and structural alterations of basement membranes. Here, we have studied these processes in a mouse mutant lacking the laminin a5 chain, a chain whose pattern of expression predicted that it might have an important role in metanephric development. Indeed, we found two defects which stem from the absence of laminin a5: renal agenesis and aberrant glomerulogenesis. The renal agenesis observed in ;20% of mutant embryos suggests a defect in branching or outgrowth of the ureteric bud from the Wolffian duct. Although we could not determine the mechanism leading to this defect, we showed that both the Wolffian duct and the ureteric bud are normally surrounded by basement membranes which contain a5, most likely as part of laminin-10 (Fig. 1 and data not shown). There are several hypotheses that could explain renal agenesis. Outgrowth of the ureteric bud is initiated by a signal emanating from the metanephric mesenchyme (Davies and Bard, 1998; Vainio and Muller, 1997). The absence of a5 from the mutant Wolffian duct basement membrane may somehow interfere with reception or slow the transduction of this signal. Indeed, laminin-1 and -2 have recently been shown to influence the localization of transmembrane receptors and the underlying cytoskeleton in cultured myotubes (Colognato et al., 1999). Laminin-10 may exhibit a similar activity in vivo, both in the Wolffian duct and in the ureteric bud. Alternatively, the budding and/or outgrowth process may
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Kidney Defects in Laminin a5 Mutant Mice
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FIG. 6. Deposition of laminin chains and entactin in developing control and Lama5 2/2 mutant kidneys. (A–H) Sections from E16.5 kidneys were doubly labeled with antibodies to laminins a1 1 a5 or to laminins a2 1 b1, as indicated. Laminin a1 has been properly eliminated from both control and mutant capillary loop stage GBM (arrows in A, C) and has been replaced by a5 in the control (B) but not in the mutant (D). In the medulla, laminin a2 is normally present only in tubular and not in collecting duct (cd) basement membranes, which all contain laminin b1 (E, F). In the mutant, a2 accumulates abundantly in the collecting duct basement membranes (G, H). This is likely due to the absence of a5, the major collecting duct laminin a chain. (I–L) Sections from E16.5 kidneys were labeled with antibodies to laminin a4 and entactin (En), as indicated. a4 is detected in both control and mutant GBM, suggesting that a4-containing laminins predominate in mutant GBM. Entactin is present in mutant GBM and is also associated with the extruded endothelial and mesangial cells (asterisk). g, developing glomerulus. Bar, 50 mm.
rely on cell/matrix interactions that are impaired by the absence of a5. The ureteric bud must extend some 200 –300 mm to reach the metanephric mesenchyme (Davies and Bard, 1998), and if outgrowth is slowed, this distance may not be covered quickly enough for successful induction of metanephrogenesis. In support of this, mutation of integrin a8 also results in the absence of kidneys, and this was correlated with slowed ureteric bud outgrowth (Muller et al., 1997), suggesting that cell/matrix interactions are critical for proper ureteric bud outgrowth.
On the other hand, the fact that an apparently normal ureter could develop in the absence of a kidney in Lama5 mutants (Fig. 2) suggests a defect in differentiation of the metanephric mesenchyme. However, if it exists, such a defect is most likely secondary to aberrant signaling by the ureteric bud, since the metanephric mesenchyme does not express detectable levels of a5 (Fig. 1C). The presence of small kidneys in the mutant also suggests a branching defect within the developing kidney. This defect was mirrored in metanephric organ culture experi-
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FIG. 7. Ultrastructural analysis of the developing GBM in control and mutant kidneys. (A and B) S-shape stage of nephrogenesis. A somewhat discontinuous basement membrane (arrowheads) underlies the podocytes (p) in both control and mutant. Note endothelial cell (e) processes making contact with the basement membrane in both cases. (C and D) Capillary loop stage glomeruli. The control maintains a basement membrane (arrowheads) underlying the podocytes, but the mutant does not have a structurally sound basement membrane, though some disorganized matrix material is evident (arrow). (E and F) Low-power view of constricting glomeruli. Podocytes (some marked by asterisks) are arranged in an essentially single-cell layer in the control but are in disarray in the mutant. Bars, (D) 1 mm, (F) 10 mm.
ments. Cultured mutant metanephroi routinely exhibited greatly reduced ureteric bud branching compared to control littermate metanephroi (Fig. 3). This suggests that the branching defect is intrinsic to the kidney and indicates a role for laminin a5 and the ureteric bud basement membrane in branching morphogenesis. In a perhaps related phenomenon, chemical and genetic interference with production of proteoglycans, which are found in all basement
membranes, also inhibits branching of the ureteric bud in vitro (Davies et al., 1995; Kispert et al., 1996) and in vivo (Bullock et al., 1998). We did not find defects in the conversion of metanephric mesenchyme to epithelium (in that nephrons were induced and did develop). This is consistent with the fact that laminin a5 is not normally found in the basement membrane initially laid down by the nascent epithelium (Miner
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Kidney Defects in Laminin a5 Mutant Mice
et al., 1997; Sorokin et al., 1997). In contrast, the nascent epithelial basement membrane does contain laminin a1, and antibodies to laminin a1 inhibit the mesenchyme to epithelium transition in vitro (Klein et al., 1988; Sorokin et al., 1992). As far as the nephron is concerned, only at the S-shape stage is there significant accumulation of a5, in both the presumptive glomerular and the tubular basement membranes (Miner et al., 1997). No defects were found in mutant S-shape bodies, but at the capillary loop and later stages, several defects became obvious. First, there was no discernible GBM lying between the podocytes and the endothelial cells. Although with antibodies we could detect components of basement membranes, such as laminin-8 (a4b1g1), type IV collagen, and entactin (Fig. 6 and data not shown), there was no ultrastructurally sound GBM (Fig. 7). This could be related to the fact that there was no fulllength laminin a chain present, and laminin-8 may not, on its own, be able to polymerize into a laminin network capable of contributing to basement membrane formation. Second, in constricting glomeruli, the podocytes were not properly configured (Figs. 5 and 7). Normally at this stage, the podocytes form what is, for the most part, a one cell layer thick epithelium lying atop the GBM (Fig. 7E). However, in the mutant, the podocytes exhibited what appears to be a stratified or pseudostratified epithelial configuration (Fig. 7F). The most straightforward interpretation is that podocytes normally maintain themselves in a single cell layer by adhering to the GBM, with which they are closely associated (Fig. 7C). Because the GBM was disrupted in the mutant (Fig. 7D), the podocytes failed to maintain their columnar epithelial arrangement. The most striking defect in the Lama5 2/2 kidney was the aborted glomerulogenesis. As glomerular constriction progressed, cells were forced out of the interior, leaving a cluster of podocytes adjacent to a group of endothelial and mesangial cells (Figs. 4 and 5). The absence of the GBM may have prevented the endothelial cells from maintaining their position within the glomerulus, due to lack of adhesive interactions, or perhaps the disorder of the podocytes altered the strength or nature of the mechanical forces generated by constriction. In either case, the results suggest that the GBM is crucial for proper vascularization and thus proper formation of glomeruli. Thus, the GBM not only is intimately involved in glomerular filtration, but also has a crucial, unique role in glomerulogenesis. The timing of the disruption of the mutant GBM correlates with the elimination of laminin a1 and the failed accumulation of laminin a5. As discussed above, we were able to detect type IV collagen, entactin/nidogen, and the components of laminin-8 (a4b1g1) in mutant constricting glomeruli (Fig. 6). However, these did not assemble into a proper basement membrane. Normally, laminin-10 (a5b1g1) is present in the GBM to fulfill the presumed requirement of a full-length a chain for laminin network self-assembly (Cheng et al., 1997). It is possible that preventing the elimination of laminin a1 (and thus laminin-1)
287 might rescue the disruption of the GBM. However, that a1 was eliminated on schedule even in the absence of a5 deposition suggests that there is an active process of basement membrane remodeling during this critical stage of glomerular development which is responsible, at least in part, for the a1 to a5 developmental switch. Of all the mutations that have been found to affect mouse kidney development, only a few result in overt defects in glomerulogenesis. These include platelet-derived growth factor-B, platelet-derived growth factor receptor b, and integrin a3 mutations. In both the Pdgfb and the Pdgfrb knockouts, there is a striking absence of mesangial cells in glomeruli, resulting in a single, distended, but otherwise normal capillary loop (Leveen et al., 1994; Soriano, 1994). In the Itga3 knockout, the glomerular capillaries are dilated and less branched, the GBM is disorganized, and podocytes fail to form foot processes (Kreidberg et al., 1996). Importantly, integrin a3 is expressed basally on podocytes at S-shape and later stages (Korhonen et al., 1990; Kreidberg et al., 1996), and it has been shown to be a receptor for laminin-10 and/or -11 (Kikkawa et al., 1998; Tani et al., 1999), both of which contain the a5 chain. That both Itga3 and Lama5 mutations affect the structure of the GBM is consistent with their gene products interacting during glomerulogenesis to (1) organize the GBM, (2) maintain the columnar arrangement of the podocyte epithelium, and (3) induce foot process formation. Another mutation which, like Lama5, affects vascularization of glomeruli, has been described in zebrafish. In cloche zebrafish mutants, endothelial cells throughout the embryo are absent (Stainier et al., 1995). Nevertheless, cloche pronephric glomeruli develop, and the podocytes lay down a GBM and form foot processes (Majumdar and Drummond, 1999). This is analogous to what occurs in mouse metanephric organ culture in vitro, in which essentially normal but avascular glomeruli form (Bernstein et al., 1981). However, it is distinctly different from the aberrant glomerulogenesis we observed in Lama5 2/2 kidneys in vivo. In conclusion, our results allow us to assign novel functions to the GBM: it is necessary for proper configuration of podocytes, for their subsequent differentiation, and for vascularization of glomeruli. Thus, the GBM is not only a major component of the glomerular filtration apparatus, but also a crucial organizer of glomerulogenesis.
ACKNOWLEDGMENTS We thank D. Abrahamson, P. Yurchenco, and A. Chung for gifts of antibodies; S. Rogers for metanephric organ culture and histology; J. Cunningham for help with electron microscopy; M. Nichol, J. Gross, C. Borgmeyer, and S. Weng for care of mice; J. Mudd for technical assistance; and M. Rauchman for comments on the manuscript. This work was supported by a George M. O’Brien Kidney Research Center grant from the NIH (P50 DK45181) to J. H. Miner.
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Koch, M., Olson, P. F., Albus, A., Jin, W., Hunter, D. D., Brunken, W. J., Burgeson, R. E., and Champliaud, M. F. (1999). Characterization and expression of the laminin g3 chain: A novel, nonbasement membrane-associated, laminin chain. J. Cell Biol. 145, 605– 618. Korhonen, M., Ylanne, J., Laitinen, L., and Virtanen, I. (1990). The a1-a6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J. Cell Biol. 111, 1245–1254. Kreidberg, J. A., Donovan, M. J., Goldstein, S. L., Rennke, H., Shepherd, K., Jones, R. C., and Jaenisch, R. (1996). Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development 122, 3537–3547. Lemmink, H. H., Mochizuki, T., van den Heuvel, P. W. J., Schroder, C. H., Barrientos, A., Monnens, L. A. H., van Oost, B. A., Brunner, H. G., Reeders, S. T., and Smeets, H. J. M. (1994). Mutations in the type IV collagen a3 (COL4A3) gene in autosomal recessive Alport syndrome. Hum. Mol. Genet. 3, 1269 –1273. Leveen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E., and Betsholtz, C. (1994). Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 8, 1875–1887. Majumdar, A., and Drummond, I. A. (1999). Podocyte differentiation in the absence of endothelial cells as revealed in the zebrafish avascular mutant, cloche. Dev. Genet. 24, 220 –229. Miner, J. H. (1998). Developmental biology of glomerular basement membrane components. Curr. Opin. Nephrol. Hypertens. 7, 13–19. Miner, J. H. (1999). Renal basement membrane components. Kidney Int. 56, 2016 –2024. Miner, J. H., Cunningham, J., and Sanes, J. R. (1998). Roles for laminin in embryogenesis: Exencephaly, syndactyly, and placentopathy in mice lacking the laminin a5 chain. J. Cell Biol. 143, 1713–1723. Miner, J. H., Patton, B. L., Lentz, S. I., Gilbert, D. J., Snider, W. D., Jenkins, N. A., Copeland, N. G., and Sanes, J. R. (1997). The laminin a chains: Expression, developmental transitions, and chromosomal locations of a1–5, identification of heterotrimeric laminins 8 –11, and cloning of a novel a3 isoform. J. Cell Biol. 137, 685–701. Miner, J. H., and Sanes, J. R. (1996). Molecular and functional defects in kidneys of mice lacking collagen a3(IV): Implications for Alport syndrome. J. Cell Biol. 135, 1403–1413. Mochizuki, T., Lemmink, H. H., Mariyama, M., Antignac, C., Gubler, M.-C., Pirson, Y., Verellen-Dumoulin, C., Chan, B., Schroder, C. H., Smeets, H. J., and Reeders, S. T. (1994). Identification of mutations in the a3(IV) and a4(IV) collagen genes in autosomal recessive Alport syndrome. Nat. Genet. 8, 77– 82. Muller, U., Wang, D., Denda, S., Meneses, J. J., Pedersen, R. A., and Reichardt, L. F. (1997). Integrin a8b1 is critically important for epithelial–mesenchymal interactions during kidney morphogenesis. Cell 88, 603– 613. Noakes, P. G., Miner, J. H., Gautam, M., Cunningham, J. M., Sanes, J. R., and Merlie, J. P. (1995). The renal glomerulus of mice lacking s-laminin/laminin b2: Nephrosis despite molecular compensation by laminin b1. Nat. Genet. 10, 400 – 406. Patterson, L. T., and Dressler, G. R. (1994). The regulation of kidney development: New insights from an old model. Curr. Opin. Genet. Dev. 4, 696 –702. Patton, B. L., Connolly, A. M., Martin, P. T., Cunningham, J. M., Mehta, S., Pestronk, A., Miner, J. H., and Sanes, J. R. (1999).
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Kidney Defects in Laminin a5 Mutant Mice
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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.