Renal collecting system growth and function depend upon embryonic γ1 laminin expression

Metanephric kidney development commences when the Wolffian duct extends out to form the ureteric bud (UB) under the inductive influence of glial-derived neurotrophic factor (GDNF) (Shakya et al., 2005). The bud undergoes growth and branching to form the collecting ducts and induces the mesenchyme to condense and form epithelial vesicles that give rise to nephrons. A number of factors are involved in the mediating UB differentiation, including heparan sulfates, morphogens or growth factors that include GDNF and fibroblast growth factors (FGF), TGFβ family members and β1 integrins (Bates, 2007; Bullock et al., 1998; Bush et al., 2004; Steer et al., 2004; Wu et al., 2009; Zhang et al., 2009). The kidney basement membranes can be conceived as solid-phase agonists capable of mediating information to cells through interactions of these factors.

Laminins are heterotrimeric glycoproteins, most sharing a γ1 subunit, involved in basement membrane assembly and function (for a review, see Yurchenco, 2011). Inactivation of the Lamc1 gene (γ1 subunit) in mice resulted in a failure of assembly of the embryonic plate basement membrane and Reichert's membrane, and developmental arrest (Smyth et al., 1999). Embryoid bodies, development of which mimics that of the peri-implantation embryo, required laminin for basement membrane assembly and epiblast polarization (Li et al., 2002; Murray and Edgar, 2000). Tissue-specific knockout of the gene in Schwann cells caused a reduction of endoneurial basement membranes, loss of myelination and increased apoptosis (Chen and Strickland, 2003; Yu et al., 2005).

Developing collecting system basement membranes share laminin γ1 and β1 subunits paired with the α1 or α5 subunit (Durbeej et al., 1996; Miner et al., 1997), making them ideal structures in which to define the role of these laminins in the morphogenesis of organs made of polarized epithelial cells. We therefore selectively inactivated the Lamc1 gene in the kidney UB and its collecting duct derivatives by crossing the conditional knockout mouse with a cre mouse under the control of the Hoxb7 promoter (Zhao et al., 2004). Inactivation of the laminin gene was found to cause a severe, yet time-limited, hypomorphic state. In some embryos, the UB failed to form, resulting in renal and ureteral agenesis. In others, the UB grew and branched slowly, resulting in small kidneys followed by hydronephrosis and diabetes insipidus. Exploration with cultured explants and collecting duct (CD) cells suggested that the growth defect results from both a loss of heparan sulfate-tethering of growth factors and reduced β1 integrin interactions acting through integrin, MAP kinase and receptor tyrosine kinase pathways.

The Lamc1 gene was inactivated in the UB by mating mice in which Lamc1 exon-2 is flanked by lox-P sites (Chen and Strickland, 2003) with mice heterozygous for a Hoxb7eGFPCre transgene (Zhao et al., 2004) that express cre recombinase and GFP in the UB from embryonic day (E) 10.5. Mice homozygous for the lox-P flanked (fl, `floxed') allele were maintained in both a C57Bl/6 and C57Bl/6-129SvEvTac backgrounds and the cre-recombinase mice were initially maintained in an FVBN/J background. Laminin-deficient (Lamc1^–/–Hoxb7) and littermate control (Lamc1^+/–Hoxb7 and Lamc1^+/fl) embryos and mice were generated by breeding males expressing the Hoxb7eGFPcre transgene and heterozygous for the laminin floxed allele with Lamc1^fl/fl females. Hoxb7 cre and floxed alleles were identified by PCR performed on genomic DNA from mice tails as described (Chen and Strickland, 2003; Zhao et al., 2004). All animal experiments were conducted according to institutional and national guidelines under an approved protocol.

Kidneys, adrenals, ureters and bladder were excised as a single block from newborn pups. Embryonic urogenital specimens were isolated at E10.5-12.5 (presence of a vaginal plug defined as E0.5) after maternal sacrifice. Whole-mount embryonic kidneys were incubated with primary antibodies (Table 1) at 4°C overnight.

For cryosections, kidneys at E12.5-14.5 were isolated, usually fixed in 3% paraformaldehyde (PFA) for 30 minutes at room temperature, embedded in OCT (Tissue-Tek, Elkhart, IN, USA), sectioned (5 μm thick) with a cryostat (Leica CM 1850) at –20°C and adhered to positively charged slides (Fisher). Sections were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and then stained with primary antibodies. For staining with α6 integrin antibody (GoH3), kidneys were frozen in liquid nitrogen, sectioned, fixed in ice-cold methanol and blocked in 50 mM Tris-HCl containing 90 mM NaCl, 5% goat serum and 0.5% BSA. Detection of bound primary antibodies was accomplished with Alexa Fluor 647 goat anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG or anti-rat IgG secondary antibodies (Molecular Probes) at 1:100 dilution and counterstained with 2 μg/ml of the nuclear stain DAPI (4′-6-diamidino-2-phenylindole). Cover slips were mounted with SlowFade reagent (Molecular Probes, S2828) to inhibit photobleaching.

Embryonic tissues and slides were viewed by indirect immunofluorescence using an inverted microscope (model IX70; Olympus) fitted with an IX-FLA fluorescence attachment and a MicroMax 5-mHz CCD camera (Princeton Instruments) controlled by IP Lab 3.5 (Scanalytics).

For electron microscopy, kidneys were fixed in 0.5% gluteraldehyde and 0.2% tannic acid in PBS for 1 hour, washed with 0.1 M sodium cacodylate buffer, transferred to modified Karnovsky's fixative, post-fixed in 1% osmium tetroxide for 1 hour, dehydrated through ethanol and embedded in Epon/SPURR resin (EM Science). Semi-thin (1 μm) and thin sections (∼90 nm) were cut with a diamond knife on a Leica model EMUC6 ultramicrotome. Semi-thin sections were stained with 1% Methylene Blue in 1% sodium borate and imaged using a Nikon Eclipse50 microscope. Thin sections were stained with saturated uranyl acetate followed by 0.2% lead citrate and imaged with a Philips CM-12 transmission electron microscope fitted with a digital camera (Hamamatsu ORCA-HR) controlled by AMT Image Capture software.

Whole-mount embryonic kidneys were cultured (37°C, 5% CO₂) on Transwell filters (Costar) within wells of a 24-well tissue culture dish containing Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics for up to 3 days. The GFP-positive fluorescent ureteric branches were photographed.

For collecting duct (CD) cell cultures, cells were isolated from Lamc1^fl/fl mice as described (Husted et al., 1988). Kidneys were removed and rinsed in DMEM and Ham's F-12. Papillae were then dissected, minced to fine pieces, placed in 0.1% collagenase in Kreb's buffer for 2.5-3 hours and suspended in DMEM:F12 medium supplemented with 10% FBS and allowed to grow. These original cell populations were transformed with SV40 at passage three. The cells were then treated with adenoCre-GFP virus, cloned (GFP-positive cells) and expanded in culture. Deletion of Lamc1 was verified by PCR genotyping. β1 integrin-deficient CD cells were prepared as described (Zhang et al., 2009). CD cells were cultured in a type I collagen (collagen-I; BD Biosciences) gel (1 mg/ml) supplemented with different concentrations of laminin-111 based on the method described by Chen et al. (Chen et al., 2004). CD cells (1.5×10³) were suspended in 100 μl of collagen-I (1 mg/ml) containing 0.1 mg/ml of laminin-111 and cultured in 96-well dishes. After polymerization for 0.5 hours at 37°C, 100 μl of DMEM containing 20 mM HEPES (pH 7.2) and 10% FBS was added to the gels. CD cells were grown for 6-7 days in the presence of 10% fetal calf serum, serum separated into heparin-binding (HB+) and non-binding (HB–) fractions by heparin-affinity chromatography and serum-free medium containing the following growth factors (GFs): fibroblast growth factor 2 (FGF2, 10 ng/ml), GDNF (100 ng/ml), transforming growth factor-α (TGFα, 1 ng/ml), FGF10 (25 ng/ml), pleotrophin (PTN, 1 μg/ml), heparin-binding epidermal growth factor (HB-EGF, 5 ng/ml), hepatocyte growth factor (HGF, 50 ng/ml), and a mixture of the above factors.

Kidney cell proliferation was measured by injecting 0.2 ml of 20 mM 5-ethynyl-2′-deoxyuridine (EdU) intraperitoneally into pregnant females followed by embryonic harvest three hours later (Zeng et al., 2010). EdU incorporation was detected in frozen sections with the Click-iT EdU Alexa Fluor 594 imaging kit (Invitrogen, Carlsbad, CA, USA) and counterstained with DAPI to detect nuclei.

To measure CD cell proliferation within collagen gels (96-well plates), cells were incubated with 10 μM EdU for one hour, washed, fixed in 3.7% PFA for 15 minutes, permeabilized with 0.1% Triton X-100, reacted with Click-iT Alexa-594 dye-conjugate, and stained with DAPI. The fraction of cells undergoing DNA synthesis was determined following imaging by manual counts of Edu-positive and DAPI-stained nuclei.

For apoptosis, TUNEL was used to detect TdT-mediated dUTP incorporation into DNA ends using the DeadEnd Colorimetric Apoptosis Detection System (Promega). Tissues were permeabilized with proteinase K and post-fixed sections with 4% PFA in PBS. Incorporation of biotinylated nucleotide catalyzed by the TdT enzyme was carried out for 1 hour at 37°C. The incorporated biotinylated nucleotides were stained using streptavidin-conjugated horseradish peroxidase (HRP) with 3,3′-diaminobenzidine (DAB) as chromogen.

CD cells cultured in collagen gels were treated with 0.2 μg/ml bacterial collagenase I (Worthington) followed by centrifugation of the liberated cell clumps. Cells were lysed and extracted with Laemmli solubilizing buffer. Equal protein loads determined by Pierce BCA assay were electrophoresed by SDS-PAGE (6-10% gels, reducing conditions) and subsequently transferred to nitrocellulose membranes. Membranes were blocked in 3% BSA in PBS with 0.1% Tween 20, incubated with primary antibodies followed by appropriate HRP-conjugated secondary antibodies. Immunoreactive bands were detected using enhanced chemiluminescence (Thermo Scientific). Antibodies used were obtained from Cell Signaling (Table 1). The light emitted from the blots were imaged with a BioRad Gel Doc 2000 and analyzed using Quantity One software (BioRad). The numerical data were then subjected to statistical analysis (Student's t-test) with the program SigmaStat (v3.5) integrated into the graphing program SigmaPlot (v10).

Osmolality of urine collected from mice was measured by freezing-point determination using a Micro Osmometer (Advanced model 3MO Plus). For determination of vasopressin response, urine was collected immediately prior to and 3 hours after injection of arginine-vasopressin (200 ng/ml solution, 2 ng/gm body weight).

RNA was isolated and stabilized from kidney with reagents provide by an RNeasy Plus Mini Kit (Qiagen). Real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to quantify transcripts encoding γ1 laminin, FGF7, FGF2, nephronectin, GDNF, RET, FGFR2, perlecan, nidogen 1, type IV collagen, WNT11 and WNT9B in embryonic kidneys, and aquaporin-2 (AQP2) and arginine vasopressin receptor 2 (AVPR2) in adult kidneys by using an ABI 7900 HT Sequence Detection System for measurement of the binding of fluorescence dye SYBR Green to double strand DNAs (DNA Core Facility). In order to normalize variations in mRNA extraction and cDNA synthesis, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured simultaneously. All primer sets were purchased from Qiagen. PCR reactions were set up in 96-well PCR tubes with each tube containing 60 ng mRNA mixed with 12.5 μl of 2× Quantitect SYBR Green RT-PCR Master Mix (Qiagen QuantiFast SYBR Green RT-PCR) and 0.3 μM primers. Triplicate aliquot reactions were carried out for each sample at 50°C for 10 minutes, initial activation at 95°C for 5 minutes followed by 40 cycles of denaturation at 95°C for 10 seconds and combined annealing/extension at 60°C for 30 seconds. A relative quantification method (Applied Biosystems `Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR') was used to compare mRNA expression in which the C_T value (threshold cycle) was calculated (ΔC_T=CT_sample – CT_GRPDH) as the mean of the triplicate samples. The difference between C_T values of samples for each target and mean C_T value of the reference for that target was then determined as ΔΔC_T (where ΔΔC_T=ΔC_T mutant sample – ΔC_T control sample) and used to establish fold difference (2^–ΔΔCt).

Mice heterozygous for a Hoxb7Cre-eGFP transgene and floxed Lamc1 were mated with mice homozygous for the floxed laminin gene. Recombination was verified by PCR as described in Materials and methods (see Fig. S1 in the supplementary material). Postnatal day (P) 0-1 pups with deficient UB laminin (Lamc1^–/–Hoxb7) either possessed small kidneys or lacked both kidneys and ureters with empty bladders when compared with littermate control (Lamc1^+/–Hoxb7 and Lamc1^fl/fl) animals (Fig. 1A-C). Mice without kidneys died shortly after birth. The defects were found in both females and males with no sex organ abnormalities. About one-third of mutant animals had absent kidneys and two-thirds had small kidneys (see Table S1 in the supplementary material).

Eleven laminin-deficient mice were examined for urinary defects starting at about 3 weeks of age when normal mice develop the ability to concentrate urine (Gattone et al., 1999). Eight of these mice (6/6 females and 2/5 males) produced large volumes of a dilute urine. Urine osmolality was noted to be lower in mutant mice compared with controls (Fig. 1D) and it did not respond to arginine-vasopressin like the normal littermates. Immunostaining of the collecting ducts using an antibody to the water channel protein aquaporin 2 revealed that the protein was less apically distributed and/or substantially decreased in overall staining in many of the collecting ducts of the laminin-deficient kidneys (Fig. 1E-G). Reduced expression of both the water channel protein aquaporin 2 and the vasopressin 2 receptor was confirmed in mutant mice relative to their control littermates by quantitative RT-PCR (Fig. 1H). The kidneys and ureters of mutant mice revealed dilated calyces without evidence for obstruction or dilation of the ureters, consistent with non-obstructive hydronephrosis (see Fig. S2 in the supplementary material).

To define the mechanisms of the developmental defects observed in the kidneys, embryos were examined between E10.5 and E12.5 (Fig. 2). In wild-type embryos, the caudal end of the Wolffian duct, detected at E10.5 with antibody to E-cadherin, was bifurcated into its ureteric bud (UB) and common nephric duct branches (Fig. 2A), whereas mutants exhibited either a broadened funnel-shaped tip without bifurcation or a single narrow tip (Fig. 2B,C), suggesting that there was a smaller UB or no UB formed at this stage. GFP fluorescence was observed in the newly formed `T-shaped' UB at the caudal end of the Wolffian duct by E11.5 (Fig. 2D) and remained strong in the developing ureter and ureteric branches in subsequent days. A single bifurcating UB coated with laminin (detected with polyclonal antibody; monoclonal antibody was insufficiently sensitive) was observed in control embryos at this stage (Fig. 2E,I). By contrast, mutant UBs were either small and unbranched with undetectable (Fig. 2F,G,J,K) or absent (Fig. 2H,L) laminin at the ureteric tip. By E12.5, control embryos possessed more than two ureteric branches, whereas null embryos possessed either a single bifurcation or no UB at all with a rudimentary blastema (Fig. 2M-O). The Wolffian duct, more rostral mesonephric tubular branches and Müllerian duct otherwise had similar appearances in control and mutant embryos. From E13.5 onwards, embryos either had kidneys with ureters or lacked kidneys and ureters altogether.

Laminin-deficient and control littermate kidneys were examined at E12.5 by brightfield (semi-thin sections) and electron microscopy (see Fig. S3 in the supplementary material). Whereas littermate control kidneys possessed UBs with more than one order of branching and adjacent nests of condensed mesenchyme, the laminin-deficient kidneys exhibited a single branch without condensed mesenchyme (see Fig. S3A-D in the supplementary material). The ultrastructure of the laminin-deficient kidneys was one of flat disordered epithelia in the ampulla (identified based on its larger lumen and cuboidal cells) with basal as well as apical tight junctions and largely absent or greatly attenuated basement membranes (see Fig. S3E-K in the supplementary material). Some laminin-deficient kidneys lacked any epithelia at all. In these, apoptotic figures were abundant (see Fig. S3L in the supplementary material) as confirmed by TUNEL staining (see Fig. S3M-O in the supplementary material).

To compare basement membrane and other components at E12.5, kidneys were evaluated by immunostaining (Fig. 3). Renal vesicles (not reactive with cytokeratin or E-cadherin antibodies) were observed in control but not in laminin-deficient kidneys. Laminin-111 subunits, γ1 laminin and β1 laminin (not shown) subunits, nidogen 1 and type IV collagen, which were strongly detected in control ureteric bud/collecting duct and renal vesicle epithelial basement membranes, were either not detected or observed at very low levels in the laminin-deficient kidney epithelia. Similarly, the heparan sulfate proteoglycans (HSPGs) perlecan, agrin and type XVIII collagen were considerably reduced in laminin-deficient kidneys. FGF2, which was detected most prominently in the basement membrane zone of the UB branches as well as within mesenchyme, was reduced throughout the mutant kidney. Attempts to detect other heparin-binding growth factors, such as FGF7 and FGF10, were unsuccessful using commercially available antibodies. α6 integrin was low to nearly absent in ureteric branches; however, differences in β1 integrin were not detected. Immunostaining for α- and β-dystroglycan was very low in control and laminin-deficient sections (images not shown). Of note, dystroglycan-null kidneys generated through conditional knockout in the UB had no obvious defect (Jarad et al., 2011). Nephronectin, reported to trigger the release of glial-derived neurotrophic factor by ligating α8β1 integrin to induce penetration of the UB (Brandenberger et al., 2001; Linton et al., 2007), was reduced in laminin-deficient UB-derived basement membranes (Fig. 3R,S).

Components of the developing kidney were also evaluated by real-time quantitative reverse transcriptase (qRT) PCR and plotted as the ratio of laminin-deficient:control mRNA levels (Fig. 4). Lamc1 mRNA was reduced to 28% of control levels. The remaining message might be due to incomplete gene inactivation (trace immunostaining for laminin was noted in the deficient kidneys) and/or to low endogenous mesenchymal expression. Fgf2, but not Fgf7, mRNA was reduced. Nephronectin, but not perlecan, type IV collagen or nidogen 1 (Fig. 4; data not shown) mRNAs were also reduced. Gdnf, its receptor Ret, and the UB factor Wnt11 (but not Wnt9b) were reduced.

Because laminins can diffuse from the site of secretion, it is possible that laminin derived from mesenchyme could assemble on UBs. This phenomenon might explain a gradual increase of laminin and other components detected in the developing collecting system as it matured. Initially, very low levels of punctate and pericellular γ1 laminin subunit were detected in E12.5 mesenchyme (Fig. 3). Furthermore, several laminin-deficient kidneys at stages later than E12.5 (where a laminin-immunostained condensed mesenchyme was present) demonstrated increased laminin staining in the UB, particularly that portion near to the laminin immunostained mesenchyme (Fig. 5). These increases, although below those of controls, were accompanied by increases in other basement membrane components, integrins and FGF2 (data not shown). Such findings support the interpretation that the laminin-deficient ureteric bud had gradually become invested with non-collecting duct laminins. By E14.5, a stage at which collecting ducts (keratin-stained), mesenchymally derived tubules (keratin-negative) and developing glomeruli were present in laminin-deficient and control littermates (Fig. 5B), γ1 laminin was detected in mutant collecting duct basement membranes, but at levels still below those of controls. The γ3 laminin subunit, absent at earlier stages of all kidneys, was found in the glomeruli of laminin-deficient and control kidneys, and to a lesser extent the collecting ducts, of all kidneys (Fig. 5B, inset). By birth, immunostaining intensities for γ1 laminin, laminin-111, perlecan, type IV collagen, α6 integrin and β1 integrin were similar in mutant and control kidneys (Fig. 5, lower panels; data not shown).

To characterize the later progression of changes of collecting duct system growth, GFP fluorescence was examined at E12.5-14.5 and at birth (Fig. 6A-H). Mutant kidneys were small and less branched relative to their control littermates at all developmental stages examined. Comparison of mutant and control branching revealed that the defective kidneys lagged in terminal branch number by about one day of development from E12.5-14.5 (Fig. 6O). When E12.5 kidneys were cultured on filter membranes, mutant kidneys showed a similar delay in UB branching, consistent with the results in vivo (Fig. 6J-N). To determine whether proliferation and/or apoptosis contributed to slow UB growth, E12.5 embryos were labeled with EdU and TUNEL. Proliferation was significantly reduced in the UB branches (72% of control) and mesenchymal cells (67% of control) of laminin-deficient kidneys (Fig. 6Q-S). Increased apoptosis was confined to the blastema in the absence of UB penetration and was almost completely absent in control and small null kidneys by TUNEL assay (see Fig. S3 in the supplementary material).

A consistent defect of laminin-deficient kidneys was the slow growth and reduced collecting duct branching. In order to address possible mechanisms underlying this defect in a relevant system that could provide sufficient cellular material for molecular analysis, laminin-deficient and β1 integrin-deficient CD cells grown within soft collagen I gels were evaluated (Fig. 7). Proliferation was assessed following labeling with EdU (Fig. 7A,B) and cell shape and branching was visualized by phalloidin-staining of F-actin (Fig. 7C). Cell elongation, branching and proliferation were observed to increase together with increasing concentrations of laminin-111. The decrease of HSPGs and loss of FGF2 in the laminin-deficient collecting ducts suggests that one basis for the growth defect was loss of growth factor-tethering by basement membrane heparan sulfates. In support of this possibility, treatment of CD cells with heparin lyases substantially reduced cell growth and branching. β1 integrin-null CD cells also grew and branched poorly. When the serum used to support the cells in culture was depleted of its heparin-binding components by heparin-affinity chromatography, cell proliferation and branching were reduced. This was restored by incubating with the heparin-binding (HB+) serum fraction. Several heparin-binding factors (GDNF, HB-EGF, FGF10, PTN, HGF, TGFα and FGF2) were also found to promote CD growth and branching in the absence of serum (data not shown).

To explore possible signaling contributions arising from growth factors and β1 integrins, CD lysates from laminin-deficient cells were immunoblotted and probed for phosphorylation of potential mediators (Fig. 8). Under the conditions used, perlecan, a basement membrane HSPG, was found to accumulate on CD cords following treatment with heparin lyases or chondroitinase ABC, but not in the absence of laminin as expected (Fig. 8A). Chondroitinase, which had no effect on growth of laminin-treated CD cells and did not alter phosphorylation of FGFR, ERK1/2 (MAPK3/MAPK1 – Mouse Genome Informatics), p38 MAP kinase (MAPK14 – Mouse Genome Informatics), AKT, paxillin or FAK (PTK2 – Mouse Genome Informatics) (data not shown), was used as a control. Reductions of FGFR, Erk1/2 and p38 MAP kinase, paxillin and FAK phosphorylation were observed in CD cells in the absence of added laminin. Following treatment with laminin and heparin lyase I+III, phosphorylation reductions in FGFR and downstream MAPK pathways (Erk1/2 and p38), but not in paxillin and FAK, were detected (Fig. 8B,C). Furthermore, when β1 integrin-null CD cells were grown for 6 days in collagen gels in the presence of laminin-111 and serum, significant decreases of phosphorylation of FAK, paxillin, ERK1/2, p38 and AKT, but only a slight (not significant) decrease of phosphorylation of FGFR1, were observed (see Fig. S4 in the supplementary material).

During metanephric development, different laminin α and β subunits join with the common γ1 subunit to generate different heterotrimers that populate the renal epithelial and vascular basement membranes (Miner et al., 1997). These subunits are important for kidney morphogenesis. For example, inactivation of the Lama5 gene affects glomerulogenesis or causes renal agenesis (Miner and Li, 2000), whereas inactivation of the Lamb2 gene, which is expressed later, affects glomerular filtration (Noakes et al., 1995). In this study, the role of γ1 laminins, which are effectively laminin-111 and laminin-511, was investigated in the context of UB development and function. The findings support the importance of these laminins for basement membrane assembly and reveal roles in collecting duct proliferation, which affects kidney size, and in emergence of postnatal competency to concentrate urine. In about a third of cases the UBs failed to protrude from the Wolffian duct to penetrate the blastema, leading to apoptosis of the renal mesenchyme and involution of the kidney. More frequently, the UB penetrated the blastema but grew and branched slowly. Very low levels of laminin were detected, arising from either a small fraction of UB cells that were not cre-inactivated, a low mesenchymal contribution or both. The bud tips, corresponding to the region found to possess the lowest level of detectable laminin at E11.5, developed a defect of polarization characterized by cuboidal epithelial cells with a disorganized distribution of tight junctions. Slow growth was due to decreased collecting duct proliferation and was accompanied by reduced mesenchymal proliferation. This was accompanied by a delay in formation of the renal vesicles.

A laminin-deficiency state was probably initiated at ∼E10.5 when a defect in morphogenesis was first detected. At E12.5, the laminin-deficient kidneys had very little detectable nidogen 1, perlecan and type IV collagen with reduced α6 integrin subunit and FGF2, components normally found in the basement membrane zone. These changes probably reflect the known requirement of laminin expression for the assembly of other basement membrane structural components. Given unaltered transcription, the non-laminin components are presumably lost through diffusion and degradation. The laminin deficiency probably persists until the mesenchyme (independent of Hoxb7 activation) condenses, expresses laminins and forms the renal vesicle with an accompanying basement membrane (Ekblom, 1981; Ekblom et al., 1980; Klein et al., 1988). By birth, the levels of laminin and associated components could not be distinguished from controls. Although the basis for two fates of agenesis and small kidneys is unclear, it is possible that there is a bilaterally regulated laminin concentration threshold above which UB penetration is enabled.

Basement membrane signals to cells are thought to be mediated through integrins and growth factors, the latter tethered to heparan sulfates and, in some cases, the core protein of perlecan (for reviews, see Iozzo, 2005; Yurchenco and Patton, 2009). As a γ1 laminin-deficiency in the developing collecting system caused an overall deficiency of basement membrane zone components and possible reduction in integrin and growth-factor signaling, the growth and branching defect might have resulted from a loss of these mediators. In support of an integrin role, β1 integrin-deficient kidneys were found to be small with reduced collecting duct branching (Zhang et al., 2009), but not as small as the kidneys examined in this study. β1 integrin-null CD cells exhibited decreased proliferation and branching. In support of a growth factor role, FGF2, a heparin-binding growth factor known to accumulate specifically in developing epithelial basement membranes (Evans et al., 2002), was reduced in the defective kidneys. Although it is unlikely that FGF2 is either the key or only reduced heparin-binding growth factor in developing kidney, it was the only heparin-binding factor that we were able to detect in control kidneys with available reagents. We suggest that a number of heparin-growth factors known to affect collecting duct growth (e.g. the heparin-binding factors and serum components we found to enhance CD proliferation and branching) are similarly reduced in laminin-deficiency because of the loss of heparan-sulfated perlecan, agrin and type XVIII collagen. Furthermore, the conclusion that a dual integrin and growth factor signaling reduction probably occurs with loss of laminin expression was supported by the finding that phosphorylation of FGFR1, mediators of integrin signaling and MAP kinase pathway mediators (common to both pathways) were all reduced in laminin-deficient CD cells.

A number of studies have revealed the importance of heparan sulfates and heparin-binding growth factors in the proliferation, branching and differentiation of the UB (Davies et al., 1995; Steer et al., 2004; Bullock et al., 1998). Targeted ablation of FGFR2 in the developing UB was found to cause aberrant UB branching and reduced proliferation (Zhao et al., 2004), and kidneys null for the heparin-binding growth factor FGF7 were small due to reduced UB growth (Qiao et al., 1999). FGF2, which binds strongly to heparin, accumulates in developing basement membranes (Evans et al., 2002) and was detected at reduced levels in the mutant kidneys. Furthermore, FGF activation of its receptor tyrosine kinase receptors is dependent upon formation of a receptor-FGF-heparan sulfate complex (Schlessinger et al., 2000). Although the in vitro analysis does not preclude a contribution of heparan sulfate arising from cell surfaces rather than from basement membranes, the mouse kidney data argue for a unique basement membrane-HSPG-specific role in this regard. Given the mouse findings, it was thought that the observed effects on the growth of CD cells would depend upon signaling pathways associated with both integrins and receptor tyrosine kinases. This expectation was supported by the observation that laminin deficiency, unlike β1 integrin- and heparan sulfate deficiencies, resulted in significantly reduced phosphorylation of mediators of both β1 integrin (by FAK and paxillin) and the FGF receptor (by auto-phosphorylation), as well as the MAP-kinase pathway, a common pathway downstream of both (for a review, see Rozengurt, 2007).

Other basement membrane zone factors were altered as well. Nephronectin, a protein expressed and secreted by the UB and its branches, was not incorporated into collecting system basement membranes in the absence of γ1 laminin expression. Its mRNA was also reduced. Genetic ablation of the nephronectin gene was found to frequently result in unilateral or bilateral renal agenesis, with evidence that nephronectin normally stimulates GDNF expression by ligating its cognate α8β1 integrin (Brandenberger et al., 2001; Linton et al., 2007). The absence of this component might have contributed to defective UB penetration. It also could be that reduced GDNF caused a partial reduction of nephronectin transcription independent of a failure of nephronectin recruitment into the UB basement membrane. Furthermore, WNT11, a promoter of branching morphogenesis expressed at the tip of the ureteric bud, might be reduced as a consequence of reduced RET (Majumdar et al., 2003). However, the bases for reduced transcription of the RET and nephronectin are unclear.

Mice with small kidneys were often unable to concentrate urine, reflecting a water transport defect resulting from reduced expression of the vasopressin receptor and aquaporin 2 transporter. An increase in the size of the cavity of the renal pelvis, thinning of the medulla and mild dilations of the proximal tubules without evidence of anatomical obstruction accompanied the defect. The histological changes might have resulted from increased outflow volume and pressure. Similar morphological changes were reported to develop in the kidneys of mice with dilute voluminous urine following inactivation of the gene for aquaporin 2 (Verkman, 2006; Yang et al., 2001). Furthermore, a similar urine concentration defect with decreased vasopressin receptor and aquaporin 2 was observed in mice following HoxB7-directed inactivation of the β1 integrin subunit (Wu et al., 2009). These findings suggest that development of a vasopressin-responsive water transport system requires β1 integrin ligation of the basement membrane. However, because in our study laminin and integrin levels were normal by birth, it is likely that the reduced expression resulted from a laminin- and integrin-dependent embryonic alteration of the collecting duct system. This might reflect an early cell fate change.

In conclusion, the findings reveal that a reduction of laminin results in a general failure of basement membrane assembly. The consequences of this structural loss supports a model of the basement membrane acting as a solid-phase agonist through two mechanistic pathways: structurally intrinsic integrin-binding ligands generated by the collecting system and basement membrane heparan sulfate-tethered growth factors provided by either the collecting system or mesenchyme, together acting to affect growth and branching. A requirement for the γ1 laminin subunit for proliferation has also been seen in developing Schwann cells (Yu et al., 2005; Yin et al., 2000). Finally, the evidence points to an embryonic basement membrane-β1 integrin interaction that is required for later maturation of a vasopressin-regulated water transport system.

We thank Carlton M. Bates (Children's Hospital of Pittsburgh of UPMC) for providing the Hoxb7GFP-cre mice for the study and for his helpful suggestions. We also thank Raj Patel (Electron Microscopy Service, Robert W. Johnson Medical School) for preparing EM sections; Evan Cadoff and Peter S. Amenta (Robert Wood Johnson Medical School) for facilitating the analysis of urine osmolality and assistance in review of histology, respectively; and Takako Sasaki (University of Erlangen, Germany), Lydia Sorokin (University of Münster, Germany) and Louis Reichardt (University of California San Francisco) for antibodies.