The ECM protein nephronectin promotes kidney development via integrin alpha8beta1-mediated stimulation of Gdnf expression

Abstract

Development of the metanephric kidney crucially depends on proper interactions between cells and the surrounding extracellular matrix. For example, we showed previously that in the absence of alpha8beta1 integrin, invasion by the ureteric bud into the metanephric mesenchyme is inhibited, resulting in renal agenesis. Here we present genetic evidence that the extracellular matrix protein nephronectin is an essential ligand that engages alpha8beta1 integrin during early kidney development. We show that embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia, which can be traced to a delay in the invasion of the metanephric mesenchyme by the ureteric bud at an early stage of kidney development. Significantly, we detected no defects in extracellular matrix organization in the nascent kidneys of the nephronectin mutants. Instead, we found that Gdnf expression was dramatically reduced in both nephronectin- and alpha8 integrin-null mutants specifically in the metanephric mesenchyme at the time of ureteric bud invasion. We show that this reduction is sufficient to explain the agenesis and hypoplasia observed in both mutants. Interestingly, the reduction in Gdnf expression is transient, and its resumption presumably enables the nephronectin-deficient ureteric buds to invade the metanephric mesenchyme and begin branching. Our results thus place nephronectin and alpha8beta1 integrin in a pathway that regulates Gdnf expression and is essential for kidney development.

Fig. 1. Generation of a Npnt-null allele

(A–F) Targeting strategy for generating Npnt mutant alleles using a BAC containing part of the Npnt locus. (A) Representation of the modified BAC (273P10) used for targeting, showing the first 11 exons (vertical bars) of the Npnt gene present in this BAC. Boxed region spans exons 1 and 2. (B,C) Illustration of the modifications that were made to the BAC DNA, including insertion of loxP sites in the introns 5′ and 3′ to the first exon, an insertion of a neomycin expression cassette flanked by frt sites, and restriction sites (asterisk). (D) Representation of the Npnt^floxneo allele, produced following homologous recombination between the modified BAC and the Npnt locus in ES cells. (E) Mice carrying Npnt^floxneo were crossed to mice expressing CRE recombinase under the β-actin promoter (Lewandoski et al., 1997) to create the Npnt^Δex1 allele. Note that this allele still contains the neo cassette. (F) Southern blot of DNA from two ES cell clones, one heterozygous for the Npnt^floxneo allele and the other wild-type at the Npnt locus. An EcoRI digest produces an 8 kb wild-type and a 4 kb mutant band. Each clone is represented by a series of three fourfold dilutions (left to right). (G) RT-PCR for Npnt and Gapdh expression in Npnt^+/+, Npnt^+/− and Npnt^−/− mice. Total RNA was extracted from spleens of newborn mice using the RNeasy mini kit (Qiagen Inc., Valencia, CA), and reverse transcribed using Superscript II and oligo(dT)_12–18 Primer (Invitrogen Corp., Carlsbad, CA). PCR was performed using forward and reverse primers that recognize sequences in Npnt exons 4 and 8, respectively, and primers that recognize a sequence in Gapdh exon 3. Control reactions without reverse transcriptase were negative for both PCR reactions (not shown). (H) Immunostain for nephronectin in kidneys from wild-type and Npnt^Δex1 homozygous (null) newborn mice using an anti-nephronectin antibody that recognizes sequences in the C-terminal region of the protein (Brandenberger et al., 2001). Primers for wild-type allele, Npnt^WT, NN-1A: 5′-AGTCCATCCTGATCACTGGCT-3′ and NN-1C: 5′-GCAACCTTCAGCGTCCC-3′, band size 279 bp. Primers for mutant allele, Npn^Δtex1, NN-1B: 5′-TATGGCTTCTGAGGCG GAA -AG AAC-3′ and NN-1F: 5′-AAGTGGAGCTTCAGGACACAG-3′, band size 509 bp.

Fig. 2. Renal agenesis in Npnt-null mice

(A–C) Urogenital tracts from newborn female littermates, shown in wholemount. (A) Npnt^+/+ urogenital tract including kidneys with adrenals, ureters, bladder and uterine horns. (B) Npnt^−/− urogenital tract with unilateral kidney agenesis. Note that other than the absence of the right kidney and ureter (asterisk), the urogenital tract appears normal. The adrenal gland on the right is attached to the dorsal mesentery, and part of the dorsal aorta is present. (C) Npnt^−/− urogenital tract with bilateral kidney agenesis (asterisks). Again, the rest of the urogenital tract appears normal. (D,E) Medial sections of Npnt^+/+ and Npnt^−/− newborn kidneys (scale bar, 1 mm). Insets show regions containing glomeruli (arrows) at higher magnification (scale bar, 100 μm), demonstrating that kidney development, including nephron formation, occurs in Npnt^−/− kidneys. (F) Percentage of Npnt heterozygous and homozygous animals with two, one or no kidneys. The percentage agenesis was determined by dividing the number of kidneys [expected (2 per animal) – observed] by the number of kidneys expected. Ad, adrenal gland; Bl, bladder; DA, dorsal arota; Ki, kidney; Ur, ureter; Ut, uterus.

Fig. 3. Developmental origin of renal agenesis in Npnt-null mice

(A–G) Embryos at the stages indicated, immunostained in wholemount for Calbindin. (A) In the Npnt^+/+ embryo at E11.0 the ureteric bud (arrowhead) has invaded the MM. (B) In the Npnt^+/+ embryo the UB (arrowhead) is similar to that in the wild-type embryo. (C) In the Npnt^+/+ embryo at E11.5 the UB has branched (open arrowheads). (D) In the Npnt^−/− embryo the UB (arrowhead) has not extended into the MM (asterisk) or branched. (E) In the Npnt^+/+ embryo at E12.5 the UB has undergone several rounds of branching (arrowheads). (F,G) Npnt^−/− embryos, showing the variable extent of branching at E12.5. (H–J) Transverse sections through E13.5 Npnt^+/+ and Npnt^−/− kidneys. (I,J) Left and right kidneys from one embryo. Note that metanephric fields have been invaded by the UB and nephron development is occurring, but the kidneys are smaller than normal in the Npnt^−/− embryo. Nephrogenesis is occurring, but is less advanced than in the wild-type littermate. (K) Transverse section of an E13.5 Npnt^−/− embryo through the region in which the kidney would normally develop. Note the bilateral kidney agenesis (arrows). (K′) Boxed region in K is shown at higher magnification. Broken line demarcates the MM. Scale bars, 100 µm. Go, gonad; In, intestine; MM, metanephric mesenchyme; UB, ureteric bud.

Fig. 4. The basement membrane is normal in Npnt-null embryos during kidney development

(A–F) Transverse sections through E11.5 Npnt^+/+ and Npnt^−/− embryos stained with antibodies against laminin (LN) or Collagen IV (COL IV). (G–L) Transverse sections through E13.5 Npnt^+/+ and Npnt^−/− embryos stained with antibodies against LN, COL IV or fibronectin (FN). Note the similar staining patterns in mutant and wild-type embryos. Scale bars, 50 µm. ND, nephric duct; UB, ureteric bud.

Fig. 5. Gdnf expression is reduced in the Npnt-null embryonic kidney at E11.5 but is normal at E10.5 and E13.5

(A–P) Transverse sections through Npnt^+/+ and Npnt^−/− embryos. (A–H) Expression at E11.5 of the genes indicated, as detected (A–F) by in situ hybridization or (G,H) by immunostaining. (A,B) Note the apparent absence of Gdnf RNA in the MM of the mutant (demarcated by dotted circles), whereas the level of Gdnf expression appears comparable in the adjacent limb bud (arrows) in mutant and wild-type embryos. (C–F) Note that expression of the Eya1 and Six2 transcription factor genes is similar in mutant and wild-type MM. (G,H) PAX2 protein is detected in both the UB (solid arrowhead) and its branches (open arrowhead), as well as in the MM. Note the lack of invasion of the UB into the MM of the Npnt mutant (asterisk). (I–L) Expression at E10.5 and (M–P) at E13.5 of the genes indicated, as detected by in situ hybridization. Note that Gdnf and Eya1 expression appears comparable in Npnt^+/+ and Npnt^−/− embryos at these stages, although the mutant embryonic kidneys are smaller than normal at E13.5. Scale bars, 100 µm. Abbreviations as in previous figures.

Fig. 6. Gdnf expression is reduced in the Itga8-null embryonic kidney at E11.5 but is normal at E10.5 and E13.5

(A–L) Transverse sections through Itga8^+/− and Itga8^−/− embryos, showing gene expression as detected by in situ hybridization. (A,B) Note the apparent absence at E11.5 of Gdnf RNA in the MM of the Itga8^−/− mutant (demarcated by dotted circles), whereas the level of Gdnf expression appears comparable in the adjacent limb buds (arrows) in Itga8^+/− and Itga8^−/− embryos. (C,D) Pax2 is expressed in both the MM and the UB (arrowhead). Note the lack of UB invasion in the Itga8^−/− MM (asterisk). (E–L) Expression at E10.5 (E–H) and at E13.5 (I–L) of the genes indicated. Note that expression of Gdnf, Pax2 and Six2 is similar in Itga8^+/− and Itga8^−/− MM at these stages. Open arrowheads point to UB branches in the MM at E13.5. Scale bars: 100 µm. Abbreviations as in previous figures.

Fig. 7. Reducing Spry1 dosage in Itga8-null embryos rescues kidney development

(A) Graph illustrating the average number of kidneys per animal at birth in animals of the genotypes indicated. Note that the percent agenesis is significantly reduced in Itga8^−/−;Spry1^+/− versus Itga8^−/−;Spry1^+/+ animals (P<0.005; see legend to Table 1). The rescue of kidney development was complete in Itga8^−/−;Spry1^−/− animals. However, we found the proportion of Itga8^−/−;Spry1^−/− animals that demonstrated a duplicated ureter phenotype did not appear to differ from the proportion of their Spry1^−/− littermates displaying that phenotype (Bason et al., 2005). (B,C) Transverse sections through embryonic kidneys of the genotypes indicated, stained with hematoxylin and eosin. Note the characteristic lack of invasion of the MM by the UB (arrowhead) at E11.5 in an Itga8^−/−;Spry1^+/+ embryonic kidney. The UB has invaded the MM at E11.5 in an Itga8^−/−;Spry1^+/− embryonic kidney. (D-G) Transverse sections through embryonic kidneys of the genotypes indicated, showing expression at E11.5 of Gdnf and Eya1, as detected by in situ hybridization. (D,E) Note the substantial reduction in the level of Gdnf expression in the MM of the Itga8^−/−;Spry1^+/− mutant compared with that in the control (Itga8^+/−;Spry1^+/−) embryo, despite invasion of the UB into the MM (arrowhead). Arrows point to Gdnf expression in the limb buds, which is similar in both genotypes. (F,G) Eya1 expression is similar in Itga8^−/−;Spry1^+/− and control (Itga8^+/−;Spry1^+/−) embryos. Note invasion of the UB into the MM of the Itga8^−/−;Spry1^+/− and control (Itga8^+/−;Spry1^+/−) embryos (arrowheads). Scale bars: 50 µm.