Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome

Abstract

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Figure 1

Gene targeting of the murine Gpc3 locus. (A) A 6.5-kb EcoRI fragment of the murine Gpc3 locus is shown (top). The SmaI fragment containing the promoter, the transcription start site (arrow), exon 1, and part of intron 1 is indicated. An EcoRI/Bg1II fragment containing the neo cassette, a neomycin gene with a PGK promoter, driving expression in the antisense direction, is shown in the middle. The mutated locus with the neo cassette replacing the SmaI fragment is shown below. Restriction enzyme sites are as follows: E, EcoRI; K, KpnI; S, SmaI. The probe (S1) hybridized to the 6.5-kb wild-type and 7.5-kb targeted allele fragments, as expected. PCR primer sites and orientation within the native and disrupted loci are indicated: a, PCRL3; b, PCR5; c, PCR2; d, PCRL2; and e, neo. (B) Southern blot analysis of EcoRI-digested genomic DNA extracted from tail biopsies of wild-type, heterozygous, and hemizygous mutant offspring (+/+, +/−, and −/, respectively). (C) Top, Northern blot analysis of GPC3 in wild-type (+/+) and (−/) E18.5 embryos. 10 μg total RNA was probed with a 2.2-kb full-length Gpc3 cDNA. Bottom, ethidium bromide staining demonstrates equal loading.

Figure 2

Histological analysis of kidney sections. Kidney sections from 4-d-old wild-type (top) and GPC3-deficient (bottom) littermates were fixed and sections stained with hematoxylin and eosin.

Figure 3

Histological analysis of wild-type (+/+) and GPC3 deficient (−/) lungs. Hematoxylin and eosin stained sections of lungs at E18, P0, and P5 are shown. Accumulation of mucus (arrowhead) and cellular debris (arrow) is indicated. Original magnification is 400.

Figure 4

Mandibular hypoplasia in Gpc3 −/ embryos. Alizarin red/Alcian blue skeletal preparations of wild-type (top) and Gpc3 −/ (bottom) E18.5 embryonic heads are shown. Note the complete lack of mandible in the mutant embryo.

Figure 5

Embryonic growth kinetics. Wild-type, heterozygous, and knockout embryos were weighed at the indicated embryonic day and at birth. Bars represent the average embryo wt + 2 SEM. Total number of embryos weighed for each group is indicated above each bar. Statistical analysis using a t test showed significant differences (P < 0.05) between mean weight of the three genotypes at each time point analyzed, with the exception of wild-type and heterozygous at P0, and heterozygous and knockout at E12.5 and E18.5.

Figure 6

Comparative analysis of IGF-II levels in serum, whole embryo, lung, liver, and kidney. (A) Circulating levels of IGF-II at different time points during development. (B) Levels of IGF-II in whole embryos at E12.5 and E14.5. Each bar represents the mean + SEM obtained by densitometric scanning of four pooled samples done in duplicate. Inset shows a representative image of the Western blot. (C) IGF-II mRNA levels were measured by Northern blot analysis and normalized with the intensity of the corresponding GAPDH band. Each bar represents the mean + SEM obtained by densitometric scanning of two different samples.

Figure 7

Kidney development in the Gpc3 −/ mouse. Paraffin-embedded tissue sections were generated from fixed tissue isolated from +/+ and −/ mice. E12.0 sections were imaged at 200×. Bar, 37.5 μm. E13.5, E16.5, and E18.5 sections were imaged at 100×. Bar, 75 μm. At E12.0, branching of the ureteric bud is markedly enhanced in the −/ versus +/+ embryo (arrowheads). By E13.5, the −/ kidney is much larger than that of its +/+ littermate and consists of histologically normal ureteric bud and mesenchymal-derived tissue elements. While these mesenchymal-derived elements (glomeruli and tubules) are present in the cortex (C) of the E16.5 −/ and +/+ kidneys, they are disorganized in the −/ cortex and the −/ medulla (M) is relatively devoid of tubular structures. Cysts (Cy) are present in −/ medulla in an irregular pattern. At E18.5, these abnormalities persist in the −/ kidney and are accompanied by total absence of the medullary tubular patterning characteristic of the +/+ kidney.

Figure 8

Analysis of cell proliferation in the renal collecting system of the GPC3-deficient mice. Incorporation of BrdU into cells of the ureteric buds or collecting ducts, identified by fluorescein-conjugated DBA, was detected in tissue sections of embryonic kidneys using an anti-BrdU peroxidase-conjugated antibody. (A) Bright-field (BF) and fluorescence (IF) images of representative kidney sections from Gpc3 +/+ and Gpc3 −/ mice at E12.5 and E16.5. The arrows mark the position of typical ureteric buds or collecting ducts, identified by fluorescein-DBA. BrdU-labeled cells (brown) are present in both the collecting system and in tissue elements derived from the metanephric blastema, and are more numerous in the cells of the collecting system of the Gpc3 −/ mice. Bar, 18.7 μm (400×). (B) Quantification of cell proliferation in the ureteric buds or collecting ducts in the kidneys of Gpc3 +/+ and Gpc3 −/ mice at E12.5, E13.5, and E16.5. Percent proliferation was calculated as the ratio of BrdU-labeled cells to the total number of ureteric bud or collecting duct cells analyzed. Bars represent the mean + SEM.