Volume 7, Issue 2, Pages 343-354 (February 2001) The Two PDGF Receptors Maintain Conserved Signaling In Vivo despite Divergent Embryological Functions  Richard A Klinghoffer, Peter F Mueting-Nelsen, Alexander Faerman, Moshe Shani, Philippe Soriano  Molecular Cell  Volume 7, Issue 2, Pages 343-354 (February 2001) DOI: 10.1016/S1097-2765(01)00182-4

Figure 1 Targeted Knockins of PDGFR cDNAs at the PDGFαR Locus and at the PDGFβR Locus (A) Schematic of the region of the wild-type PDGFαR genomic locus designated for targeting with either the wild-type αR cDNA or αβ cDNA knockin vectors. The only difference between the construct used to derive the αKI lines and αβKI lines is in the PDGFR cDNA inserted within the cDNA cassette. SP, I, II, and III represent the approximate positions of exons encoding the signal peptide, first, second, and third immunoglobulin domains, respectively. SA, splice acceptor; 3pA, triple polyadenylation sequence. (B and C) Southern blot analysis of wild-type (++) and (B) αKI-targeted (+/α) or (C) αβKI-targeted (+/αβ) ES cell DNA digested with NheI. The probe used to detect targeting events (shaded box in [A]) is derived from a region of PDGFαR cDNA encoding the extracellular portion of the receptor and thus hybridizes to the cDNA insert cassette (8.7 kb fragment) as well as to an extra 4 kb fragment indicative of correct targeting. (D) Schematic of the region of the gene encoding the PDGFβR designated for targeting with either the wild-type βR cDNA or βα knockin vectors. (E and F) Southern blot analysis of wild-type (++) and (E) βKI-targeted or (F) βαKI-targeted ES cell DNA digested with SacI. As above, the probe used to detect targeting events (shaded box in [D]) is derived from a region of PDGFβR cDNA encoding the extracellular portion of the receptor. Therefore, it hybridizes to the cDNA insert cassette (1.4 kb fragment for wild-type β and 5.4 kb fragment for βα) as well as to an extra 9.5 kb fragment indicative of correct targeting. A, ApaI; Bg, BglII; S, SacII; N, NheI; B, BamHI; RI, EcoRI; Sm, SmaI; X, XbaI; RV, EcoRV Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 2 Western Blot Analysis of PDGFR Expression and Activation (A–C) For targeting at the PDGFαR locus, lysates were run in triplicate and blotted for (A) the PDGFαR extracellular domain (ECD), (B) the carboxy-terminal tail (C-tail), and (C) an anti-RasGAP antibody, to control for equal loading of protein. Note that the slower migration of the αβ band in (A) is due to the larger size of the chimeric fusion protein than the wild-type PDGFαR. For targeting at the PDGFβR locus, sample lysates were also run in triplicate. (D) Expression of the βα PDGFR chimera was analyzed by blotting with an antibody that recognizes the C-terminal tail of the PDGFαR. The intensity of the PDGFR band in the βα lane was 2.7-fold and 2.4-fold greater than wild-type and β cDNA the control lanes, respectively, suggesting that the βα PDGFR is expressed in place of the endogenous PDGFβR. These values were determined using NIH image software. (E) Blotting for the C-terminal tail of the PDGFβR. (F) Blotting for RasGAP as a protein loading control. (G–I) To test the integrity of PDGF-induced activation by the chimeric PDGFRs, cells derived from homozygous mutant and control embryos were analyzed. (G) PDGF-dependent tyrosine phosphorylation. Cells were left resting or were stimulated with 30 ng/ml PDGFBB. Cells lysates were then immunoprecipitated with an antibody against RasGAP and assayed for phosphotyrosine content by Western blotting. (H) The blot shown in (G) was stripped and reprobed with a RasGAP antibody. (I) To further test the ability of the βα PDGFR to respond to PDGFBB, cells expressing this chimeric receptor were compared to wild-type controls in an assay for PDGF-induced DNA synthesis. The data are means of triplicate assay ± SD Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 3 Defective Embryonic Kidney Development in βα/− Hemizygotes (A and B) Whole kidneys removed from βα/+ (A) and βα/− (B) embryos. Note the speckled appearance of the βα/− kidney, which is due to erythrocyte-filled glomeruli. (C–F) Sections through wild-type (C), βα/βα (D), and βα/− (E and F) kidneys stained with periodic acid-Schiff's reagent. Note the highly distended capillaries within the glomeruli of βα/− embryos. (G and H) αSMA labeling (black) for embryonic mesangial cells by immunohistochemistry. Photographed under an oil lens at 63× Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 4 Glomerulosclerosis in Adult βα/− Kidneys (A and B) Periodic acid-Schiff's (PAS) staining of βα/− (A) and βα/+ (B) kidneys. The βα/− glomerulus displays focal segmental glomerulosclerosis as observed by the large matrix-filled pink area that is now devoid of cells. (C–F) Masson Trichrome (MTC) staining of βα/− (C), βα/βα (D), wild-type (E), and β cDNA/− kidneys for collagen deposition (blue). Photographed under an oil lens at 63× Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 5 Thrombus Formation in βα/βα Glomeruli from Mice Injected with Anti-GBM Antibody Wild-type (WT) and βα/βα (βα) mice were injected with either sheep anti-glomerular basement membrane antisera (αGBM) or normal sheep serum control as described in the Experimental Procedures. Kidneys were sectioned and stained with PAS. The arrows denote the limit of the glomerulus at the basement membrane. Following injection of αGBM, both wild-type and βα glomeruli develop multilayered cell crescents between the capillary tuft and the basement membrane. However, thrombus formation only occurs in βα glomeruli. Crescents and thrombi were not observed in animals injected with control serum. Photographed under an oil lens at 63× Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 6 βα/− Mice Develop a Proliferative Retinopathy Due to Loss of Pericytes at the Retinal Microvasculature (A) Gross appearance of the eye of a βα/− mouse. Note the cotton-like opacity in the normally black eye. (B and C) Hematoxylin/eosin-stained sections of a βα/− eye (B) and a βα/+ eye (C). (E–G) Retinas from wild-type mice expressing the pericyte/lacZ transgene. (H–J) Retinas from βα/− mice expressing the pericyte/lacZ transgene. (E and H) Whole mounts. (F and I) Flat mounts photographed at the center of the retina. (G and J) Flat mounts photographed at the periphery of the retina Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 7 Heart Enlargement in βα Knockin Mice (A) Hearts from 10-week-old mice. (B) To quantify the heart enlargement, heart weight (wet) to body weight ratios were derived: WT (n = 16) average = 0.0060 ± 0.0009, βα/+ (n = 11) average = 0.0061 ± 0.0008, βα/βα (n = 34) average = 0.0081 ± 0.0011, βα/− (n = 9) average = 0.0132 ± 0.0040, β cDNA/β cDNA (n = 6) average = 0.0058 ± 0.0007, β cDNA/− (n = 13) average = 0.0057 ± 0.0008. (C–F) Heart sections from βα/− (C and E) and heterozygous littermate (D and F) mice Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)

Figure 8 Decreased Duration of PDGF-Dependent MAPK Phosphorylation in βα PDGFR–Expressing Cells βα and control cells were stimulated with the indicated concentrations of PDGFBB, and cell lysates were subjected to anti-phospho MAP kinase Western blot. All blots were striped and reprobed with an anti-MAP kinase antibody to ensure that equal levels of protein were loaded (data not shown). (A) Dose response of MAPK phosphorylation after 5 min of stimulation with PDGFBB. (B) The kinetics of MAPK phosphorylation in response to stimulation with 30 ng/ml PDGFBB. (C) The kinetics of MAPK phosphorylation in wild-type fibroblasts expressing both PDGFRs, in response to stimulation by PDGFAA or PDGFBB Molecular Cell 2001 7, 343-354DOI: (10.1016/S1097-2765(01)00182-4)