Comparative Study
doi: 10.1016/j.ydbio.2006.11.033. Epub 2006 Dec 2.Defects in eye development in transgenic mice overexpressing the heparan sulfate proteoglycan agrin
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- PMID:17196957
- PMCID: PMC1831846
- DOI: 10.1016/j.ydbio.2006.11.033
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Comparative Study
Defects in eye development in transgenic mice overexpressing the heparan sulfate proteoglycan agrin
Peter G Fuerst et al. Dev Biol..
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Abstract
The importance of heparan sulfate proteoglycans (HSPGs) in neurodevelopment is becoming increasingly clear. However, studies on HSPGs are hampered by pleiotropic effects when synthesis or modification of heparan sulfate itself is targeted, and by redundancy when the core proteins are altered. Gain-of-function experiments can sometimes circumvent these issues. Here we establish that transgenic mice overexpressing the HSPG agrin have severe ocular dysgenesis. The defects occur through a gain-of-function mechanism and penetrance is dependent on agrin dosage. The agrin-induced developmental defects are highly variable, and include anophthalmia, persistence of vitreous vessels, and fusion of anterior chamber structures. A frequently observed defect is an optic stalk coloboma leading to the misdifferentiation of the optic stalk as retina, which becomes continuous with the forebrain. The defects in optic-stalk differentiation correlate with reduced sonic hedgehog immunoreactivity and overexpansion of the PAX6 domain from the retina into the optic stalk. The ocular phenotypes associated with agrin overexpression are dependent on genetic background, occurring with high penetrance in inbred C57BL/6J mice. Distinct loci sensitizing C57BL/6J mice to agrin-induced dysgenesis were identified. These results indicate that agrin overexpression will provide a tool to explore the molecular interactions of the extracellular matrix and cell surface in eye development, and provide a means for identifying modifier loci that sensitize mice to developmental eye defects.
Figures
Agrin transgenic mice. A) Agrin is a forty-exon gene spanning approximately 40 kb of genomic DNA. The agrin protein is almost 2,000 amino acids long, with either a transmembrane N-terminus (SN-agrin) or an N-terminus with a signal peptide for secretion (LN-agrin). In the N-terminal half of the protein, there are nine-follistatin repeats (F), a laminin-like domain (L), two serine/threonine-rich regions (ST), and an SEA domain. In the C-terminal half, there are four EGF-like repeats (EGF) and three laminin-type globular domains (G). There are also at least three sites of alternative splicing (X, Y, Z). The sites of glycosaminoglycan (GAG) addition are in the N-terminal half of agrin. The agrin transgene construct was a BAC containing the entire agrin gene, in some cases modified to fuse cyan fluorescent protein (CFP) onto the C-terminus of agrin. B) TheAgrnCFP transgene was functional and rescued the neonatal lethality caused by the deletion of agrin by homologous recombination. Mice homozygous for an agrin deletion, but carrying the transgene, had neuromuscular junctions (NMJs) that were indistinguishable from controls (C). The nerve, stained for neurofilament (green) overlapped the postsynaptic acetylcholine receptors labeled with α-bungarotoxin (red). The NMJs shown were from P12 mice. (D) The genotypes of mice rescued by theAgrnCFP2R9 andAgrnCFP6R16 transgenic strains were confirmed by Southern blotting. Digestion of genomic DNA withNcoI yielded the anticipated 2 kb band in the null allele, a 5 kb band in the wild-type allele, and a 6 kb band in theAgrnCFP transgene (cause by the CFP insertion). Lane 1AgrnDel/Del rescued by theAgrnCFP2R9 insertion, compared to anAgrnDel/+ littermate (lane 2). Lane 3AgrnDel/Del rescued by theAgrnCFP6R16 transgene, compared to anAgrnDel/+ (lane 4). Lane 5 was a wild-type control. Notice the higher copy number of theAgrnCFP insertion in the 2R9 strain (dark 6 kb band). (E) Northern blotting confirmed that the only agrin transcripts generated in the rescued mice were theAgrnCFP from the transgene. The transgenic transcript was shifted by 1 kb by the CFP insertion. The same blots were hybridized with probes against Agrin, CFP, and β-actin as a loading control. Densitometry using a phosphorimager indicated that agrin was overexpressed approximately 2.5-fold in the 2R9 strain (shown in (E) at P12). F) The agrin-CFP protein was detectable by western blotting using an anti-GFP antibody. The antibody did not recognize protein from non-transgenic mice (lane1), but recognized a high-molecular-weight smear in theAgrnCFP transgenic strains (arrowhead at 250 kDa). A smaller degradation or cleavage product was also evident, as previously reported for the endogenous protein. Relative expression levels of theAgrnCFP strains are shown, with MAP2 as a loading control shown below. G) The protein smear was reduced to a sharp band by treatment with heparatinase, and only partially affected by treatment with Chondroitinase ABC, consistent with the transgenic protein also being a heparansulfate proteoglycan.
Distribution of agrin and agrin-CFP in the adult mouse eye. (A) Cross sections of wild type retinas stained with anti-agrin antibodies (GR14, red) revealed agrin to be localized to cells on the edges of the inner nuclear layer (INL) and outer plexiform layer (OPL), which were identified as blood vessels, and along the internal limiting membrane (ILM). The photoreceptors of the outer nuclear layer (ONL) were not labeled. (B) Cross sections ofAgrnCFP6R16 retinas labeled using an anti-GFP antibody to recognize the transgenic protein (red), showed agrin-CFP localization to be identical to anti-agrin on wild-type sections. (C) Transverse sections of wild-type eyes stained with anti-GFP antibodies showed no staining, establishing that the antibody is specific for the transgenic agrin-CFP protein. (D) Sections of transgenic retinas stained with the GR14 anti-agrin (red) revealed the same localization. (E) Transverse sections of transgenic retinas probed with antibodies to GFP (red) and PAX6 (green) revealed that agrin-CFP does not co-localize with PAX6. (F) In the cornea, agrin-CFP is localized to Descemet’s membrane (DM) and Bowman’s layer (BL). (G, H) In the anterior chamber of the eye, agrin-CFP is localized to the iris (I), ciliary bodies (CB) and lens capsule (LC). (I) Sections of optic nerve fromAgrnCFP6R16 mice showed agrin-CFP localization to the outer sheath (OS) of the nerve, which is continuous with the blood brain barrier. Retinal sections are oriented with the retinal ganglion layer (RGL) up. All sections are counter-stained with DAPI (blue) to visualize nuclei. The measurement bar located in the lower right panel is equivalent to 75 μm in A, B, C, F and H, 225 μm in D and E, and 37.5 μm in G and I.
Agrin and agrin-CFP localization in the developing eye. The presence of agrin in the developing eye was assayed at four times relevant to the onset of developmental defects observed in dysgenic eyes. (A) In E9.5AgrnCFP6R16 embryos stained with antibodies to GFP, agrin-CFP is localized to the ECM separating the neural and surface ectoderm (NE and SE). (B) In E10.5 transgenic embryos, agrin-CFP is localized to the ECM of the interior optic cup (OC) and surrounding the invaginating lens (L), as well as along the exterior optic stalk (OS). (C) In E12 transgenic embryos, agrin-CFP is present in the ECM of the optic cup (OC), lens capsule (L) and inner and outer optic stalk (OS). (D) In E14 transgenic embryos agrin-CFP is localized to the vitreous (V) and internal limiting membrane (ILM) of the retina (R). Sections of wild-type eyes at E12 (E) and E14 (F) were stained with an anti-agrin antibody (GR14, red). The distribution of agrin in the wild-type developing eye is the same as the distribution of transgenic agrin-CFP inAgrnCFP6R16 transgenic mice. All sections are counterstained with DAPI (blue) to visualize the nuclei. The measurement bar located in the lower right panel is equivalent to 165 μm in A, 210 μm in B and 250 μm in C-F.
Agrin-CFP localization in the dysgenic eye. (A) In E11.5AgrnCFP2R9 eyes stained with antibodies to GFP (red), agrin-CFP is localized throughout the lens stalk (LS), as well as along the basal lamina of the surface epithelium and lens, and the internal limiting membrane (ILM) of the optic cup (OC). (B) Transverse sections through the ventral optic cup of E14AgrnCFP2R9 eye revealed agrin-CFP to be localized to the persistent fetal fissure (FF). (C) Transverse sections through E16AgrnCFP2R9 embryos stained with hematoxylin and eosin revealed misdifferentiation of retinal tissue along the length of the optic nerve. The retina (R) formed in place of the dorsal portion of the optic nerve (ON), and is continuous with the forebrain (FB). (D) E15AgrnCFP2R9 optic nerve sections were stained with antibodies to GFP, to detect agrin-CFP (green), and CHX10 (red), a marker of retinal differentiation. The retinal pigment epithelium, visualized with brightfield microscopy, is marked on the image (arrow). CHX10 labeled the misdifferentiated tissue, and agrin-CFP is localized to the ECM surrounding the optic nerve. (E) Sagittal sections of E15AgrnCFP2R9 optic nerves were stained with antibodies to PAX6 (green) and PAX2 (red), PAX6 localized to the dorsal half of the optic nerve, while PAX2 localized to the ventral half. The retinal pigment epithelium is again marked (arrow). (F) In P0AgrnCFP2R9 eyes double-labeled with anti-GFP to detect agrin-CFP (red) and dystroglycan (green), agrin-CFP co-localizes with dystroglycan in acellular ECM connecting the lens and cornea. (G) In P0AgrnCFP2R9 eyes with infiltration of fibrovascular tissue, agrin-CFP localized to hyaloid vessels (V) adhering to folds of the retina (R). (H) In P20AgrnCFP2R9 eyes with lens-cornea fusions, agrin-CFP localized to the basal lamina of the cornea (arrow heads) and to tissue connecting the lens and cornea (arrow). (I) In P20AgrnCFP2R9 eyes with irideocorneal fusion, agrin-CFP is localized to the points of adhesion between the iris and cornea (arrowhead). The measurement bar located in the lower right panel is equivalent to 90μm in A, 250 μm in B, 1.1 mm in C, 220 μm in D and E , 105 μm in F, 180 μm in G, 75 μm in H, and 35 μm in I.
PAX2 and PAX6 localization inAgrnCFP2R9 and wild-type eyes during development. Embryonic eyes at E12.5 and E14 were labeled with antibodies to PAX2 (red) and PAX6 (green). Mildly affected and severely affected examples ofAgrnCFP2R9 eyes are shown in the middle column and right column respectively. (A) In wild-type embryos at developmental day 12.5, PAX6 is localized to the peripheral optic cup, while PAX2 is limited to the posterior margin of the optic cup. (B and C) InAgrnCFP2R9 eyes at developmental day E12.5, PAX2 and PAX6 localization is similar to that of the wild-type eye; PAX6 is limited to the optic cup and PAX2 is localized to the posterior margins of the optic cup and optic stalk, regardless of the degree of dysgenesis. (D) At E14, PAX6 is localized to the optic cup, while PAX2 is localized to the optic nerve in wild-type eyes. (E) In mildly dysgenicAgrnCFP2R9 eyes at E14, PAX6 and PAX2 localization was similar to wild-type, with PAX6 limited to the optic cup, and PAX2 limited to the optic nerve. (F) However, in severely dysgenicAgrnCFP2R9 eyes with optic nerve colobomas, PAX6 localization expanded medially along the dorsal optic nerve, while PAX2 localized to the ventral margin of the medial optic nerve. The measurement bar located in the lower right panel is equivalent to 215 μm in A, B and C and 415 μm in D, E and F.
SHH and optic nerve development inAgrnCFP2R9 mice. The schematics in the left column represent the planes of section and regions of interest for each pair of images, shown in relation to the developing eyes and optic nerve. (A and B) Transverse sections of wild-type andAgrnCFP2R9 E11 embryos stained with antibodies to SHH (green) revealed a similar midline and parasagittal staining pattern in the telencephalon. (C and D) Transverse sections of E12.5 embryos stained with antibodies to SHH (red) showed a similar staining intensity inAgrnCFP2R9 mice compared to controls. (E and F) Wild-type andAgrnCFP2R9 E14 embryos sectioned at the optic disc and double-labeled with antibodies to SHH (green) and neurofilament (NF, red) showed varying levels of SHH inAgrnCF2R9P mice, ranging from nearly wild type to vastly reduced (an intermediate example is shown in F). (G-J) Sagittal sections at the optic disc (G, H) or in the developing optic nerve (I, J) from E12.5 embryos were stained with antibodies to NF (green) and PAX2 (red) to visualize the retinal ganglion cell axons and the cells of the optic stalk, respectively. NF staining was reduced in theAgrnCFP2R9 transgenic optic nerve compared to wild-type. The cross-section of the optic nerve ofAgrnCFP2R9 embryos was also much larger compared to wild type controls, reflecting the failure of the optic stalk to fuse. The measurement bar located in the lower right panel is equivalent to 280 μm in A and B, 98 μm in C and D, 95 μm in E and F, 70 μm in G and H and 50 μm in I and J.
QTL maps of C57BL/6J loci associated with ocular dysgenesis. A plot of the log of odds (lod) that a given chromosomal location would segregate in the cross at observed levels in dysgenic mice. Chromosome numbers are plotted along the X-axis with the centromeric end to the left. The log of odds is plotted on the Y-axis. (A) Modeling the various types of ocular dysgenesis observed inAgrnCFP2R9 mice as a continuous spectrum of a single phenotype identified a single suggestive locus on Chromosome 13. The ocular defects observed inAgrnCFP2R9 mice were therefore also modeled as distinct phenotypes. (B) A highly significant QTL, correlating with anophthalmia and microphthalmia, was detected in the middle of Chromosome 2, centered at 35 cM. (C) A suggestive QTL, correlating with coloboma of the fetal fissure, was detected on Chromosome 4. (D) A suggestive QTL, correlating with corneal lenticular adhesions, was detected on the proximal portion of Chromosome 2. (E) A suggestive QTL, correlating iridocorneal adhesions, was detected on Chromosome 12. (F) A suggestive QTL, correlating with PHPV, was detected on Chromosome 13.
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