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Requirement for TGFß receptor signaling during terminal lens fiber differentiation

¹ Department of Anatomy and Histology, The University of Sydney, NSW 2006, Australia
² Save Sight Institute, The University of Sydney, NSW 2006, Australia
³ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
⁴ Department of Medicine, Baylor College of Medicine, Houston, TX, USA
⁵ Children’s Medical Research Institute, Westmead Hospital, Westmead NSW 2145, Australia

View larger version (25K): [in a new window]   Fig. 1. Dominant-negative TßR transgenes. Diagrams of type I (A) and type II (B) TGFß receptors showing the secretory, extracellular, transmembrane and intracellular kinase domains of both forms of receptors. In addition, the type I receptor has a glycine-serine rich domain (GS domain). PCR was used to generate truncated, kinase-deficient forms (k) of TßRI and TßRII. kTßRI (A) was a 534 bp cDNA that included the signal peptide, extracellular domain, transmembrane domain and 13 residues of the intracellular domain. kTßRII (B) was a 940 bp cDNA, that included the signal peptide, extracellular domain, transmembrane domain and part of the intracellular domain. Both sequences were cloned between the A-crystallin promoter, and the small t intron and early region polyadenylation sequences of SV40 for microinjection. Transgenic mice were screened with primers specific for the SV40 sequence (arrows) and riboprobes for in situ hybridization were generated from a cDNA (bar) encoding part of the SV40 region of the transgenes.

View larger version (84K): [in a new window]   Fig. 2. Phenotype of kTßRI and kTßRII transgenic mice. Photographs showing eyes of non-transgenic (A,C) and transgenic OVE591 (B), R20 (D) and OVE 550 (E,F) mice at P21 (A,B), 8 weeks (C,D) and P5 (E,F). (A) Non-transgenic eye at P21. (B) The kTßRII transgenic (OVE591) eye at P21 is slightly smaller than the non transgenic eye and has a distinct opacity in the lens. (C) Non-transgenic eye at 8 weeks of age. (D) The kTßRI transgenic (R20) eye at 8 weeks is of similar size to the non transgenic but has a distinct opacity in the lens. (E) Dissected eyes from P5 non-transgenic and transgenic kTßRII (OVE550) mice showing the prominent nuclear cataract in the transgenic eye. (F) Further dissection of lenses from the globes clearly revealed the nuclear cataract in the smaller transgenic lens. The clear wild-type lens is able to focus a light source (arrowhead); however, the cataractous, transgenic lens blocks the light (arrow). Scale bar: 3 mm.

View larger version (115K): [in a new window]   Fig. 3. Fiber-specific expression of transgenes in embryonic lenses. Micrographs showing bright-field (A,C,E) and dark-field (B,D,F) images of in situ hybridization for expression of transgene mRNA in heterozygous E15.5 embryos from lines OVE550 (A,B), OVE591 (C,D) and R20 (E,F). The embryonic pattern of transgene expression was similar in all three lines. There was no detectable expression in the anterior epithelia (arrows). Expression commenced at the equator in early fibers (arrowheads) and was detectable throughout the fiber mass. Strongest expression of the transgene was detected in line OVE550, followed by R20 and OVE591. Scale bar: 100 µm.

View larger version (197K): [in a new window]   Fig. 4. Histology of transgenic eyes. Hematoxylin and Phloxine-stained sections of non-transgenic (C,D,G) compared with transgenic OVE591 (A,E,F), OVE550 (A',B) and R20 (H) eyes. (A) At P1, transgenic OVE591 (A) eyes and lenses showed normal morphology and were similar in size to the non-transgenic (not shown). However the OVE550 eyes and lenses were markedly smaller (A') and showed disrupted morphology. (B) Higher magnification view of the OVE550 eye showing the distinctly abnormal morphology. The anterior epithelium, which is normally a monolayer (A, arrowhead), was multi-layered (upper inset) and, while fibers in the transitional zone and outer cortex appeared relatively normal, the bow zone was abnormal with aberrant posterior positioning of nuclei (open arrow) within the fiber cell. There was marked disruption of inner cortical and nuclear fibers (*) with evidence of pyknotic nuclei (lower inset). (C,D) At P21, non-transgenic eyes and lenses showed normal morphology with a normal monolayer of epithelial cells (C, arrowhead), tightly packed, concentric layers of secondary fiber cells and a distinct ‘bow zone’ (D, open arrow). (E,F) The OVE591 transgenic lenses were slightly smaller then wild type and there was distinct degeneration of inner cortical and nuclear fibers (*) and multilayering of the adjacent epithelium at the anterior pole (E, inset). Cortical secondary fibers were less densely packed and the ‘bow zone’ was disrupted (F, open arrow). In the inner cortical fibers there was accumulation of eosinophilic material (arrows), and evidence of fiber swelling and pyknotic nuclei that persisted in the degenerate nuclear fibers (arrowheads). (G) Normal lens morphology of non-transgenic lenses at P120, showing the normal arrangement of anterior epithelium, differentiating fibers in the transitional and bow zones, and the uniformly stained mass of denucleated mature fiber cells that form the lens nucleus (asterisk). (H) The transgenic R20 lens shows accumulation of eosinophilic material in the cortical fibers (arrows) and disruption of the lens nucleus (asterisk). Pyknotic nuclei were also evident in the inner cortical fibers (arrowhead, inset). Scale bar: 100 µm in A,A’,D,F; 50 µm in B,G,H; 200 µm in C,E; 25 µm in other insets.

View larger version (148K): [in a new window]   Fig. 5. Histology of transgenic lenses. Toluidine Blue stained resin sections (1 µm) of non-transgenic (A,C) and transgenic OVE550 (B,D) lenses. (A) Equatorial region of an E18 wild-type lens, showing the normal sequence of events of epithelial cell elongation and differentiation into fibers. (B) Equatorial region of the transgenic (OVE550) lens, showing disrupted fiber differentiation. There is an accumulation of fiber cell nuclei (black arrowhead) in the transitional zone. Fibers in this region and the cortex have not elongated fully and do not contact the epithelial layer. Instead they have formed junctions with the lateral surfaces of the nuclear fibers (large arrow). Abnormal stellate cells (white arrowhead) were present between fibers and epithelial cells in the germinative zone. Disrupted positioning of nuclei (small arrow) in cortical fibers results in loss of the bow zone. In the lens nucleus the fibers are grossly disorganized and degenerate (asterisk). (C) Equatorial region of the P2 wild-type lens showing normal fiber cell elongation and terminal differentiation. (D) Equatorial region of the P2 transgenic (OVE550) lens showing more marked disruption of terminal fiber differentiation. Initial differentiation in the transitional zone and cortex appeared similar to the wild type. However, in the inner cortex, fibers appeared to swell and degenerate rapidly (arrows), forming a large, central, cellular debris-filled vacuole. The anterior epithelium, overlying the degenerate fibers was multi-layered (inset). Scale bar: 100 µm in A-D; 50 µm in inset.

View larger version (84K): [in a new window]   Fig. 6. Fiber cell apoptosis. Hoechst dye staining (A,C,E) and TUNEL reaction (B,D,F) on sections of P2 non-transgenic (A,B) and transgenic OVE550 (C-F) lenses at P2. (A) In non-transgenic lenses Hoechst staining revealed the normal monolayer of epithelial cells and the ‘bow-like’ distribution of nuclei in the lens fibers. (B) No TUNEL-positive cells were seen in non-transgenic lenses. Occasional labeled cells (arrowheads) were detected in cornea and blood vessels lining the inner limiting membrane of the retina. (C) Hoechst staining of transgenic lens revealed a multilayered anterior epithelium and disrupted formation of the ‘bow zone’. In the inner cortex, nuclei appear to be positioned abnormally within the fiber cells and formed an aberrant ‘bow zone’. Many of these fiber nuclei were condensed and pyknotic (boxed). Occasional Hoechst-stained nuclei, representing aberrantly migrating epithelial cells, were found localized along the posterior capsule (arrowheads). (D) TUNEL reaction revealed labeling of the pyknotic nuclei in the inner cortex and nucleus of the lens and also occasional labeled cells in the multilayered epithelium (arrows). TUNEL-positive nuclei were also observed at the equator and in the cells that had migrated posteriorly along the posterior capsule (arrowheads). (E) Higher magnification view of boxed region in C, showing condensed and pyknotic nuclei (arrows) of inner cortical fibers. These fiber nuclei also showed an abnormal distribution pattern within the fiber cells, resulting in an abnormal ‘bow zone’. (F) TUNEL reaction of the same region shown in E, showing that the pyknotic nuclei are TUNEL positive (arrows). Scale bar: 100 µm in A-D; 65 µm in E,F.

View larger version (108K): [in a new window]   Fig. 7. Crystallin expression. Expression of B-crystallin (D-F) and ß-crystallin (G-I) in non-transgenic (A,D,G) and transgenic OVE591 (B,E,H) and OVE550 (C,F,I) lenses at P1. (A-C) Hematoxylin-stained bright-field images of D-F showing fiber degeneration has not yet occurred in OVE591 (B) lens but is clearly evident (*) in OVE550 (C) lens. (D) In wild-type lens, B-crystallin was strongly expressed in the anterior epithelium and in cortical fibers, but little or no expression was detected in nuclear fibers. (E) In the OVE591 lens, a similar level of expression was detected in the lens epithelium but increased expression was detected in the cortical and nuclear fibers. (F) In the anterior epithelium and in the cortical fibers of the OVE550 lens, there was stronger expression of B-crystallin compared with the non-transgenic control, but expression was absent in the degenerate nuclear fibers (*). (G) Expression of ß-crystallin in wild-type lenses was restricted to cortical fiber cells. (H) The OVE591 lens showed similar level and pattern of expression to the wild type. (I) The OVE550 lens line showed increased expression in the outer cortical fibers that persisted to the inner cortical and nuclear fibers, but expression was absent in the degenerate nuclear fibers (*). Scale bar: 240 µm.

View larger version (106K): [in a new window]   Fig. 8. MIP expression. Expression of MIP in non-transgenic (A,D) and transgenic OVE591 (B,E,G,H) and OVE550 (C,F) lenses at P1 (A-F) and P3 (G,H). (A-C) Bright-field images of D-F showing wild-type (A) OVE591 (B) and OVE550 (C) lenses. (D) In the wild-type lens, expression of MIP was initiated in the transitional zone (arrowhead) and strongest in the outer cortical fibers. Expression decreased in the inner cortical fibers and was absent from the nuclear fibers. (E) Similarly, in the OVE591 lens there was initiation of expression in the transitional zone (arrowhead) but there was enhanced expression of MIP in the cortical fibers compared with the wild-type lens. (F) In the OVE550 lens, MIP expression commenced in the transitional zone and was increased in the cortical fibers, particularly in fibers (black asterisk) adjacent to the degenerative zone (white asterisk). No expression was detectable in the degenerated nuclear fibers (white asterisk). (G) In the OVE591 lens at P3, when fiber degeneration became apparent, MIP expression was initiated normally in the transitional zone but was strongly expressed in the cortical fibers, particularly in the regions preceding the zone of degeneration where fiber cells were swollen and disorganized (see Fig. 5D). Expression ceased abruptly in the degenerative zone (boxed). (H) Higher magnification bright-field view of the boxed region in G. Loss of MIP expression signal (arrowheads) in the cortical fibers precedes the occurrence of the nuclear pyknosis (arrows). Scale bar: 225 µm in A-F; 250 µm in G; 50 µm in H.

View larger version (113K): [in a new window]   Fig. 9. Expression of the intermediate filaments filensin (CP115) and phakinin (CP49). Immunofluorescence for CP115 (A-C) and CP49 (D-F) in wild-type (A,C) and OVE591 (B,C,E,F) lenses at P1 (A,B,D,E) and P3 (C,F). (A) Patchy reactivity for CP115 was detected in fiber cells of the non-transgenic lens at P1, particularly the inner cortical fibers (arrowheads). No reactivity was present in the epithelium (arrow) or lens nucleus. (B) Increased reactivity for CP115 was present in the inner cortical fibers (arrowheads) and also the nucleus of the transgenic OVE591 lens at P1. (C) At P3 in the OVE591 lens, the central fibers are degenerate (asterisk). Distinct reactivity for CP115 was present in a band of cortical fibers (arrowheads) and in fibers that adjoin the zone of degeneration (arrows). (D) At P1, CP49 was localized diffusely in fibers of the wild-type lens and was also detected along apico-lateral membranes of the epithelial cells (arrow). (E) Increased reactivity for CP49 was detected in fibers of the OVE591 lens at P1, particularly in a band of fibers in the deeper cortex (arrowheads). (F) At P3, the fibers that were highly reactive for CP49 at P1 (E) have degenerated. Only occasional cells at the edge of the degenerate fiber region (asterisk) show intense reactivity for CP49 (arrows). Scale bar: 100 µm.

View larger version (124K): [in a new window]   Fig. 10. Migration response on a laminin substratum. Phase contrast (A-D) and fluorescence images, showing labeling of filamentous actin and nuclei (E,F), of migrating non-transgenic (A,C,E) or transgenic OVE550 (B,D,F) lens epithelial cells cultured for 4 days on laminin without FGF2 (A,B) or with 100 ng/ml FGF2 (C-F). (A) Wild-type lens epithelial cells (arrowheads), cultured without FGF2, migrated from the capsule (arrow) onto the laminin substratum. (B) Transgenic (OVE550) lens epithelial cells (arrowheads) showed a similar migration response away from the explant edge (arrow), when cultured without FGF2. (C) In the presence of a fiber-differentiation dose of FGF2, wild-type epithelial cells showed an augmented migratory response (arrowheads). (D) Transgenic (OVE550) epithelial cells showed inhibition of cell migration when cultured with FGF2, with very few cells (arrowhead) migrating from the edge of the capsule (arrow). (E) Staining of filamentous actin with phalloidin-rhodamine, in wild-type cells cultured with FGF-2, revealed labeling of stress fibers (red) in the actively migrating cells (arrowheads). Cell nuclei have been stained with Hoechst dye (blue). (F) Phalloidin-rhodamine staining of FGF2-treated transgenic (OVE550) cells showed a complete absence of stress fibers in epithelial cells (arrowheads) that have remained in close association with the capsule (arrow). Scale bar: 100 µm in A-D; 20 µm in E,F.