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The molecular basis of defective lens development in the Iberian mole.

Carmona FD, Jiménez R, Collinson JM - BMC Biol. (2008)

Bottom Line: Fossorial mammals face natural selection pressures that differ from those acting on surface dwelling animals, and these may lead to reduced visual system development.PAX6 is not down-regulated in developing lens fibre nuclei, as it is in other species, and there is ectopic expression of FOXE3, a putative downstream effector of PAX6, in some, but not all lens fibres.The undifferentiated status of the anterior epithelial cells was compromised, and most of them undergo apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. d.carmona@abdn.ac.uk

ABSTRACT

Background: Fossorial mammals face natural selection pressures that differ from those acting on surface dwelling animals, and these may lead to reduced visual system development. We have studied eye development in a species of true mole, the Iberian mole Talpa occidentalis, and present the molecular basis of abnormal lens development. This is the first embryological developmental study of the eyes of any fossorial mammal at the molecular level.

Results: Lens fibre differentiation is not completed in the Iberian mole. Although eye development starts normally (similar to other model species), defects are seen after closure of the lens vesicle. PAX6 is not down-regulated in developing lens fibre nuclei, as it is in other species, and there is ectopic expression of FOXE3, a putative downstream effector of PAX6, in some, but not all lens fibres. FOXE3-positive lens fibres continue to proliferate within the posterior compartment of the embryonic lens, but unlike in the mouse, no proliferation was detected anywhere in the postnatal mole lens. The undifferentiated status of the anterior epithelial cells was compromised, and most of them undergo apoptosis. Furthermore, beta-crystallin and PROX1 expression patterns are abnormal and our data suggest that genes encoding beta-crystallins are not directly regulated by PAX6, c-MAF and PROX1 in the Iberian mole, as they are in other model vertebrates.

Conclusion: In other model vertebrates, genetic pathways controlling lens development robustly compartmentalise the lens into a simple, undifferentiated, proliferative anterior epithelium, and quiescent, anuclear, terminally differentiated posterior lens fibres. These pathways are not as robust in the mole, and lead to loss of the anterior epithelial phenotype and only partial differentiation of the lens fibres, which continue to express 'epithelial' genes. Paradigms of genetic regulatory networks developed in other vertebrates appear not to hold true for the Iberian mole.

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β-crystallin genes are expressed in PAX6-positive cells, including epithelial cells, in the lens of the Iberian mole. (A) The lens epithelium remains β-crystallin-negative at the s7 stage, but a weak β-crystallin expression is observed at the s8 stage. From s9 to adulthood, both fibre and epithelial cells show a similar β-crystallin-immunofluorescence. (B) In E18 mice samples, β-crystallin (red) is detected in the cytoplasm of lens fibres and Pax6 (green) in the nucleus of epithelial cells. In s5c mole embryos, the lens epithelium appeared PAX6-positive and β-crystallin-negative, but the fibre cells were positive for both proteins. At s10, all the lens cells, including the epithelial cells, showed immunoreactivity against both anti-PAX6 and anti-β-crystallin antibodies. The nuclei are stained blue with 4'6-diamidino-2-phenylindole stain. Photomicrographs were taken using a single bandpass fluorescence mirror unit and merged with 'The Gimp' software. Scale bar represents 100 μm in all figures of both A and B. LE, lens epithelium; FC, fibre cells.
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Figure 10: β-crystallin genes are expressed in PAX6-positive cells, including epithelial cells, in the lens of the Iberian mole. (A) The lens epithelium remains β-crystallin-negative at the s7 stage, but a weak β-crystallin expression is observed at the s8 stage. From s9 to adulthood, both fibre and epithelial cells show a similar β-crystallin-immunofluorescence. (B) In E18 mice samples, β-crystallin (red) is detected in the cytoplasm of lens fibres and Pax6 (green) in the nucleus of epithelial cells. In s5c mole embryos, the lens epithelium appeared PAX6-positive and β-crystallin-negative, but the fibre cells were positive for both proteins. At s10, all the lens cells, including the epithelial cells, showed immunoreactivity against both anti-PAX6 and anti-β-crystallin antibodies. The nuclei are stained blue with 4'6-diamidino-2-phenylindole stain. Photomicrographs were taken using a single bandpass fluorescence mirror unit and merged with 'The Gimp' software. Scale bar represents 100 μm in all figures of both A and B. LE, lens epithelium; FC, fibre cells.

Mentions: The function of fibre cells in mole lenses was investigated using β-crystallin as a marker for terminal differentiation. In mice and chickens, β-crystallin genes are expressed only in lens fibre cells that have down-regulated Pax6, but not in epithelial cells where Pax6 expression is maintained. Pax6 is reported to be a transcriptional repressor of genes encoding β-crystallins [27,28,36]. In the mouse, using an antibody that recognises all β-crystallin isoforms, we confirmed that expression of Pax6 and β-crystallin is mutually exclusive – lens fibres were β-crystallin-positive and Pax6-negative, whereas all lens epithelial cells were Pax6-positive and β-crystallin-negative. During mole lens development, β-crystallins were first detected at the s5c stage (Figure 10), but not at previous stages (not shown). These proteins were located in the cytoplasm of the elongating posterior cells: the lens epithelium was β-crystallin-negative until stage s8. Hence, at stages s5c-s7, when the first defects of molecular patterning of the lens were being noted (the altered patterns of PAX6, FOXE3 and PROX1 expression described above), the expression of β-crystallins was normal, with expression in lens fibre cells but not the epithelium. Nevertheless, weak immunoreactivity against the anti-β-crystallin antibody was observed later, in epithelial cells at the last prenatal stage (s8) (Figure 10A), and similar high levels of β-crystallin expression were detected in both the epithelial cells and the lens fibres from the s9 stage on (lower rows in Figure 10A and 10B). Figure 10B shows that, in contrast to the mutually exclusive patterns of expression observed in wild-type mouse lenses, all the cells expressing β-crystallins are PAX6-positive. Only those cells located in the lens epithelium between stages s5c and s7, are immunoreactive for PAX6 but not for β-crystallins (N ≥ 4 lenses/stage). These data suggest a breakdown of the inhibitory effect of PAX6 expression on lens fibre differentiation, consistent with the results above.


The molecular basis of defective lens development in the Iberian mole.

Carmona FD, Jiménez R, Collinson JM - BMC Biol. (2008)

β-crystallin genes are expressed in PAX6-positive cells, including epithelial cells, in the lens of the Iberian mole. (A) The lens epithelium remains β-crystallin-negative at the s7 stage, but a weak β-crystallin expression is observed at the s8 stage. From s9 to adulthood, both fibre and epithelial cells show a similar β-crystallin-immunofluorescence. (B) In E18 mice samples, β-crystallin (red) is detected in the cytoplasm of lens fibres and Pax6 (green) in the nucleus of epithelial cells. In s5c mole embryos, the lens epithelium appeared PAX6-positive and β-crystallin-negative, but the fibre cells were positive for both proteins. At s10, all the lens cells, including the epithelial cells, showed immunoreactivity against both anti-PAX6 and anti-β-crystallin antibodies. The nuclei are stained blue with 4'6-diamidino-2-phenylindole stain. Photomicrographs were taken using a single bandpass fluorescence mirror unit and merged with 'The Gimp' software. Scale bar represents 100 μm in all figures of both A and B. LE, lens epithelium; FC, fibre cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 10: β-crystallin genes are expressed in PAX6-positive cells, including epithelial cells, in the lens of the Iberian mole. (A) The lens epithelium remains β-crystallin-negative at the s7 stage, but a weak β-crystallin expression is observed at the s8 stage. From s9 to adulthood, both fibre and epithelial cells show a similar β-crystallin-immunofluorescence. (B) In E18 mice samples, β-crystallin (red) is detected in the cytoplasm of lens fibres and Pax6 (green) in the nucleus of epithelial cells. In s5c mole embryos, the lens epithelium appeared PAX6-positive and β-crystallin-negative, but the fibre cells were positive for both proteins. At s10, all the lens cells, including the epithelial cells, showed immunoreactivity against both anti-PAX6 and anti-β-crystallin antibodies. The nuclei are stained blue with 4'6-diamidino-2-phenylindole stain. Photomicrographs were taken using a single bandpass fluorescence mirror unit and merged with 'The Gimp' software. Scale bar represents 100 μm in all figures of both A and B. LE, lens epithelium; FC, fibre cells.
Mentions: The function of fibre cells in mole lenses was investigated using β-crystallin as a marker for terminal differentiation. In mice and chickens, β-crystallin genes are expressed only in lens fibre cells that have down-regulated Pax6, but not in epithelial cells where Pax6 expression is maintained. Pax6 is reported to be a transcriptional repressor of genes encoding β-crystallins [27,28,36]. In the mouse, using an antibody that recognises all β-crystallin isoforms, we confirmed that expression of Pax6 and β-crystallin is mutually exclusive – lens fibres were β-crystallin-positive and Pax6-negative, whereas all lens epithelial cells were Pax6-positive and β-crystallin-negative. During mole lens development, β-crystallins were first detected at the s5c stage (Figure 10), but not at previous stages (not shown). These proteins were located in the cytoplasm of the elongating posterior cells: the lens epithelium was β-crystallin-negative until stage s8. Hence, at stages s5c-s7, when the first defects of molecular patterning of the lens were being noted (the altered patterns of PAX6, FOXE3 and PROX1 expression described above), the expression of β-crystallins was normal, with expression in lens fibre cells but not the epithelium. Nevertheless, weak immunoreactivity against the anti-β-crystallin antibody was observed later, in epithelial cells at the last prenatal stage (s8) (Figure 10A), and similar high levels of β-crystallin expression were detected in both the epithelial cells and the lens fibres from the s9 stage on (lower rows in Figure 10A and 10B). Figure 10B shows that, in contrast to the mutually exclusive patterns of expression observed in wild-type mouse lenses, all the cells expressing β-crystallins are PAX6-positive. Only those cells located in the lens epithelium between stages s5c and s7, are immunoreactive for PAX6 but not for β-crystallins (N ≥ 4 lenses/stage). These data suggest a breakdown of the inhibitory effect of PAX6 expression on lens fibre differentiation, consistent with the results above.

Bottom Line: Fossorial mammals face natural selection pressures that differ from those acting on surface dwelling animals, and these may lead to reduced visual system development.PAX6 is not down-regulated in developing lens fibre nuclei, as it is in other species, and there is ectopic expression of FOXE3, a putative downstream effector of PAX6, in some, but not all lens fibres.The undifferentiated status of the anterior epithelial cells was compromised, and most of them undergo apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. d.carmona@abdn.ac.uk

ABSTRACT

Background: Fossorial mammals face natural selection pressures that differ from those acting on surface dwelling animals, and these may lead to reduced visual system development. We have studied eye development in a species of true mole, the Iberian mole Talpa occidentalis, and present the molecular basis of abnormal lens development. This is the first embryological developmental study of the eyes of any fossorial mammal at the molecular level.

Results: Lens fibre differentiation is not completed in the Iberian mole. Although eye development starts normally (similar to other model species), defects are seen after closure of the lens vesicle. PAX6 is not down-regulated in developing lens fibre nuclei, as it is in other species, and there is ectopic expression of FOXE3, a putative downstream effector of PAX6, in some, but not all lens fibres. FOXE3-positive lens fibres continue to proliferate within the posterior compartment of the embryonic lens, but unlike in the mouse, no proliferation was detected anywhere in the postnatal mole lens. The undifferentiated status of the anterior epithelial cells was compromised, and most of them undergo apoptosis. Furthermore, beta-crystallin and PROX1 expression patterns are abnormal and our data suggest that genes encoding beta-crystallins are not directly regulated by PAX6, c-MAF and PROX1 in the Iberian mole, as they are in other model vertebrates.

Conclusion: In other model vertebrates, genetic pathways controlling lens development robustly compartmentalise the lens into a simple, undifferentiated, proliferative anterior epithelium, and quiescent, anuclear, terminally differentiated posterior lens fibres. These pathways are not as robust in the mole, and lead to loss of the anterior epithelial phenotype and only partial differentiation of the lens fibres, which continue to express 'epithelial' genes. Paradigms of genetic regulatory networks developed in other vertebrates appear not to hold true for the Iberian mole.

Show MeSH
Related in: MedlinePlus