Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance.
BACKGROUND
The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey) and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections.


RESULTS
Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection.


CONCLUSIONS
Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6+/- heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm.
Manuel
Martine
M
Genes and Development Group, Centres for Integrative Physiology and Neuroscience Research, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD, UK. Martine.Manuel@ed.ac.uk
Pratt
Thomas
T
Liu
Min
M
Jeffery
Glen
G
Price
David J
DJ
D000818:Animals; D001369:Axons; D002452:Cell Count; D004622:Embryo, Mammalian; D005136:Eye Proteins / Q000235:genetics*; D018628:Gene Dosage; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics; D008850:Microphthalmos / Q000196:embryology / Q000235:genetics*; D009024:Morphogenesis; D009897:Optic Chiasm / Q000196:embryology; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D015792:Retinal Dysplasia / Q000196:embryology / Q000235:genetics*; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005136:Eye Proteins; D018398:Homeodomain Proteins; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins