Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors.
BACKGROUND
The paired homeobox protein Pax6 is essential for proliferation and pluripotency of retinal progenitors. However, temporal changes in Pax6 protein expression associated with the generation of various retinal neurons have not been characterized with regard to the cell cycle. Here, we examine the dynamic changes of Pax6 expression among chicken retinal progenitors as they progress through the neurogenic cell cycle, and determine the effects of altered Pax6 levels on retinogenesis.


RESULTS
We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates.


CONCLUSIONS
These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase re-entry, whereas rapid accumulation or reduction of Pax6 protein during the G2/M phase of the cell cycle may be required for specific neuronal fates. These findings thus provide novel insights on the dynamic regulation of Pax6 protein among neurogenic progenitors and the temporal frame of neuronal fate determination.
Hsieh
Yi-Wen
YW
Jules Stein Eye Institute and Department of Ophthalmology, Molecular Biology Institute, University of California, David Geffen School of Medicine, Stein Plaza, Los Angeles, CA 90095, USA. evanhsieh07@gmail.com
Yang
Xian-Jie
XJ
D000367:Age Factors; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D016923:Cell Death / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D002642:Chick Embryo / Q000166:cytology; D053595:Embryonic Stem Cells / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism; D055495:Neurogenesis / Q000502:physiology*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000166:cytology* / Q000196:embryology; D014162:Transfection / Q000379:methods
D005136:Eye Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D018809:Proliferating Cell Nuclear Antigen; D012097:Repressor Proteins; D001973:Bromodeoxyuridine
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