Abstract
Purpose: :
Pax6 is broadly expressed in retinal progenitor cells, but becomes progressively restricted to prospective ganglion, amacrine and horizontal cells, while disappearing from prospective photoreceptors (Belecky-Adams et al, 1997). Pax6 over-expression in vitro leads to inhibition of photoreceptor differentiation and increases in neuronal differentiation (Toy et al, 2000). The experiments reported here further investigate the hypothesis that Pax6 is both a positive regulator of neuronal differentiation and a repressor of photoreceptor differentiation.
Methods: :
On embryonic day (ED) 2-3, the right eye of chick embryos was electroporated with constructs expressing either Pax6 (experimental group) or a control gene (control group). Vectors were RCASBP(A) retrovirus or a pcDNA3 expression plasmid; electroporated cells were identified by immunodetection of a viral protein or by GFP expression from a co-electroporated plasmid, respectively. On ED6, progenitor differentiation within the retinal microenvironment in vivo was investigated by immunocytochemistry for Pax6 and markers for different retinal cell types, both in retinal sections and in freshly dissociated cell suspensions. The capacity of the progenitors to differentiate cell-autonomously into neurons or photoreceptors was also tested in low density cultures of electroporated retinas, isolated on ED6 and cultured for 4 days in vitro.
Results: :
Embryos from both experimental and control groups showed normal macroscopic and histological appearance. Pax6 over-expressing cells were seen in all retinal layers. Markers for ganglion (Islet1 and Brn3a) and horizontal (Lim1) cells showed no differences between experimental and control embryos. On the other hand, ONL cells over-expressing Pax6 showed ectopic expression of AP-2α (an amacrine cell marker), but did not express visinin (a photoreceptor marker). When tested in low density cultures, progenitors electroporated with Pax6 in vivo showed a strong bias towards differentiation as non-photoreceptor neurons, concomitant to a marked inhibition of their differentiation as photoreceptors. Similar results were obtained by morphological analysis andby immunocytochemistry for AP2α, Lim1 and RA4 (specific for amacrine, horizontal and ganglion cells, respectively).
Conclusions: :
Taken together these results suggest that Pax6 expression/repression in retinal progenitors represents a critical step in retinal cell differentiation during normal development, and that Pax6 may inhibit retinal progenitor cells from choosing the photoreceptor pathway.
Keywords: transcription factors • photoreceptors • differentiation