Abstract
Purpose: :
Although mature photoreceptor subtypes can be readily recognized using structural and molecular criteria, it has not been possible to identify their undifferentiated progenitors, or to isolate them for molecular analysis. To overcome this limitation, we have taken advantage of the laminar distribution of the progenitors for different retinal cell types, to devise a strategy based on the use of laser capture micro–dissection (LCM) to isolate morphologically undifferentiated photoreceptor progenitors located in the outer nuclear layer (ONL) of the developing retina. ONL samples are processed for cDNA or aRNA synthesis, and compared with samples from other retinal layers and/or other developmental stages.
Methods: :
On embryonic day (ED) 8 and 18, unfixed retinas were cryoprotected and frozen, and 7–10µm sections were collected on PEN foil slides. ONL and inner retina (IR) layers were isolated with a Leica LMD6000 system, using an optically guided laser. Samples were lysed and processed for cDNA synthesis with PCR–based global amplification, or for aRNA synthesis. Expression of candidate genes was investigated by PCR and QPCR ("real time" PCR).
Results: :
At ED8, when photoreceptor progenitors are still undifferentiated, the laminar pattern of the retina and the ONL/IR boundary are well established; boundaries were respected during isolation of ONL and IR cells by LCM. Lack of cross–contamination was corroborated by PCR analysis. Transcription factors known to be restricted to the inner retina, such as Pax6, Chx10, NeuroM, and NeuroB, were detected in IR samples but not in ONL samples which, however, had signals for Crx, Rax2 and NeuroD. Muller cell markers were restricted to the IR at E8, but were also detected in ONL samples on ED18, probably reflecting the presence of Muller cell processes in that layer. The red, green, and rod–opsin visual pigment mRNAs were detected only in cDNA from the ONL, and were particularly abundant at ED 18.
Conclusions: :
The studies demonstrate the usefulness of LCM for the isolation of specific layers of the chick embryo retina for molecular analysis, opening a new avenue for the characterization of photoreceptor progenitor cells.
Keywords: photoreceptors • gene/expression • transcription factors