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Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation

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Abstract

The chicken retina contains five visual photoreceptor subtypes, based on the specific opsin gene they express. In addition to the central role they play in vision, some or all of these photoreceptors translate photoperiodic information into a day–night rhythm of melatonin production. This indolic hormone plays an important role in the photoperiodic regulation of retinal physiology. Previous studies have stopped short of establishing whether melatonin synthesis takes place in all the photoreceptor spectral subtypes. Another issue that has been left unsettled by previous studies is when during development are retinal precursor cells committed to a specific photoreceptor subtype and to a melatoninergic phenotype? To address the first question, in situ hybridization of the five opsins was combined with immunofluorescent detection of the melatonin-synthesizing enzyme hydroxyindole O-methyltransferase (HIOMT, EC.2.1.1.4). Confocal microscopy clearly indicated that all photoreceptor spectral subtypes are involved in melatonin synthesis. To tackle the second question, retinal precursor cells were dissociated between embryonic day 6 (E6) and E13 and cultured in serum-free medium for 4 days to examine their ability to autonomously activate the expression of opsins and HIOMT. Real-time PCR on cultured precursors indicated that red-, green- and violet-sensitive cones are committed at E6, rods at E10 and blue-sensitive cones at E12. HIOMT gene expression was programmed at E6, probably reflecting the differentiation of early cones. The present study provides a better characterization of photoreceptor subtypes in the chicken retina and describes a combination of serum-free culture and real-time PCR that should facilitate further developmental studies.

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Acknowledgements

We would like to thank Mrs L. Cousin for her technical help at multiple steps of this work. We appreciate the technical assistance of the Service Commun de Publication Assistée par Ordinateur (Mr J. Habrioux).

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  1. Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS, 1 rue Georges Bonnet, Poitiers, 86022, France

    Pierre Voisin, V. Cailleau, N. Naud, A. Cantereau & M. Bernard

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  1. Pierre Voisin
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  2. V. Cailleau
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  3. N. Naud
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  4. A. Cantereau
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  5. M. Bernard
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Correspondence to Pierre Voisin.

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This work was supported by the CNRS (UMR 6187) and Retina France.

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Fig. S1

Northern blot validation of the probes used in ISH. Total RNA (20 μg) from adult chicken retina was electrophoresed on agarose gel, transferred on nitrocellulose membrane and hybridized under high-stringency conditions with 32P-labeled DNA probes specific for each of the five chicken opsins mRNAs. The probes were obtained by excising the inserts of the pGEMTeasy® plasmids used for generating the ISH cRNA probes. The radioactive blots were analyzed on a PhosphorImager. The sizes of the transcripts were estimated by comparing their migration with those of ribosomal RNAs (28S: 4 kb; 18S: 1.8 kb): RH1, 1.6 kb (major band) and 3 kb (minor band, possibly corresponding to unprocessed nuclear RNA); LWS, 1.6 kb; RH2, 2.8 kb; SWS2, 3.1 kb and SWS1, 3.7 kb (JPEG 21 kb)

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Fig. S2

Cell viability of retinal precursors cultured in serum-free medium. Retinal precursors dissociated at different incubation times (from E6 to E13) were cultured for 4 days in serum-free medium. Cell viability was measured with CellTiter96® on the starting day (t0) and after 4 days in culture. Results are presented as percentage of cell viability relative to t0. This shows that the absence of serum did not affect cell survival at any developmental stage (JPEG 30 kb)

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Voisin, P., Cailleau, V., Naud, N. et al. Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation. Cell Tissue Res 348, 417–427 (2012). https://doi.org/10.1007/s00441-012-1374-z

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  • DOI: https://doi.org/10.1007/s00441-012-1374-z

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