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Updated: Jul 2, 2026
Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs
Published on: May 18, 2022
Microarray analysis of retinal gene expression in chicks during imposed myopic defocus
Ruth Schippert1, Frank Schaeffel, Marita Pauline Feldkaemper
1Institute for Ophthalmic Research, Section of Neurobiology of the Eye, University Eye Hospital Tuebingen, Tuebingen, Germany.
Purpose:
The retina plays an important regulatory role in ocular growth. To screen for new retinal candidate genes that could be involved in the inhibition of ocular growth, we used chick microarrays to analyze the changes in retinal mRNA expression after myopic defocus was imposed by positive lens wear.
Methods:
Four male white leghorn chicks, aged nine days, wore +6.9D spectacle lenses over both eyes for 24 h. Four untreated age-matched male chicks from the same batch served as controls. The chicks were euthanized, and retinas from both eyes of each chick were pooled. RNA was isolated and labeled cRNA was prepared. These samples were hybridized to Affymetrix GeneChip Chicken Genome arrays with more than 28,000 characterized genes. After comparison of multiple normalization methods, GC-RMA and a false-discovery rate of 6% was chosen for normalization of the data. The expression of 16 candidate genes was further studied, using semiquantitative real-time RT-PCR. In addition, the expression of the mRNA of some of these candidate genes was assessed in chicks that wore either +6.9D lenses for 4 h or -7D lenses for 24 h.
Results:
123 transcripts were found to be differentially expressed (p<0.05; at least 1.5-fold change in expression level), with an absolute mean fold-change of 1.97+/-1.16 (mean+/-standard deviation). Nine of the sixteen genes that were examined by real-time RT-PCR were validated. Regardless of whether positive or negative lenses were worn, six of these nine genes were regulated in the same direction after 24 h: arginyltransferase 1 (ATE1), E74-like factor 1 (ELF1), growth factor receptor-bound protein 2 (GRB2), SHQ1 homolog (S. cerevisiae) (SHQ1), spectrin, beta, non-erythrocytic 1 (SPTBN1), prepro-urotensin II-related peptide (pp-URP). Three genes responded differently to positive and negative lens treatment after 24 h: ATP-binding cassette, sub-family C, member 10 (ABCC10), CD226 molecule (CD226) and oxysterol binding protein 2 (OSBP2).
Conclusions:
The validated genes that were regulated only by myopic defocus may represent elements in a pathway generating a "stop-signal" for eye growth. Some of the genes identified in this study have so far not been described in the retina. Further investigation of their function may improve the understanding of the signaling cascades in emmetropization. More general, published microarray data are variable among different animal models (mouse, chick, monkeys), tissues (retina, retina/retinal pigment epithelium), treatments (diffusers, lenses, lid-suture), as well as different treatment durations (hours, days), and comparisons remain difficult. That only a small number of common genes were found emphasizes the need for careful normalization of the experimental parameters.
Insights
This study identified novel retinal genes involved in inhibiting eye growth by analyzing gene expression changes in chicks exposed to myopic defocus. These findings offer new insights into the signaling pathways regulating emmetropization.
Area of Science:
- Ophthalmology
- Genetics
- Molecular Biology
Background:
- The retina is crucial for regulating eye growth.
- Identifying genes that inhibit ocular growth is essential for understanding emmetropization.
Purpose of the Study:
- To screen for novel retinal candidate genes involved in inhibiting ocular growth.
- To analyze changes in retinal mRNA expression in response to myopic defocus induced by positive lens wear in chicks.
Main Methods:
- White leghorn chicks wore +6.9D spectacle lenses for 24 hours; untreated chicks served as controls.
- Retinal mRNA expression was analyzed using Affymetrix GeneChip Chicken Genome arrays.
- Gene expression was further validated using semiquantitative real-time RT-PCR, with additional experiments using +6.9D and -7D lenses for varying durations.
Main Results:
- 123 transcripts showed differential expression (p<0.05, ≥1.5-fold change).
- Nine out of sixteen examined genes were validated by RT-PCR.
- Six genes (ATE1, ELF1, GRB2, SHQ1, SPTBN1, pp-URP) were consistently regulated by myopic defocus after 24 hours, while three genes (ABCC10, CD226, OSBP2) showed differential responses to positive and negative lens treatments.
Conclusions:
- Validated genes regulated solely by myopic defocus may constitute a 'stop-signal' pathway for eye growth.
- Several identified genes have not been previously described in the retina, offering new avenues for emmetropization research.
- Variability in microarray data across different models and treatments highlights the need for standardized experimental parameters.
