Animals and operations
Adult male newts Cynops pyrrhogaster were collected in Okazaki, Japan in 1994. At the time, we estimate that they were at least 14 years of age: one of us (G.E., unpublished observations) has determined that it takes more than 14 years for the body of a male newt to attain 90% the size of a full-grown newt (average body length: 11.6±2 cm). When collected the average body length of our specimens was equivalent to that of full-grown male newts. Thus by the end of our experiments these animals were likely almost 30 years old. The first fourteen lentectomies were performed every April 15 and October 15 from 1996 to 2002. Lentectomies number 15 and number 16 were performed on the same dates in 2008. Lentectomies number 17 and number 18, which provided tissues for this analysis were performed on July 9, 2009 and February 2, 2010, respectively. Lenses from newts of similar size that had never been lentectomized served as controls. Control newts were born in 1996 from eggs collected in the laboratory, and at the time of lens collection for RNA analysis, they were about 14-years-old. Usage of animals complied with the institutional regulations.
For lentectomies, newts were anaesthetized in 0.1% ethyl 3-aminobenzoate (Sigma) prepared in fresh water. Using a sharp blade, an incision was made in the cornea along the nasal temporal axis. The lens was then removed in its entirety with fine forceps.
Histology and immunofluorescence
Whole lenses were fixed and kept in 1% PFA. After paraffin embedding and before sectioning, tissue blocks were soaked in cold water, allowing the lens to soften and improving tissue morphology. 10 μm sections were stained with mouse β5-crystallin antibody to visualize lens fibre arrangement.
RNA isolation reverse transcription and first-strand cDNA synthesis
Total RNA was extracted from carefully isolated regenerated lenses after lentectomy using the Qiagen RNeasy Plus purification kit (Qiagen). Samples were then flash-frozen in liquid nitrogen and homogenized using lysis buffer from the kit, and RNA isolation was performed according to the manufacturer's instructions. Briefly, samples were lysed and homogenized in a highly denaturing guanidine-isothiocyanate–containing buffer. The lysate is then passed through a gDNA eliminator spin column to remove genomic DNA. Ethanol is added to the flow-through to provide appropriate binding conditions for RNA, and the sample is then applied to an RNeasy spin column, where total RNA binds to the membrane and contaminants are washed away. High-quality RNA is then eluted in 30 μl, or more, of water.
Quantities of 100 ng of total RNA were reverse transcribed using Invitrogen Superscript II reverse transcriptase according to the manufacturer's instructions (Invitrogen). First-strand complimentary DNA synthesis was performed using SuperScript II RT.
Quantitative PCR was performed using a Stratagene MX300 machine (Agilent). The SYBR GreenER (Invitrogen) qPCR reagent system was used to amplify cDNA. The following primers were: Gapdh forward, 5′-GCATGCTGTGACTGCTACACAAAAG-3′ and reverse, 5′-GCTGGAATGATATTCTGGTTTGCAC-3′; Ef1a forward, 5′-GCACCACGAGGCGCTGGT-3′ and reverse, 5′-CAACACAGGAGCGTATCCCTG-3′; alphaA-Crystallin forward, 5′-TCACCGGAAGACCTAAGTGTC-3′ and reverse, 5′-GGTCAGCATGCCATCAGTGG-3′; betaB1-Crystallin forward, 5′-GGATACCTGGTCTAACAG-3′ and reverse, 5′-GCCACTGCATGTCCCTG-3′; gamma-Crystallin forward, 5′-CCTATGAGTGCAGCACTGAGT-3′ and reverse, 5′-GTCATTGAAGCCCATCCAGTG-3′; Delta1 forward, 5′-CCGACCGGCTCATCAGTCGT-3′ and reverse, 5′-CCCCGCAGGTGAAGTGCC-3′; Mafb forward, 5′-AGAGCACCACGCCTCGGA-3′ and reverse, 5′-GACAATCCCCAACACAAC-3′; Pax6 forward, 5′-AGGCCTCCTCCTACTCTTGC-3′ and reverse, 5′-GGGAAATGAGACCTGTCGAA-3′; Prox1 forward, 5′-ACATGTGCAGCAACTCTTCG-3′ and reverse, 5′-CATCCCTCGGATGATGTTCT-3′; Sox1 forward, 5′-CGCCCTGTCCGCAGAGG-3′ and reverse, 5′-GCTAGGATAGCCGCATGTTC-3′; and Sox2 forward, 5′-AAGTTTCGCCAACTTCC-3′ and reverse, 5′-GGAGTTAAGAATGCCGGTG-3′. Steps used to perform qPCR analysis included a hold step at 50 °C for 2 min to activate uracil-DNA glycosylase, followed by another hold at 95 °C for 10 min. Samples then underwent 38 cycles of 95 °C for 30 s followed by 58–60 °C for 45 s. Subsequently, melt analysis was performed by increasing the temperature from 65 to 95 °C. Relative expression levels were calculated using the ΔΔCt-method in which the dCt value for each gene was first calculated by subtracting the Ct value (lowest) of a sample from rest of the samples Ct values. Then the Relative Unit (RU) was calculated as log2 of the Ct value. RU values of all genes were then normalized to the RU value of the housekeeping gene (EF1a or GAPDH) for each sample by dividing the RU of a sample with that of the corresponding housekeeping gene. Average RUs from four control and four experimental samples were then calculated and plotted in individual graphs. A t-test was used to calculate P-values for significance.