Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka, Oryzias latipes

Abstract

Medaka is an attractive model to study epimorphic regeneration. The fins have remarkable regenerative capacity and are replaced about 14 days after amputation. The formation of blastema, a mass of undifferentiated cells, is essential for regeneration; however, the molecular mechanisms are incompletely defined. To identify the genes required for fin regeneration, especially for blastema formation, we constructed cDNA libraries from fin regenerates at 3 days postamputation and 10 days postamputation. A total of 16,866 expression sequence tags (ESTs) were sequenced and subjected to BLASTX analysis. The result revealed that about 60% of them showed strong matches to previously identified proteins, and major signaling molecules related to development, including FGF, BMP, Wnt, Notch/Delta, and Ephrin/Eph signaling pathways were isolated. To identify novel genes that showed specific expression during fin regeneration, cDNA microarray was generated based on 2900 independent ESTs from each library which had no sequence similarity to known proteins. We obtained 6 candidate genes associated with blastema formation by gene expression pattern screening in competitive hybridization analyses and in situ hybridization. Olrfe16d23 and olrfe14k04 were expressed only in early regenerating stages when blastema formation was induced. The expression of olrf5n23, which encodes a novel signal peptide, was detected in wound epidermis throughout regeneration. Olrfe23l22, olrfe20n22, and olrfe24i02 were expressed notably in the blastema region. Our study has thus identified the gene expression profiles and some novel candidate genes to facilitate elucidation of the molecular mechanisms of fin regeneration.

Global analysis of gene expression inXenopushindlimbs during stage‐dependent complete and incomplete regeneration

“…Consistent with Katogi et al (2004) who found SOCS3 (identified as olrf30i08) expressed at higher levels at day 3 postamputation vs. day 10 postamputation in regenerating Medaka fins, we find that the induced expression of SOCS3 declines 14-fold from 1dPA to 5dPA at the regeneration-complete stage. However, at the regeneration-incomplete stage, the decline in expression is only 1.3-fold (Katogi et al, 2004).…”

“…The results of eight previously published regeneration-related genomic analyses were selected for comparison to the microarray results (Ishino et al, 2003;King et al, 2003;Summan et al, 2003;Katogi et al, 2004;Swamy et al, 2004;Wolfe et al, 2004;Reddien et al, 2005;Schnapp et al, 2005;Tazaki et al, 2005). Due to the variety of organisms studied in these articles and the information provided, different approaches were taken to compare them to the Xenopus data.…”

“…However, at the regeneration-incomplete stage, the decline in expression is only 1.3-fold (Katogi et al, 2004). Continued expression of SOCS3 at the regeneration-incomplete stage compared to the regeneration-complete stage may indicate an attempt to negatively regulate the chronic cytokineinduced inflammation of st57 5dPA pseudoblastemas relative to st53 blastemas (Schreiber et al, 2002).…”

“…This work has been conducted using many different organisms, including Planaria, fish, frogs, salamanders, mice, and humans (Ishino et al, 2003;King et al, 2003;Summan et al, 2003;Katogi et al, 2004;Swamy et al, 2004;Wolfe et al, 2004;Reddien et al, 2005;Schnapp et al, 2005;Tazaki et al, 2005). Through various approaches (see the Experimental Procedures section), we compared our microarray results to the reported results of eight other published works (Tables 2-5).…”

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb

“…Given that the efficiency of knock-down varies greatly between morpholinos, it would be difficult to argue for using this technique to compare which of two genes has a greater effect on fin outgrowth. However, as the expression of new molecules during regeneration are being identified in microarray or differential display screens, this technique does permit placing these molecules into known regeneration pathways and revealing the functional relevance of these genes for regeneration (Katogi et al, 2004;Padhi et al, 2004). For example, knock-down of Msxb did not effect msxc expression at 3 days post-amputation, but both msxb and msxc expression were greatly reduced or absent when Fgfr1 levels were knocked-down (Poss et al, 2000; Fig.…”