Yuki Nakatani

Cellular and molecular processes of regeneration, with special emphasis on fish fins.

The phenomenon of ‘epimorphic regeneration’, a complete reformation of lost tissues and organs from adult differentiated cells, has been fascinating many biologists for many years. While most vertebrate species including humans do not have a remarkable ability for regeneration, the lower vertebrates such as urodeles and fish have exceptionally high regeneration abilities. In particular, the teleost fish has a high ability to regenerate a variety of tissues and organs including scales, muscles, spinal cord and heart among vertebrate species. Hence, an understanding of the regeneration mechanism in teleosts will provide an essential knowledge base for rational approaches to tissue and organ regeneration in mammals. In the last decade, small teleost fish such as the zebrafish and medaka have emerged as powerful animal models in which a variety of developmental, genetic and molecular approaches are applicable. In addition, rapid progress in the development of genome resources such as expressed sequence tags and genome sequences has accelerated the speed of the molecular analysis of regeneration. This review summarizes the current status of our understanding of the cellular and molecular basis of regeneration, particularly that regarding fish fins.

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

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.

Ras is an essential component for notochord formation during ascidian embryogenesis

In ascidian embryos, inductive interactions are necessary for the fate specification of notochord cells. Previous studies have shown that notochord induction occurs at the 32-cell stage and that basic fibroblast growth factor (bFGF) has notochord-inducing activity in ascidian embryos. In vertebrate, it is known that bFGF receptors have tyrosine kinase domain and the signaling pathway is mediated by the small-GTP binding protein, Ras. To study the role of Ras in ascidian embryos, we injected dominant negative Ras (RasN17) into fertilized eggs. RasN17 inhibited the formation of notochord, suggesting that the Ras signaling pathway is involved in signal transduction in the induction of notochord cells. When the presumptive-notochord (A6.2) blastomere was co-isolated with the inducer (A6.1) blastomere and then RasN17 was injected into the A6.2 blastomere, notochord differentiation was suppressed. The presumptive-notochord blastomeres injected with RasN17 were treated with bFGF. Many of them failed to develop notochord-specific features. Next, we examined the effect of injecting constitutively active Ras (RasV12) into the A6.2 blastomeres. However, microinjection of RasV12 into these cells did not bypass notochord induction. These results suggest that the Ras signaling pathway is essential for the formation of notochord and that another signaling pathway also must be activated simultaneously in notochord formation during ascidian embryogenesis.

Identification of novel markers expressed during fin regeneration by microarray analysis in medaka fish

Urodeles and fish have a remarkable ability to regenerate lost body parts, whereas many higher vertebrates, including mammals, retain only a limited capacity. It is known that the formation of specialized cell populations such as the wound epidermis or blastema is crucial for regeneration; however, the molecular basis for their formation has not been elucidated. Recently, approaches using differential display and microarray have been done in zebrafish for searching molecules involved in regeneration. Here, we used the medaka fish, a distantly diverged fish species, for microarray screening of transcripts up‐regulated during regeneration. By setting criteria for selecting transcripts that are reliably and reproducibly up‐regulated during regeneration, we identified 140 transcripts. Of them, localized in situ expression of 12 transcripts of 22 tested was detected either in differentiating cartilage, basal wound epidermis, or blastema. Our results provide useful molecular markers for dissecting the regeneration process at a fine cellular resolution. Developmental Dynamics 236:2685–2693, 2007.

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3‐kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.

Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis.

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32‐cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32‐cell stage and is completed by the 64‐cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32‐cell embryos, and were heterochronously recombined at various stages ranging from the early 32‐cell stage to the 64‐cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32‐cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64‐cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.

Development of the lateral plate mesoderm in medaka Oryzias latipes and Nile tilapia Oreochromis niloticus: insight into the diversification of pelvic fin position.

The position of the pelvic fins among teleost fishes has tended to shift rostrally during evolution. This positional shift seems to have led to the diversification of feeding behavior and allowed adaptation to new environments. To understand the developmental basis of this shift in pelvic fin position among teleosts, we investigated the embryonic development of the lateral plate mesoderm, which gives rise to the pelvic fins, at histological levels in the medaka Oryzias latipes (abdominal pelvic fins) and Nile tilapia Oreochromis niloticus (thoracic pelvic fins). Our histological analyses revealed that the lateral plate mesodermal cells expand not only ventrally but also rostrally to cover the yolk during embryogenesis of both medaka and Nile tilapia. In medaka, we also found that the lateral plate mesoderm completely covered the yolk prior to the initiation of the pelvic fin buds, whereas in Nile tilapia the pelvic fin buds appeared in the body wall from the lateral plate mesoderm at the thoracic level when the lateral plate mesodermal cells only covered one‐third of the yolk. We discuss the relevance of such differences in the rate of the lateral plate mesoderm expansion on the yolk surface and the position of the pelvic fins.