Background: Zebrafish have the ability for heart regeneration. However, another teleost animal model, the medaka, had not yet been investigated for this capacity. Results: Compared with zebrafish, the medaka heart responded differently to an injury: An excessive fibrotic response occurred in the medaka heart, and existing cardiomyocytes or cardiac progenitor cells remained dormant, resulting in no numerical difference between the uncut and injured heart with respect to the number of EdUincorporated cardiomyocytes. The results obtained from the analysis of the medaka raldh2-GFP transgenic line showed a lack of raldh2 expression in the endocardium. Regarding periostin expression, the localization of medaka periostin-b, a marker of fibrillogenesis, in the medaka heart remained at the wound site at 30 dpa; whereas zebrafish periostin-b was no longer localized at the wound but was detected in the epicardium at that time. Conclusions: Compared with zebrafish heart regeneration, the medaka heart phenotypes suggest the possibility that the medaka could hardly regenerate its heart tissue or that these phenotypes for heart regeneration showed a delay.

The vertebral column is a defined feature of vertebrates. In birds and mammals, the sclerotome yields cartilaginous material for the vertebral column. In teleosts, however, it remains uncertain whether the sclerotome participates in vertebral column formation. To investigate osteoblast development in the teleost, we established transgenic systems that allow in vivo observation of osteoblasts and their progenitors marked by fluorescence of DsRed and enhanced green fluorescent protein (EGFP), respectively. In twist-EGFP transgenic medaka, EGFP-positive cells first appeared in the ventromedial portion of respective somites corresponding to the sclerotome, migrated dorsally around the notochord, and concentrated in the intervertebral regions. Ultrastructural analysis of the intervertebral regions revealed that some of these cells were directly located on the osteoidal surface of the perichordal centrum, and enriched with rough endoplasmic reticulum in their cytoplasm. By using the double transgenic medaka of twist-EGFP and osteocalcin-DsRed, we clarified that the EGFP-positive cells in the intervertebral region differentiated into mature osteoblasts expressing the DsRed. In vivo bone labeling in fact confirmed active matrix formation and mineralization of the perichordal centrum exclusively in the intervertebral region of zebrafish larvae as well as medaka larvae. These findings strongly suggest that the teleost intervertebral region acts as a growth center of the perichordal centrum, where the sclerotome-derived cells differentiate into osteoblasts. Developmental Dynamics 236:3031-3046, 2007.

The vertebrate mineralized skeleton is known to have first emerged as an exoskeleton that extensively covered the fossil jawless fish. The evolutionary origin of this exoskeleton has long been attributed to the emergence of the neural crest, but experimental evaluation for this is still poor. Here we determine the embryonic origin of scales and fin rays of medaka (teleost trunk exoskeletons) by applying long-term cell labelling methods, and demonstrate that both tissues are mesodermal in origin. Neural crest cells, however, fail to contribute to these tissues. This result suggests that the trunk neural crest has no skeletogenic capability in fish, instead highlighting the dominant role of the mesoderm in the evolution of the trunk skeleton. This further implies that the role of the neural crest in skeletogenesis has been predominant in the cephalic region from the early stage of vertebrate evolution.

Hatching enzyme is an enzyme that digests an egg envelope at the time of embryo hatching. Fish hatching enzymes have been purified from several fish species [1][2][3][4][5]. Among them, the hatching enzyme of medaka Oryzias latipes has been extensively studied, and its study field was extended not only to characterization of the enzyme itself, but also to the mechanism of its egg envelope digestion [6,7]. The hatching of There are two hatching enzyme homologues in the zebrafish genome: zebrafish hatching enzyme ZHE1 and ZHE2. Northern blot and RT-PCR analysis revealed that ZHE1 was mainly expressed in pre-hatching embryos, whereas ZHE2 was rarely expressed. This was consistent with the results obtained in an experiment conducted at the protein level, which demonstrated that one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid. Therefore, the hatching of zebrafish embryo is performed by a single enzyme, different from the finding that the medaka hatching enzyme is an enzyme system composed of two enzymes, medaka high choriolytic enzyme (MHCE) and medaka low choriolytic enzyme (MLCE), which cooperatively digest the egg envelope. The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. The digestion manner of ZHE1 appears to be highly analogous to that of MHCE, which partially digests the egg envelope and swells the envelope. The cross-species digestion using enzymes and substrates of zebrafish and medaka revealed that both ZHE1 and MHCE cleaved the same sites of the egg envelope proteins of two species, suggesting that the substrate specificity of ZHE1 is quite similar to that of MHCE. However, MLCE did not show such similarity. Because HCE and LCE are the result of gene duplication in the evolutionary pathway of Teleostei, the present study suggests that ZHE1 and MHCE maintain the character of an ancestral hatching enzyme, and that MLCE acquires a new function, such as promoting the complete digestion of the egg envelope swollen by MHCE.Abbreviations MCA, 7-amino-4-methylcoumarin; MHCE, medaka high choriolytic enzyme; MLCE, medaka low choriolytic enzyme; ZHE, zebrafish hatching enzyme; ZPD, ZP domain.

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