Evolutionary pathway of pseudogenization of globin genes, α5 and ß5, in genus Oryzias.

Hemoglobin transports oxygen in many organisms and consists of α- and ß-globin chains. Previously, using molecular phylogenetic analysis, we proposed that both α- and ß-globins of teleost could be classified into four groups. We also showed that the Hd-rR strain of medaka (Oryzias latipes) inhabiting southern Japan had all four groups of globin genes but that the α- and ß-globin genes of group III were pseudogenized (α5(ψα), ß5(ψß)). Based on the small degree of nucleotide variations, the pseudogenization of ß5 was assumed to have occurred at a relatively late stage of evolution. Here, we compared the α5(ψα)-ß5(ψß) of two other strains of O. latipes and found that both α5(ψα) and ß5(ψß) of the northern Japanese and Korean strains were pseudogenized similar to those of Hd-rR. In a Philippine population (Oryzias luzonensis), α5(ψα) was also pseudogenized, but the structure was different from that of O. latipes, and ß5(ψß) was almost deleted. Interestingly, an Indonesian population (Oryzias celebensis) had α5 and ß5 genes that were deduced to be functional. Indeed, they were expressed from the young to adult development stages, and this expression pattern was consistent with the expression of α2 and ad.α1 in Hd-rR. Because α2 and ad.α1 in Hd-rR were assigned to groups I and II, respectively, we speculate that their expression patterns might be altered by pseudogenization of group III genes. These results provide a basis for further investigations of recruiting and changing expression patterns of one globin gene after pseudogenization of other globin genes during evolution.

Molecular co-evolution of a protease and its substrate elucidated by analysis of the activity of predicted ancestral hatching enzyme.

BACKGROUND: Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. We have comprehensively studied the molecular mechanisms of the enzyme action to its substrate egg envelope, and determined the gene/protein structure and phylogenetic relationships. Because the hatching enzyme must have evolved while maintaining its ability to digest the egg envelope, the hatching enzyme-egg envelope protein pair is a good model for studying molecular co-evolution of a protease and its substrate. RESULTS: Hatching enzymes from medaka (Oryzias latipes) and killifish (Fundulus heteroclitus) showed species-specific egg envelope digestion. We found that by introducing four medaka-type residue amino acid substitutions into recombinant killifish hatching enzyme, the mutant killifish hatching enzyme could digest medaka egg envelope. Further, we studied the participation of the cleavage site of the substrate in the species-specificity of hatching enzyme. A P2-site single amino acid substitution was responsible for the species-specificity. Estimation of the activity of the predicted ancestral enzymes towards various types of cleavage sites along with prediction of the evolutionary timing of substitutions allowed prediction of a possible evolutionary pathway, as follows: ancestral hatching enzyme, which had relatively strict substrate specificity, developed broader specificity as a result of four amino acid substitutions in the active site cleft of the enzyme. Subsequently, a single substitution occurred within the cleavage site of the substrate, and the recent feature of species-specificity was established in the hatching enzyme-egg envelope system. CONCLUSIONS: The present study clearly provides an ideal model for protease-substrate co-evolution. The evolutionary process giving rise to species-specific egg envelope digestion of hatching enzyme was initiated by amino acid substitutions in the enzyme, resulting in altered substrate specificity, which later allowed an amino acid substitution in the substrate.

Adaptive evolution of fish hatching enzyme: one amino acid substitution results in differential salt dependency of the enzyme.

Embryos of medaka Oryzias latipes hatch in freshwater, while those of killifish Fundulus heteroclitus hatch in brackish water. Medaka and Fundulus possess two kinds of hatching enzymes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), which cooperatively digest their egg envelope at the time of hatching. Optimal salinity of medaka HCE was found in 0 mol l(-1) NaCl, and activity decreased with increasing salt concentrations. One of the two Fundulus HCEs, FHCE1, showed the highest activity in 0 mol l(-1) NaCl, and the other, FHCE2, showed the highest activity in 0.125 mol l(-1) NaCl. The results suggest that the salt dependencies of HCEs are well adapted to each salinity at the time of hatching. Different from HCE, LCEs of both species maintained the activity sufficient for egg envelope digestion in various salinities. The difference in amino acid sequence between FHCE1 and FHCE2 was found at only a single site at position 36 (Gly/Arg), suggesting that this single substitution causes the different salt dependency between the two enzymes. Superimposition of FHCE1 and FHCE2 with the 3-D structure model of medaka HCE revealed that position 36 was located on the surface of HCE molecule, far from its active site cleft. The results suggest a hypothesis that position 36 influences salt-dependent activity of HCE, not with recognition of primary structure around the cleavage site, but with recognition of higher ordered structure of egg envelope protein.

1kbp 5' upstream sequence enables developmental stage-specific expressions of globin genes in the fish, medaka Oryzias latipes.

Hemoglobin of bony fish and higher vertebrates is a tetrameric protein constructed by 2 α- and 2 ß-globins, which are expressed in a developmental stage-specific manner. The genomic organization of genes for embryonic and adult α- and ß-globin varies from species to species. In fish, it is known that there is a unique genomic organization of globin genes, that is, α- and ß-globin genes are arranged in a bi-directional and head-to-head orientation with respect to transcription start sites. In medaka, we have demonstrated that 14 globin genes are located in 2 different clusters, and 5 pairs of the α- and ß-globin genes were found to be organized in a head-to-head orientation. The developmental expression patterns of the 11 globin genes were classified into 4 types. To clarify how their developmental stage-specific expressions are regulated, we produced 4 types of GFP- or RFP-transgenic medaka. Such transgenic medaka revealed that each of the 1-1.7 kbp 5' upstream sequences from respective globin genes possesses the ability to regulate the developmental stage-specific globin gene expression. In particular, the intervals between head-to-head α3 and ß3, and α4 and ß4 genes controlled the synchronized expression of the globin genes located at both sides of the intervals, which is significant to understand the mechanism by which equal amounts of α- and ß-globins are expressed in erythroid cells. We also demonstrated that the head-to-head intervals can control the expression of the globin genes located at both sides. These findings are significant to understand the mechanism by which α- and ß-globins are equally expressed in erythroid cells.

Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism.

We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei.

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved.

Evolution of the teleostean zona pellucida gene inferred from the egg envelope protein genes of the Japanese eel, Anguilla japonica.

A fish egg envelope is composed of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among vertebrate species. Euteleost fishes synthesize ZP proteins in the liver, while otocephalans synthesize them in the growing oocyte. We investigated ZP proteins of the Japanese eel, Anguilla japonica, belonging to Elopomorpha, which diverged earlier than Euteleostei and Otocephala. Five major components of the egg envelope were purified and their partial amino acid sequences were determined by sequencing. cDNA cloning revealed that the eel egg envelope was composed of four ZPC homologues and one ZPB homologue. Four of the five eel ZP (eZP) proteins possessed a transmembrane domain, which is not found in the ZP proteins of Euteleostei and Otocephala that diverged later, but is found in most other vertebrate ZP proteins. This result suggests that fish ZP proteins originally possessed a transmembrane domain and lost it during evolution. Northern blotting and RT-PCR revealed that all of the eZP transcripts were present in the ovary, but not in the liver. Phylogenetic analyses of fish zp genes showed that ezps formed a group with other fish zp genes that are expressed in the ovary, and which are distinct from the group of genes expressed in the liver. Our results support the hypothesis that fish ZP proteins were originally synthesized in the ovary, and then the site of synthesis was switched to the liver during the evolutionary pathway to Euteleostei.

Intron-loss evolution of hatching enzyme genes in Teleostei.

BACKGROUND: Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon-intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution. RESULTS: We investigated the evolutionary pathway of intron-losses in hatching enzyme genes of 27 species of Teleostei. Hatching enzyme genes of basal teleosts are of only one type, which conserves the 9-exon-8-intron structure of an assumed ancestor. On the other hand, otocephalans and euteleosts possess two types of hatching enzyme genes, suggesting a gene duplication event in the common ancestor of otocephalans and euteleosts. The duplicated genes were classified into two clades, clades I and II, based on phylogenetic analysis. In otocephalans and euteleosts, clade I genes developed a phylogeny-specific structure, such as an 8-exon-7-intron, 5-exon-4-intron, 4-exon-3-intron or intron-less structure. In contrast to the clade I genes, the structures of clade II genes were relatively stable in their configuration, and were similar to that of the ancestral genes. Expression analyses revealed that hatching enzyme genes were high-expression genes, when compared to that of housekeeping genes. When expression levels were compared between clade I and II genes, clade I genes tends to be expressed more highly than clade II genes. CONCLUSIONS: Hatching enzyme genes evolved to lose their introns, and the intron-loss events occurred at the specific points of teleostean phylogeny. We propose that the high-expression hatching enzyme genes frequently lost their introns during the evolution of teleosts, while the low-expression genes maintained the exon-intron structure of the ancestral gene.

Mechanism of egg envelope digestion by hatching enzymes, HCE and LCE in medaka, Oryzias latipes.

Hatching of medaka embryos from the fertilized egg envelope involves two enzymes, HCE and LCE. HCE swells the envelope and then LCE completely dissolves it. We determined HCE and LCE cleavage sites on the egg envelope that are primarily constructed of two groups of subunit proteins, ZI-1,2 and ZI-3. HCE and LCE cleaved different target sequences on the egg envelope proteins but shared one common cleavage site. HCE cleaved the N-terminal region of ZI-1,2 and ZI-3, mainly the Pro-Xaa-Yaa repeat sequence of ZI-1,2 into hexapeptides, but not the site within a zona pellucida (ZP) domain that is considered to be the core structure of the egg envelope. The cleavage of these N-terminal regions results in swelling and softening of the envelope. LCE cleaved the middle of the ZP domain of ZI-1,2, in addition to the upstream of the trefoil domain of ZI-1,2 and the ZP domain of ZI-3. This middle site is in the intervening sequence connecting two subdomains of the ZP domain. Cleaving this site would result in the solubilization of the swollen egg envelope by the disruption of the filamentous structure that is thought to be formed by the non-covalent polymerization of ZP domains.

Crystal structure of zebrafish hatching enzyme 1 from the zebrafish Danio rerio.

Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 Å resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3(10)-helices, three α-helices, and two ß-sheets. The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1' contributes the most and that the residues at P4-P2' also contribute to the recognition of the major substrate peptide by ZHE1, whereas GluP3' and the hydrophobic residues at P4-P2, P2', and P5' contribute significantly to the recognition of the minor substrate peptide by ZHE1. Molecular models of these two substrate peptides bound to ZHE1 have been built based on the crystal structure of a transition-state analog inhibitor bound to astacin. In substrate-recognition models, the AspP1' in the major substrate peptide forms a salt bridge with Arg182 of ZHE1, while the GluP3' in the minor substrate peptide instead forms a salt bridge with Arg182. Thus, these two substrate peptides would be differently recognized by ZHE1. The shapes and electrostatic potentials of the substrate-binding clefts of ZHE1 and the structurally similar proteins astacin and bone morphogenetic protein 1 are significantly dissimilar due to different side chains, which would confer their distinctive substrate preferences.

Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio.

The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0-1.80 and 50.0-1.14 A were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0-1.14 A. The space group was assigned as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 A. The crystal contained one ZHE1 molecule in the asymmetric unit.

Different hatching strategies in embryos of two species, pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, that belong to the same order Clupeiformes, and their environmental adaptation.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis. They were expressed specifically in hatching gland cells (HGCs), which differentiated from the pillow and migrated to the edge of the head in both species. HGCs of Japanese anchovy stopped migration at that place, whereas those of Pacific herring continued to migrate dorsally and distributed widely all over the head region. During evolution, the program for the HGC migration would be varied to adapt to different hatching timing. Analysis of the gene expression revealed that Pacific herring embryos synthesized a large amount of hatching enzyme when compared with Japanese anchovy. Chorion of Pacific herring embryo was about 7.5 times thicker than that of Japanese anchovy embryo. Thus, the difference in their gene expression levels between two species is correlated with the difference in the thickness of chorion. These results suggest that the hatching system of each fish adapted to its respective hatching environment. Finally, hatching enzyme genes were cloned from each genomic DNA. The exon-intron structure of group B genes basically conserved that of the ancestral gene, whereas group A genes lost one intron. Several gene-specific changes of the exon-intron structure owing to nucleotide insertion and/or duplication were found in Japanese anchovy genes.

Purification and characterization of zebrafish hatching enzyme - an evolutionary aspect of the mechanism of egg envelope digestion.

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.

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene.

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.

Globin gene enhancer activity of a DNase-I hypersensitive site-40 homolog in medaka, Oryzias latipes.

In medaka, we found a C16orf35-like gene in the region within 1 Kbp 3' downstream of the psibeta end of the 36-Kbp embryonic globin gene cluster ((5')alpha0(3')-(3')beta1(5')-(5')alpha1(3')-(5')beta2(3')-(5')alpha2(3')-(3')alpha3(5')-(5')beta3(3')-(3')beta4(5')-(5')alpha4(3')-(3')psialpha(5')-(5')psibeta(3')). Intron 5 of the gene contained a region having NF-E2 binding sites located between GATA boxes. The region was homologous to human HS-40 in terms of the existence and structure of characteristic transcription-factor binding sites and was named Ol-HS-40. Injection of the fusion gene construct Ol-HS-40-alpha0(up-2)GFP, consisting of Ol-HS-40, a 5' upstream 200-bp minimum promoter for alpha0, and green fluorescent protein (GFP), showed that Ol-HS-40, as in human HS-40, had the ability to strongly enhance GFP expression in erythroid cells of embryos. Further analysis using transgenic technology revealed that Ol-HS-40 had the ability to change the type of the GFP expression from embryo-to-young fish to embryo-to-adult. In addition, the results suggest that Ol-HS-40, although its natural function remains unclear, has strong enhancer activity for the expression of not only the alpha-globin gene but also the beta-globin gene.

Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis.

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.

Analysis of the exon-intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei.

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution.

Evolution of teleostean hatching enzyme genes and their paralogous genes.

We isolated genes for hatching enzymes and their paralogs having two cysteine residues at their N-terminal regions in addition to four cysteines conserved in all the astacin family proteases. Genes for such six-cysteine-containing astacin proteases (C6AST) were searched out in the medaka genome database. Five genes for MC6AST1 to 5 were found in addition to embryo-specific hatching enzyme genes. RT-PCR and whole-mount in situ hybridization evidenced that MC6AST1 was expressed in embryos and epidermis of almost all adult tissues examined, while MC6AST2 and 3 were in mesenterium, intestine, and testis. MC6AST4 and 5 were specifically expressed in jaw. In addition, we cloned C6AST cDNA homologs from zebrafish, ayu, and fugu. The MC6AST1 to 5 genes were classified into three groups in the phylogenetic positions, and the expression patterns and hatching enzymes were clearly discriminated from other C6ASTs. Analysis of the exon-intron structures clarified that genes for hatching enzymes MHCE and MAHCE were intron-less, while other MC6AST genes were basically the same as the gene for another hatching enzyme MLCE. In the basal Teleost, the C6AST genes having the ancestral exon-intron structure (nine exon/eight intron structure) first appeared by duplication and chromosomal translocation. Thereafter, maintaining such ancestral exon-intron structure, the LCE gene was newly diversified in Euteleostei, and the MC6AST1 to 5 gene orthologs were duplicated and diversified independently in respective fish lineages. The HCE gene lost all introns in Euteleostei, whereas in the lineage to zebrafish, it was translocated from chromosome to chromosome and lost some of its introns.

Thyroid hormone-induced expression of a bZip-containing transcription factor activates epithelial cell proliferation during Xenopus larval-to-adult intestinal remodeling.

In the intestine during amphibian metamorphosis, stem cells appear, actively proliferate, and differentiate into an adult epithelium analogous to the mammalian counterpart. To clarify the molecular mechanisms regulating this process, we focused on a bZip-containing transcription factor (TH/bZip). We previously isolated TH/bZip from the Xenopus intestine as one of the candidate genes involved in adult epithelial development. Northern blot and in situ hybridization analyses showed that the transient and region-dependent expression of TH/bZip mRNA correlates well with the growth of adult epithelial primordia originating from the stem cells throughout the Xenopus intestine. To investigate its role in the adult epithelial development, we established an in vitro gene transfer system by using electroporation and organ culture techniques, and we overexpressed TH/bZip in the epithelium of Xenopus tadpole intestines. In the presence of thyroid hormone (TH) where the adult epithelial primordia appeared after 3 days of cultivation, overexpression of TH/bZip significantly increased their proliferating activity. On the other hand, in the absence of TH where the epithelium remained as larval-type without any metamorphic changes, ectopic expression of TH/bZip significantly increased the proliferating activity of the larval epithelium but had no effects on its differentiated state. These results indicate that TH/bZip functions as a growth activator during amphibian intestinal remodeling, although TH/bZip expression in the epithelium alone is not sufficient for inducing the stem cells.

Purification and gene cloning of Fundulus heteroclitus hatching enzyme. A hatching enzyme system composed of high choriolytic enzyme and low choriolytic enzyme is conserved between two different teleosts, Fundulus heteroclitus and medaka Oryzias latipes.

Two cDNA homologues of medaka hatching enzyme -- high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE) -- were cloned from Fundulus heteroclitus embryos. Amino acid sequences of the mature forms of Fundulus HCE (FHCE) and LCE (FLCE) were 77.9% and 63.3% identical to those of medaka HCE and LCE, respectively. In addition, phylogenetic analysis clearly showed that FHCE and FLCE belonged to the clades of HCE and LCE, respectively. Exon-intron structures of FHCE and FLCE genes were similar to those of medaka HCE (intronless) and LCE (8-exon-7-intron) genes, respectively. Northern blotting and whole-mount in situ hybridization showed that both genes were concurrently expressed in hatching gland cells. Their spatio-temporal expression pattern was basically similar to that of medaka hatching enzyme genes. We separately purified two isoforms of FHCE, FHCE1 and FHCE2, from hatching liquid through gel filtration and cation exchange column chromatography in the HPLC system. The two isoforms, slightly different in molecular weight and in MCA-peptide-cleaving activity, swelled the inner layer of chorion by their limited proteolysis, like the medaka HCE isoforms. In addition, we identified FLCE by TOF-MS. Similar to the medaka LCE, FLCE hardly digested intact chorion. FHCE and FLCE together, when incubated with chorion, rapidly and completely digested the chorion, suggesting their synergistic effect in chorion digestion. Such a cooperative digestion was confirmed by electron microscopic observation. The results suggest that a hatching enzyme system composed of HCE and LCE is conserved between two different teleosts Fundulus and medaka.