Cloning, Phylogenetic Analysis And Expression Patterns Of Agat, Gamt And Ct1 In Amphioxus And Zebrafish And The Function Of Gamt In Zebrafish

The cephalochordate amphioxus belongs to an invertebrate group closest to mammalian. The study of vertebrate development of the mechanism in animal model amphioxus is an important part of embryology and developmental biology and amphioxus soon become a new favorite for molecular biologists to carry out scientific research. The role of amphioxus in molecular phylogenetic study will be more essential with the compelet of the whole genome sequencing of florida amphixus.The advantages of high fecundity, in vitro embryo development and transparency make Zebrafish the best material for studying embryo development. Genetic mutation screening showed that the mutant phenotype of zebrafish shared similar characteristics with the clinical symptoms of many human associated diseases. The genome sequence data has revealed the zebrafish genes and the corresponding genes causing human disease have high homology. Therefore, zebrafish as a model is becoming the most powerful tool for vertebrate animals biology, physiology and human diseases.The Cr / phosphocreatine (P-Cr) / creatine kinase (CK) system is essential for the buffering and transport of high energy phosphates. Cr is taken up by food, or synthesized endogenously by a two-step mechanism involving L-arginine:glycine amidinotransferase (AGAT) and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT). Cr is taken up by cells through CT1, a specific Cr transporter belonging to the Na+-dependent neurotransmitter transporter family. According to Cr metabolic pathways, abnormal synthesis or transportation of Cr can cause three kinds of creatine deficiency syndrome (CDS). The common clinical feature of creatine deficiency syndromes is the neurological symptoms and severe neurological development delay during childhood. GAMT and AGAT deficiency are treatable by oral creatine supplementation, while patients with CRTR deficiency do not respond to this type of treatment. Despite the patients with AGAT and GAMT can recover partly by taking creatine, the brain development and intellectual barriers are still left. The CNS toxicity of GAMT deficiency may be caused by the accumulation of the GAA.We obtained the 1817bp agat (DQ521270) cDNA sequence in amphioxus for the first time. The predicted protein of the amphioxus AGAT is 426 amino acids in length, with theoretical molecular weight of 49.3654 kDa and isoelectric point of 6.65. Intron PCR results showed that there are at least two agat gene copies in amphioxus.Phylogenetic analysis showed that Qingdao amphioxus AGAT we cloned and two Florida amphioxus AGAT (924 and 927) formed a single clade, independent of the single species clade of sea urchin and another large clade of the 15 species of mammalian. Interestingly, Florida AGAT 927 and Qingdao amphioxus AGAT constitute a subclade, while Florida AGAT 924 alone constitute another subclade. Zebrafish AGAT is located in the top of mammalian clade, alone constitutes a subclase. The phylogenetic tree lined with the generally accepted animal evolutionary relationships.There is no transcript of agat during the cleavage and blastula stage. In mid-gastrula, there is faint expression signal of agat in the mesendoderm. Then in neurala I stage, the transcripts are collected in the paraxial mesoderm. And in the following development neurala and knife-shape larvae stage, the expression of agat is in the myogenic somites and the signal of expression is stronger than that in the earlier developmental stages.We obtained the 1542bp gamt (DQ174309) cDNA sequence in amphioxus for the first time. The predicted protein of the amphioxus GAMT is 236 amino acids in length, with theoretical molecular weight of 26.8616 kDa and isoelectric point of 5.20. Intron PCR results showed that there are at least two gamt gene copies in amphioxus.Phylogenetic tree of GAMT protein was similar to AGAT. As no other lower invertebrate AGAT was reported, amphioxus AGAT was used as a root. A copy of Florida GAMT 924 formed a clade with GAMT from Qingdao amphioxus, while another copy of GAMT 399 Florida am formed an independent one. zebrafish GAMT also located on the top of vertebrate clade, but shared a same clade with Xenopus GAMT. The expression patterns of amphioxus gamt have a high degree of consistency with agat in time or space. No gamt was found before blasta stage. The gamt expression first appeared in the whole mesendoderm. gamt then expressed specific in the araxial mesoderm and the somites and continued this pattern to knife-shaped larvae. In addition, gastrointestinal epithelial cells also detected a weak gamt expression. The differences between gamt and agat expression patterns are that gamt expression is stronger than agat in muscle and gamt has broader expression areas, inthe digestive tract besides muscle.We obtained the 426bp ct1 cDNA sequence in amphioxus for the first time. The predicted protein of the Florida amphioxus GAMT is 631 amino acids in length, with theoretical molecular weight of 70.7913 kDa and isoelectric point of 5.06.Phylogenetic tree of CT1 protein was different from those of AGAT and GAMT. Florida CT1 was used as a root. Qingdao amphioxus CT1 and zebrafish CT1 formed a common clade. That may suggest that zebrafish CT1 has a higher homology with Qingdao amphioxus CT1 than with other mammalian.The expression pattern of amphioxus ct1 is vastly different from agat and gamt. From cleavage to blasta stage, ct1 was strongly expressed. In mid-gastrula, ct1 was slightly expressed in the mesendoderm. In early neurala, the expression of ct1 was upregulated in the notochord mesoderm, somatic mesoderm and endoderm. Later, ct1 expression was found in somites, the formatting notochord and the digestive tract. In the formatted notochord, ct1 expression disappeared. In late neurala and knife-shaped larvae, the sustained expression of ct1 was detected in muscle and the digestive tract.During amphioxus development, creatine synthesis is found mainly in the somites (including the paraxial mesoderm). The transit sites is broader, including somites (including paraxial mesoderm), the formatting notochord (including notochord mesoderm), and gut (including endoderm).First, these three genes are all expressed in the somites (including the paraxial mesoderm). agat expression was relatively weak. In early neurala somites, agat expression was weakest and ct1 strongest, ct1 subsequently appeared in various tissues of the average trend, relatively speaking, and the expression of ct1 in the somites decreased. From mid- neurala to knife-shaped larvae, gamt is expressed strongest in the somites. This suggested that both creatine synthesis and transfer can occur in amphioxus somites and different proportion of creatine mount from synthesis and transfer was found at the different stages of amphioxus development.Second, the expressions of three genes are not in exoderm. During the early development of amphioxus, three genes are expressed in mesendoderm. Later, three genes were expressed in somites, only gamt and ct1 in the digestive tract and only ct1 in the notochord. And these organs are derived from mesendoderm.Third, the expressed sites of ct1 are the widest, of gamt are less, and of agat are least, ct1 expressed in the somites, the notochord and digestive tract, gamt expressed in the somites and digestive tract (although weakly), while ct1 only expressed in the somites.In adult amphioxus, both agat and gamt are expressed in the nerve cord, connective tissue under epidermis, epibranchial groove, nephridia, gill epithelium, endostyle and enveloped membrane of mature oocytes On the other hand, ct1 was only in gut epithelium and the epibranchial groove (weaker). In addition, none of three gene was expressed in the muscle, notochord and epidermis.Zebrafish agat is formed by eight exons. The predicted protein of zebrafish AGAT is 422 amino acids long, with theoretical molecular weight of 48.0668 kDa and isoelectric point of 8.03. There is only one agat copy in the genome, located on the 18th chromosome.The temporal and spatial expression pattern of agat was determined from fertilization to 48 hpf. agat transcripts first appeared in the yolk syncytial layer (YSL) at gastrula stage. At early segmentation stage, strong agat expression persisted in the YSL and a weak expression also appeared in the mature somites. As development proceeds, agat still expressed strongly in the YSL and weakly in the somites. By 48 hpf, the expression of agat disappeared in the somites; however, strong expression was detected in the liver.In adult zebrafish, agat was strongly expressed in the intestine and ovaries. The positions where agat was not/weak expressed included retina, lens, heart, small intestine (mucosa, the lamina), liver and pancreas.Zebrafish gamt is formed by six exons. The predicted protein of zebrafish GAMT is 234 amino acids long, with theoretical molecular weight of 26.7538 kDa and isoelectric point of 6.29. gamt have two copies of the complete genes and a part gene copy of 4, 5 and 6 exon and they were in tandem on the 11th chromosome.Transcripts of gamt were first detected in the central area of the yolk from cleavage to gastrula stage. They were progressively expanded in the YSL at bud and early segmentation stages, while no transcript was detected in other tissues. At about 16 hpf, gamt expression could be detected in the mature somites besides the YSL. The expression in the somites was gradually up regulated from 17 hpf to 22 hpf stage. Like agat, gamt expression in the somites disappeared at 48 hpf, but persisted in the YSL and appeared in the liver. However, gamt mRNA was also detected in the epithelial cells of the gut where no agat mRNA was found.Strong gamt signals were found in adult zebrafish in retina, heart, liver, intestinal goblet cell, pancreas, ovary, of which gamt in the liver is strongest, especially in hilar, followed by heart and pancreas, gamt was not expressed in local parts of three organ, lens fibers, small intestine (lamina propria, lamina muscularis and serous membrane)and Phase III oocytes.Zebrafish ct1 is formed by thirteen exons. The predicted protein of zebrafish CT1 is 652 amino acids long, with theoretical molecular weight of 73.0075 kDa and isoelectric point of 5.07. There are two complete copies of ct1 in the genome, which are located on chromosome 8 and 23 respectively.ctl exhibited overlapping and distinct expression pattern compared with agat and gamt, ct1 was initially expressed ubiquitously in the embryo from cleavage to early segmentation stage. Then it was gradually restricted in the somites, the optic vesicles and the central nervous system (CNS). By prim-5 and prim-15 stage, ct1 expression was strongly expressed in both the newly formed and mature somites. By 48 hpf, the ct1 transcripts decreased to a much weaker extent in the somites, but strongly appeared in the foregut. ct1 expression has been reported in CNS, choroid plexus, dorsal aorta, hepatic primordium, skeletal muscles, epidermis and dermis, kidney, lung, stomach and intestine epithelial cells of rat embryos. However, ct1 expression in zebrafish embryos was restricted to a fewer tissues, including somites, CNS, optic vesicle and gut.Strong expressions of ct1 were detected in many tissues of adult zebrafish, including the retina, lens, heart, small intestine, pancreas, and ovary, ct1 was not found only in lens fibers, liver and intestinal lamina propria.Several interesting differences were noticed between the expression patterns of agat, gamt and ct1 in zebrafish embryos. First, unlike agat and/or gamt, no transcripts of ct1 were detected in the yolk, the YSL or the liver. Second, ct1 expression appeared in the CNS while agat and gamt did not. Finally, high level of ct1 expression was detected in all the somites, while weak agat and gamt expressions were detected only in the mature somites. Thus, our results show that agat, gamt and ct1 genes exhibit unique expression pattern during zebrafish embryogenesis. The expressions of agat and gamt in zebrafish are detected in YSL before 48 hpf and in the liver after 48 hpf. This may suggest that zebrafish YSL and liver at different developmental stages are important tissues related to creatine synthesis. In addition, the somites during segmentation stage may be also involved in this process. In zebrafish, the expression of ct1 is detected in CNS while agat and/or gamt are not. It implies that fish CNS may not function in creatine synthesis but just transport creatine to supply itself for energy.Compared with their homologues during rat embryonic development, there is a similar expression pattern of agat, gamt and ct1 between zebrafish and rat embryos. They are all expressed in skeletal muscle. In addition, agat and gamt are expressed in liver. On the other side, there are differences of these gene expression patterns between two species. For example, three genes are expressed in the CNS of rat embryo while only ct1 is expressed in the CNS of zebrafish embryo, agat gene is strongly expressed in rat kidney and pancreas, while no expression is found in the same organs of zebrafish embryo. Among these three genes related to creatine metabolism, the spatiotemporal expression of ct1 shows a remarkable conservation between fish and mammal. It implies that the similar mechanism of creatine transport may occur in fish and mammal.The expression patterns of agat, gamt and ct1 in adult zebrafish are similar to that in adult mammalian. For example, agat are expressed in brain, liver and pancreas; gamt are expressed in the liver and pancreas; ct1 are expressed in the heart and small intestine and not expressed in the liver. Differences are also found. First, agat has a very high level in the liver and pancreas in mammalian while it is very low in the corresponding organs of zebrafish. Second, gamt has low/no expression in the myocardium of mammalian, but was in strongly expressed in zebrafish heart. Third, ct1 has weak expression in the mammalian pancreas while it is strong in zebrafish pancreas.Although there are the reports on the amphioxus and zebrafish CK, no conclusion about the source of creatine has reached. This study on agat, gamt and ct1 is for the first time to confirm that Cr synthesis and transit system as mammals similar type exists in amphioxus and zebrafish. Because of the special status of amphioxus in the evolutionary, this study about amphioxus agat, gamt and ct1 will provide some clues on the evolution of creatine metabolic pathways and the origin of human CDS. In recent years, zebrafish has been used as a model in many diseases research because it is easy to cultivate, operate, observe, plus the genome of zebrafish and human have many similarities. Therefore, the further studies in zebrafish agat, gamt and ct1 will provide new evidence for pathogenic mechanism, diagnosis and treatment of the human CDS.