| The fins are divided into median fins and paired fins,which are distributed in pairs along the two sides of the fish body,including pectoral fins and pelvic fins.Median fin grows along the midline of the body and include dorsal,caudal,anal,etc.Median fin predates paired fins in evolutionary history and share similar morphological features.Studies have found that the limbs of fish quadrupeds are homologous,and related genes such as fgf,Wnt and Hox have similar expression patterns and functions during the development of the two.Although the developmental biology of the paired fin is relatively well understood,its origin remains a mystery.At present,there are three hypotheses on the origin of median fins:the origin from gill arch,the origin from fin fold,and the common selection of median fin genetic model modules.A new study in medaka shows that a conserved cis-acting element of the SHH gene（ZRS enhancer）is involved in both pectoral and dorsal fin development,which is the latest evidence supporting the same developmental origin of paired and median fins.These results suggest that further research on the mechanism of dorsal fin development not only can provide new connotation for appendage development of fish but also help to reveal the evolutionary origin of dual fin.Since there are both breeds with and without dorsal fins in goldfish,goldfish are ideal materials for studying dorsal fin development of fish.In this study,we firstly observed and compared the embryonic development process of Ryukin and Celestial,and then screened some unique transcripts and differentially expressed genes of Ryukin and Celestial by full-length transcriptome sequencing and comparative transcriptome analysis.On this basis,the expression patterns and functions of XXIV collagen al（col24a1）,fibroblast growth factor 7(fg（7）and its receptor（fgfr2）in the embryonic development of three different goldfish breeds,i.e.,Ryukin,Celestial and common goldfish,were studied.The main results are as follows:1.The root cause of dorsal fin failure in egg breed goldfish is the failure to develop the dorsal fin ruff.The goldfish were artificially inseminated and incubated at 24℃.Microscopic observations were made and photographed according to the stages and timing of embryonic and post-embryonic development in common goldfish.The rate of embryonic development of Ryukin and Celestial was found to be comparable to that of common goldfish,and the characteristics of each developmental stage were very consistent,but the development of the dorsal fin fold of Celestial was completely different from that of common goldfish and Ryukin.During the OVC stage of development,the dorsal fin fold extends from the tail and terminates at the eighth body segment;during the OVC stage,the dorsal fin fold does not extend but terminates only at the 22nd body segment of the tail.During subsequent embryonic development,dorsal fin fold of Ryukin fades away after the dorsal fin stripe is formed.Neither the dorsal fin fold nor the dorsal fin bar of Celestial is ever developed.Therefore,we hypothesize that the development of goldfish dorsal fin is predicated on the development of dorsal fin fold,and that the underlying cause of dorsal fin loss in egg breeding goldfish may be due to mutations in certain genes that prevent the dorsal fin ruff from developing properly.2.Full-length transcriptome sequencing provides a large number of gene transcripts for the study of developmental biology of goldfish embryos.Based on microscopic observation of Ryukin and Celestial embryo development,total RNA was extracted from fertilized eggs,14 somite stage and 35%OVC stage embryos of Ryukin and Celestial,respectively,and mixed in equal amounts for full-length transcriptome sequencing using the PacBio Sequl platform.After clustering to remove redundancy and correction,Ryukin and Celestial were sequenced to produce 122,576 and 117,201 concordant sequences respectively,and both had over 95%concordant sequences compared to the reference genome.On this basis,further correction and clustering to remove redundancy yielded 54106 and 54218 high-quality homologous iso forms,of which over 90%were new transcripts of known genes or from new genes.The common isomers of Ryukin and Celestial only accounted for 28%of their respective isomers,and most of the isomers were unique to the two cultivars.This is partly related to the lack of saturation of the PacBio Sequl platform sequencing,but also reflects the large number of transcript sequence differences between Ryukin and Celestial.Full-length transcriptome sequencing yields longer,more structurally complete transcript sequences and predicts a large number of variable shear events and variable polyadenylation sites compared to the annotated transcripts of the reference genome.This provides a very valuable resource of gene transcript sequences for research on the developmental biology of goldfish embryos.3.The differentially expressed genes of Ryukin and Celestial embryos were identified by comparative transcriptome analysis.According to Pearson correlation coefficient analysis,it was found that the correlation between repetitions of the same variety at the same period was very high,and the correlation between different varieties at the same time period was also very high,but the correlation between different periods of the same variety was very low,indicating that a large number of genes were expressed in time sequence with the development of goldfish embryos.At the same developmental stage,there were a large number of differentially expressed genes between Ryukin and Celestial.For example,compared with Ryukin,3339 genes were up-regulated and 3306 genes were down-regulated.Compared with Celestial,1643 genes were up-regulated and 3162 genes down-regulated on 35%vesicolar closure expectation day.No significantly enriched GO items were found in enrichment analysis（P>0.05）,but significantly enriched in several pathways such as metabolic process and extracellular matrix（ECM）receptor interaction（P<0.05）.Since ECM plays an important role in cell migration and transformation,we further studied the differentially expressed genes in ECM.Col24a1 gene,fgf7 and its receptor fgfr2 may play important roles in the development of dorsal fin folds.There were at least three XXIV type collagen αl chain genes（col24a1）in goldfish,which were named as col24al-1,col24a1-2 and col24a1-3 respectively in this study.Sequence comparison showed that the three col24a1 genes were significantly different in sequence.The 2188bp,2920bp and 800bp gene fragments were obtained by core cloning and rapid end-sequence amplification（RACE）,respectively.The expression levels of col24a1-1 and col24a1-2 were higher in Ryukin,Celestial and Common goldfish,while the expression levels of col24a1-3 were lower.The expression level of col24a1-1 was mainly expressed in the early stage of embryo development of Ryukin and Common goldfish,while the expression level of col24a1-2 was higher in the middle and late stage of embryo development of three goldfish.In contrast,the expression pattern of col24a1 gene was more similar to that of Pyrroid and Pyrroid.Fgf7 and fgfr2 were cloned to obtain 1373 bp and 3466 bp gene fragments,respectively.The former had variable splicing preserving intron,while the latter had variable polyadenylate cyclization.The expression patterns offgf7 and fgfr2 showed a trend of high and then low expression,and the highest expression levels were found from the segment stage to the early stage of vesicular closure.Since the development of the dorsal fin fold in goldfish begins at the late segment stage and grows rapidly during vesicular closure,we hypothesized that fgf7 and its receptorfgfr2 may play an important role in the development of the dorsal fin fold.Small interfering RNAs were designed and synthesized for microinjection in Ryukin zygote stage,and negative control group was set up.Microscopic observation of embryo development showed that compared with the negative control group,the embryos withfgfr2 interference died more severely,and all of them died during vesicle closure.The effect offgfr2 gene silencing on dorsal fin fold development was not observed.Microinjection of small interfering RNA targeting col24a1 significantly reduced the expression of the target gene,and a lucid embryo with undeveloped dorsal fin folds was observed,while embryos in the negative control group developed normally.These results suggest that fgfr2 gene plays an irreplaceable role in the development of goldfish embryos,while col24a1 gene may be involved in the development of dorsal fin folds of goldfish.However,goldfish only breed in the spring of each year and the eggs are sticky,which is not conducive to study the function of genes in embryonic development by microinjection of SMALL interference RNA.Subsequent experiments can further study the role of col24a1 in embryonic development in zebrafish or medaka.In summary,the dorsal fin fold of goldfish begins to develop in the late somites and grows rapidly during the optic vesicle closure period,and the failure of the dorsal fin fold to develop properly in egg breeds is the root cause of dorsal fin loss.A large number of full-length transcripts were obtained by transcriptome sequencing and a large number of differentially expressed genes of Ryukin and Celestial were identified.The expression of fgf7 and its receptor fgfr2 gene was the highest in the late segment and vesicle closure stage of goldfish embryos,and the expression level in the gold and gold was significantly higher than that in the gold and gold,while the silencing offgfr2 gene resulted in serious retarded development of goldfish embryos.Microinjection of small interfering RNA targeting col24a1-1 significantly reduced the expression level of col24a1 gene,and embryos without dorsal fin folds were observed.These results lay a foundation for the excavation and functional identification of genes related to dorsal fin development. |
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