The Role Of Hira Gene In The Development Of Zebrafish

The zebrafish, as a model of vertebrate,uaually used in studies of developmental biology, because of its short breeding cycle, spawning quantity, embryo morphology than whole transparent, piss in observation and operation, easy feeding etc. Our laboratory have studied the role of Hira gene in gibel carp’s embryo development, The results show that the Hira gene play an important role in the early embryo development of gibel carp. Besides, we have studied the spatial and temporal expression characteristics of Hira gene in the gibel carp and color crucian carp, and the expression characteristics during oogenesis. Because of gibel carp breedingseason is short and material is not very convenient, in order to further enrich the study about the role of Hira gene in the fish, this research using the model organism zebrafish as the research object, study the role of Hira in the embryo development of zebrafish by real-time fluorescence quantitative PCR, Morpholino knock down and whole-mount in situ hybridization.The results of qRT-PCR show that the expression of Hira is strongest in unfertilized egg and the expression then decreases after fertilize30min,but the expression levels can return at tail bud stages. After fertilization, the expression of Hira gene in low quantities, may be the mRNA of Hira gene for the early development of the embryo is required for some protein synthesis. Prior research results show that Hira gene plays an important role in the process of sperm nucleus decondensation. Maybe the Hira gene has the same effect in the zerbrafish,besides, may have some effect on other life activity. The expression level of Hira then decreases at the later development stage, Indicates that the homozygous transcription of Hira gene begains and may have some effects on the later development in zebrafish. Analysis of Hira gene in different tissues of zebrafish revealed that the strongest expression in ovary and testis, and there was some expression in muscle, brain, heart, and liver. This suggests that the Hira gene may play an important regulatory effect in the development of germ system, muscle tissue, nervous system and circulatory system in zebrafish. situ hybridization results showed that significant hybridization signals can be observed in the early developmental embryo, This indicated that Hira may effect the early development of zebrafish embryo.besides, the hybridization signals aslo can be observed at head ane somite in tail bud and muscle affection stage embryo,This indicated that Hira may have a certain role on the nervous system and body axis development.Knock down of Hira gene result in the zebrafish embryo with unnormal development, such as the slowing of epiboly in gastrulation, about three hours delay; many embryos died before hatching larvae; the inhibition of yolk utility and short body axis; pericardial cavity hydrops; the vitality of Hira-MO embrys declines after hatching larvae. These results indicated that Hira gene may play an important role in the normal development in zebrafish.Besides, gibel carp (Carassius auratus gibelio) is excellent material for the occurrence of germ cells in the developmental biology and fertilization mechanisms, because of its unique mode of reproduction, gibel carp exercise two mode of reproduction, sexual reproduction and gynogenetic. That’s because the gibel carp in the fertilization process have a different response mechanisms of homologous and heterologous sperm nucleus:when the egg of gibel carp inseminated with homologous sperm(gibel carp sperm), the sperm can condensation during fertilization and form into male pronucleus, although the most offspring by this mode of reproduction are female,but aslo can produce a certain proportion of male offspring; But when inseminated with heterologous sperm(non gibel carp sperm),the heterologous sperm undergoes decondensation,therefor the male pronucleus cannot be formed, so the genetic material of sperm can not participate in the offspring’s development process, the offsprings of this reproduction are females. Now there has been some research about the response mechanism of fertilization of gibel carp. Our laboratory have studied the role of histone chaperone HIRA in sperm decondensation of fish. The research showed that,when the HIRA protein was removed from the egg extracts by immunodepletion, the sperm nucleus didn’t decondense for both gibel carp or color crucian carp,which showed that the HIRA was an important protein in the role of sperm decondensation offish. Then, our laboratory studied the role of Maternal histone chaperones HIRA and histone H2a variant H2aflo in the sperm decondensation of fish by immunofluorescence colocalization, The research showed that the fluorescence signal of HIRA and H2aflo could be observed in decondensed sperm nuclei, however, in the sperm of color crucian carp which couldn’t decondense in the egg extracts of gibel carp, there was only the fluorescence signal of HIRA could be observed, the signal of H2aflo couldn’t be observed.which indicated that Maternal histone H2aflo could not be integrated into the heterologous sperm nucleus, was one of the reason that the heterologous sperm couldn’t decondense in the gibel carp egg extarcts. In order to study whether there was other maternal histone participate the decondensation of sperm nuclues, We studied the role of acetylated histone H4in the decondensation of fish sperm nucleus by immunofluorescence colocalization in vitro system. Our reseach showed that there was not fluorescence signals of acetylated histone H4in the condensed sperm nucleus of color crucian carp which were incubationed in the gibel carp egg extarcts, but the fluorescence signals can be observed on the homologous decondensation sperm nuclues.besides, the heterologous sperm(gibel carp sperm) inhubationed in color crucian carp egg extarcts can decondensate, become more fluffy, and the fluorescence signals of acetylated histone H4aslo can be observed. This indicated that Maternal acetylated histone H4could not be integrated into the heterologous sperm nucleus, was one of the reason that the heterologous sperm couldn’t decondense in the gibel carp egg extarcts.