| Lampreys is one of the extant members of the Agnathans,the most primitive ancestral group of the living vertebrates.Due to their phylogenetic position,critical stages closely related to other living vertebrates and invertebrates,they are of considerable model organism to subject of vertebrate evolution and developmental biology.Manipulation of gene expression in lamprey tissues is required to perform functional studies.Here,firstly,we present the protocol on efficient housing and handling,performing fertilization,culturing larva through to the desired developmental stage.And morphological and histological features are studied during larval development of lamprey.The study provides an experimental research platform for studies on embryo,larva and ammocoete.Secondly,micromanipulation of gene expression in lamprey is studied by morpholino knock-down technique and RAN interference technique.And genes knockdown are performed by microinjection of morpholino oligonucleotides(MO)(L-HMGB1 MO、LIP(lamprey immune protein)MO and VLRB MO)in embryo and cerebroventricular microinjection of L-HMGB1si-RNA in the adult,which developed and validated gene knock-down technique for genes function research in lamprey.And gene expression profiles of L-HMGB1 knock-down morphant is analyzed by transcriptome,and gene expression profile of LIP and VLRB knock-down morphant is analyzed by microarray,respectively.Based on the transcriptome analysis,c-fos,tp63,PSMC5,ef and TLR5 were identified and validated by Real-time PCR in L-HMGB1 knock-down morphant.Furthermore,expression of both CDA and VLR is strongly inhibited by L-HMGB1 MO,and co-injection of the L-Hmgb1 protein with the L-HMGB1 MO causes a significant rescue effect.Our findings suggest VLR gene assembly mediated by CDA,which is also assisted by L-HMGB1 in lamprey.The results are as follows:1.Research platform for studies on embryo,larva and ammocoete.The body weight of Lampetra morii is significantly correlated with total length,head length,eye length and head length behind eye,and the body weight of Lampetra japonica is significantly correlated with total length,snout length and head length behind eye.The study the relationship between body weight and morphological attributes provided essential theoretical basis and ideal measure for selective breeding of lamprey.During breeding period,L.japonica and L.morii displayed the secondary sexual characteristics and the spawning behaviors are similar.The secondary sexual characteristics varied between males and females.The study of reproductive biology of lamprey provided basis for artificial fertilization.There are no significant difference in fertilization rate and hatching rate of artificial fertilization between L.japonica and L.morii(P>0.05).The method of artificial fertilization is the technical basis for the early developmental research.The cleavage of lamprey zygote is holoblastic cleavage and the embryology had been subdivided into cleavage,blastula,gastrula,neural plate and groove,head,prehatching and hatching.The embryo breaks through the fertilization membrane at 11-12 day-post-fertilization.The allometric growth pattern of lamprey yolk-sac larvae enabled rapid development of organisms.The ammocoete stage is reached when yolk is extruded from the anus and the digestive tract is complete at 15 day-post-hatch.The total length and body weight of ammocoete generally increased over time.The melanophores were extensively distributed and increased in ammocoete.The early development of lamprey is the basis for the research platform for micromanipualtion.2.Research platform for micromanipulation of gene expression in lampreyL-HMGB1、LIP and VLRB were expressed in every stages of embryonic development in the lamprey.A surprising number of immune-related genes(VLRB、VLRC、LIP、C3、C1q、Galectin、Syk、TCRL、CD45、TLR5、TLR7、TLR10、TLR18、CXCR4、CCR9、IL-18R、IL-17R、L-HMGB1、MIF、IL-8、IL-17、c-Rel、AHR、BCL11b、Artemis and CDA1)were expressed in larvae and adults.Comparison of expression of a select panel of immune-related between larvae and adults revealed different gene expression profiles,and the distinctions were particularly informative for the larvae and adults.Examination of gross morphology of L-HMGB1 MO-injected embryos revealed prominent changes starting at gastrula stages.The embryo show severe morphological defects that have different outlook as the control.And the L-HMGB1 knock-down morphant dies within 24 hours.The Real-time PCR and immunohistochemical analysis identified down-regulated L-HMGB1 in the knock-down morphant(P<0.01).The LIP MO-injected embryos develop normally and the gross morphology of LIP MO-injected embryos have the same outlook as the control one.And the embryo breaks through the fertilization membrane at 11～12 day-post-fertilization.The Real-time PCR analysis identified down-regulated LIP in the LIP knock-down morphant(P<0.01).The VLRB MO-injected embryos develop normally and the gross morphology of VLRB MO-injected embryos have the same outlook as control one.And the embryo breaks through the fertilization membrane at 11～12 day-post-fertilization.The Real-time PCR analysis identified down-regulated VLRB in the VLRB knock-down morphant(P<0.05).L-HMGB1 knockdown is performed by cerebroventricular micro injection of L-HMGB1 si-RNA in the adult.The Real-time PCR analysis identified down-regulated L-HMGB1 in L-HMGB1si-RNA-1 injected lamprey,L-HMGB1 si RNA-2 injected lamprey and L-HMGB1 si RNA-3 injected lamprey,respectivelu(P<0.05).3.Transcriptional analysis of L-HMGB1、LIP and VLRB knockdown by transcriptome and microarrayThe transcriptome analysis indicate that 2556 genes were differentially expressed in L-HMGB1 knock-down morphant.232 pathways were affected by the L-HAMGB1 knockdown,and 22 unigenes(up-regulated genes)and 4 unigenes(down-regulated genes)affect apoptosis pathway.And 13 pathways are related the apoptosis(MAPK signaling pathway、Hedgehog signaling pathway、AMPK signaling pathway,etc.).The microarray analysis indicate that 4593 genes were differentially expressed in LIP knock-down morphant,and 240 pathways were affected by the LIP knockdown.And differentially expressed unigenes are enriched in 18 significantly enriched pathways(Amyotrophic lateral sclerosis、Ribosome、Tryptophan metabolism,etc).The microarray analysis indicate that 5006 genes were differentially expressed in VLRB knock-down morphant,and 236 pathways were affected by the VLRB knockdown.And differentially expressed unigenes are enriched in 8 significantly enriched pathways(Ribosome、Ribosome biogenesis in eukaryotes、Rheumatoid arthritis,etc).4.The expression of development and immune-related genes in L-HMGB1 knock-down morphantc-fos,tp63,PSMC5 and ef were identified and validated by Real-time PCR.tp63,PSMC5 and ef genes were down-regulated(P<0.01).And c-fos were up-regulated(P<0.05).The expression of CDA1 and CDA2 are down-regulated(P<0.01),and the expression of VLRA、VLRB and VLRC are down-regulated(P<0.01),and the VLRB protein is down-regulated in L-HMGB1 knock-down morphant.Co-injection of the rL-Hmgb1 protein with the L-HMGB1 MO causes a significant rescue of the VLRB protein expression.And the deformity rate is lower than that of L-HMGB1 knock-down morphant.Our findings suggest VLR gene assembly mediated by CDA,which is also assisted by L-HMGB1 in lamprey. |
