S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Master's Degree_농생명공학부)
(A) study on acquisition of pluripotency in early developmental stage of chick blastoderm
초기 발달단계에서 닭 배아의 다능성 획득에 관한 연구
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부 바이오모듈레이션전공, 2016. 2. 한재용.
- Pluripotency refers the ability that can differentiate into any of the three germ layers
endoderm (gastrointestianl tract and lung), mesoderm (muscle, blood and bone) and ectoderm (nervous system and epidermal tissues). In mammals, embryogenesis is accompanied with a gradually loss of developmental capacity from a zygote which has totipotency. The zygote take series of cleavage to form cluster of cells for further development. The cluster of cells differentiate into ICM which has pluripotency and will makes body proper and trophectoderm which will differentiate into extraembryonic lineage cells.
In vivo, pluripotency is gradually acquired from zygote through ZGA and pluripotent gene network. Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4
official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Notwithstanding usage in development biology, the studies for pluripotency in avian embryos is still remains largely unknown. Thus, to compare and analyze the acquisition of pluripotency between mammals and aves, we select chicken as a model of aves.
We studied early development of intrauterine period of chick blastoderm. We described the general development of chick embryo. To investigate the novel phenomenon of chick development, firstly, we hypothesized that the gravity is one of the reason for shedding mechanism. We conducted ex vivo culture and the results indicate that gravity has no or little impact on the shedding phenomenon of chick blastoderm. Secondly, we conducted TUNEL assay with EGK.VIII blastoderms to detect programmed cell death (apopotosis). But, we cannot detect any TUNEL positive signals in shedding cells. Taken together, cell shedding, the novel mechanism of chick blastoderm during area pellucida formation, is not a passive moving of cells which is occurred by gravity or apoptosis.
Next, we investigated the similarities of amino acid sequences of core pluripotent genes cPOUV and cNanog. As results, DNA binding domains of cPOUV and cNanog are highly conserved among the species, but the full length similarities of amino acid are low. Then, we focused on Nanog homeodomain of mouse and chicken were analyzed using PHYRE2. The results indicate that Nanog homeodomain of mouse and chicken have similar secondary structure and DNA binding sites. According to DNA binding domain similarity, we can assume that the function of chicken Nanog is similar to mouse.
To investigate the expression of pluripotency related gene, we harvested chick blastoderms of intrauterine period by abdominal massage and classified the blastoderms according to Eyal-Giladi and Kochavs criteria. White Leghorn (WL) hens (54-56 weeks old) were used for the collection of intrauterine eggs. To collect oocytes, WL hens were sacrificed and the follicles were detached from the ovaries. Then, spatial and temporal expression patterns of pluripotent genes were observed during intrauterine stages of chick using PCR and in situ hybridization.
RT- and Real time-PCR were conducted to investigate the expression of cPOUV, cSOX2, cNANOG and ENS-1. As results, EGK.X which is homologue of mouse blastocyst, has all the pluripotency related gene expression except cSOX2. However, expression dynamics is differ from those of mouse. Mouse Oct4 and Sox2 are maternally inherited and Nanog is upregulated by Oct4- Sox2 transcriptional complex in mouse embryo. But, in chicken, no cPOUV transcripts are found in the oocytes and cNANOG is expressed from the oocyte to EGK.X instead of cPOUV. cSOX2 is not exist until EGK.X in which pluripotency is fully acquired. Taken together, The lineage specific expression pattern of chick pluripotent genes are conserved. But their expression dynamics are differ from what occur in mammals and the underlying mechanisms could be different from mammals. Also, our results revealed that pluripotency seems to be related to cell shedding which is novel mechanism of chick early development. This work will serve as a foundation for the better elucidation of the mechanism of pluripotency formation and lineage segregation, as well as for the comparative information of the evo-devo among the species.