Hyung Chul Lee

MicroRNA-mediated posttranscriptional regulation is required for maintaining undifferentiated properties of blastoderm and primordial germ cells in chickens

MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.

Cleavage Events and Sperm Dynamics in Chick Intrauterine Embryos

This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

DAZL Expression Explains Origin and Central Formation of Primordial Germ Cells in Chickens

The timing and biological events associated with germ cell specification in chickens have not been determined yet. In this study, we report the origin of primordial germ cells (PGCs) and germ plasm dynamics through investigation of the expression of the chicken homolog of deleted in azoospermia-like (cDAZL) gene during germ cell specification. Asymmetric localization of germ plasm in the center of oocytes from preovulatory follicle stages leads to PGCs being formed in the center. During cleavage stages, DAZL expression pattern changes from a subcellular localization to a diffuse form before and after zygotic genome activation. Meanwhile, PGCs exhibit transcriptional active status during their specification. In addition, knockdown studies of cDAZL, which result in reduced proliferation, aberrant gene expression profiles, and PGC apoptosis in vitro, suggest its possible roles for PGC formation in chicken. In conclusion, DAZL expression reveals formation and initial positioning of PGCs in chickens.

Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation. Summary: Early chick embryos share previously unappreciated features with anamniote embryos such as the timing of zygotic gene activation and yolk syncytial layer formation.

Physiological Effects of Diethylstilbestrol Exposure on the Development of the Chicken Oviduct

Estrogen has dramatic effects on the development and function of the reproductive tract in mammals. Although diethylstilbestrol (DES) triggers the development of reproductive organs in immature animals, continued exposure to DES induces dysfunction of the female reproductive tract in mice. To investigate the effects of neonatal estrogen exposure on the reproductive tract of female chickens, we implanted DES pellets into the abdominal region of immature female chicks and then examined the effects of DES on the oviducts of both immature chicks and sexually mature chickens (30 weeks old). DES induced mass growth and differentiation of the oviduct in immature chicks. The chick oviduct increased by 2.7- and 29-fold in length and weight, respectively, following primary DES stimulation. In secondary DES stimulation, the length and weight of the chick oviduct increased by 4.5- and 74-fold, respectively. Additionally, DES treatments caused abnormal development of the infundibulum and magnum in hen oviducts. Furthermore, infundibulum abnormality gave rise to unusual ovulation of follicles and resulted in infertility and dysfunction of the magnum, such as less production of egg white proteins. Our results indicate that DES exposure during early developmental stages in chickens has detrimental effects on the development and maintenance of the female reproductive tract after sexual maturation.

Site-specific recombination in the chicken genome using Flipase recombinase–mediated cassette exchange

Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome‐editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase‐mediated gene cassette exchange (RMCE), one of the site‐specific recombination technologies, and confirmed RMCE in TG chicken‐derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken‐derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis‐element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.—Lee, H. J., Lee, H. C., Kim, Y. M., Hwang, Y. S., Park, Y. H., Park, T. S., Han, J. Y. Site‐specific recombination in the chicken genome using Flipase recombinase‐mediated cassette exchange. FASEB J. 30, 555‐563 (2016). www.fasebj.org

Compensatory proliferation of endogenous chicken primordial germ cells after elimination by busulfan treatment

IntroductionPrimordial germ cells (PGCs) are the major population of cells in the developing bilateral embryonic gonads. Little is known about the cellular responses of PGCs after treatment with toxic chemicals such as busulfan during embryo development. In this study, we investigated the elimination, restorative ability, and cell cycle status of endogenous chicken PGCs after busulfan treatment.MethodsBusulfan was emulsified in sesame oil by a dispersion-emulsifying system and injected into the chick blastoderm (embryonic stage X). Subsequently, we conducted flow cytometry analysis to evaluate changes in the PGC population and cell cycle status, and immunohistochemistry to examine the germ cell proliferation.ResultsResults of flow cytometry and immunohistochemistry analyses after busulfan treatment showed that the proportion of male PGCs at embryonic day 9 and female PGCs at embryonic day 7 were increased by approximately 60% when compared with embryonic day 5.5. This result suggests the existence of a compensatory mechanism in PGCs in response to the cytotoxic effects of busulfan. Results of cell cycling analysis showed that the germ cells in the G0/G1 phase were significantly decreased, while S/G2/M-phase germ cells were significantly increased in the treatment group compared with the untreated control group in both 9-day-old male and female embryos. In addition, in the proliferation analysis with 5-ethynyl-2′-deoxyuridine (EdU) incorporation, we found that the proportion of EdU-positive cells among VASA homolog-positive cells in the 9-day embryonic gonads of the busulfan-treated group was significantly higher than in the control group.ConclusionsWe conclude that PGCs enter a restoration pathway by promoting their cell cycle after experiencing a cytotoxic effect.

Germline Modification and Engineering in Avian Species.

Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through long-term culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies.

Germline-competent stem cell in avian species and its application

Germ cells are the only cell type in the body that can transfer genetic information to the next generation. Germline-competent stem cells can self-renew and contribute to the germ cell lineage giving rise to pluripotent stem cells under specific conditions. Hence far, studies on germline-competent stem cells have contributed to the generation of avian model systems and the conservation of avian genetic resources. In this review, we focus on previous studies on germline-competent stem cells from avian species, mainly chicken germline-competent stem cells, which have been well established and characterized. We discuss different sources of germline-competent stem cells and recent advances for the future applications in birds.

Cell-Specific and Temporal Aspects of Gene Expression in the Chicken Oviduct at Different Stages of the Laying Cycle

ABSTRACT Egg formation and embryonic development occur as the yolk passes through the magnum, isthmus, and shell gland of the oviduct before oviposition in hens. The present study identified candidate genes associated with secretory function of the chicken oviduct after ovulation and contributing to egg formation and oviposition. Hens (nu2009=u20095 per time point) were euthanized to recover the reproductive tract when the egg was in the magnum (3 h after ovulation) and the shell gland (20 h after ovulation). Total RNA was extracted from each segment of the oviducts and subjected to Affymetrix chicken GeneChip analysis. Quantitative PCR and in situ hybridization analyses of selected genes confirmed the validity of the gene expression patterns detected using microarray analysis. In particular, ACP1, CALB1, CYP26A1, PENK, RCAN1 and SPP1 expression increased significantly in the shell gland between 3 h and 20 h postovulation, whereas only RCNA1 expression increased significantly in the magnum between 3 h and 20 h postovulation. Results of the high-throughput analysis revealed cell-specific and temporal changes in gene expression in the oviduct at 3 h and 20 h postovulation in laying hens provide novel insight into changes at the molecular and cellular levels of candidate genes related to formation of the egg and oviposition.