Single-cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway-related gene expression profiles during mitotic arrest.

Mitotic arrest is necessary for the embryonic development of germ cells, and thus, it is important to understand the signaling pathways that regulate mitotic arrest. Here, we investigated the signaling pathway dynamics of male embryonic chicken germ cells during mitotic arrest by single-cell transcriptome analysis using germ-cell tracing models. We identified signaling pathways that change at the transcriptional level during chicken male germ-cell development after sex determination. We found that several components of the BMP, Notch, and JAK-STAT signaling pathways were downregulated at the mitotic-arrest stage and were reactivated 1 week after hatching when all germ cells are quiescent after entering mitotic arrest. In addition, the transcriptional levels of components of the MAPK, Hedgehog, and thyroid-hormone signaling pathways were steadily upregulated after mitotic arrest. This suggests the cooperation of multiple signaling pathways during entry into mitotic arrest and subsequent quiescence of chicken male germ cells.

Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 +/- and RAG1 -/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 +/- chickens, lymphatic organs in 3-week old RAG1 -/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 +/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens.

BACKGROUND: Germ cell mitotic arrest is conserved in many vertebrates, including birds, although the time of entry or exit into quiescence phase differs. Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch. However, mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage. RESULTS: We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens. Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages (E12, E16, and hatch), we found that metabolic and signaling pathways were regulated, and that the epigenome was reprogrammed during mitotic arrest. In particular, we found that histone H3K9 and H3K14 acetylation (by HDAC2) and DNA demethylation (by DNMT3B and HELLS) led to a transcriptionally permissive chromatin state. Furthermore, we found that global DNA demethylation occurred gradually after the onset of mitotic arrest, indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome. DNA hypomethylation persisted after hatching, and methylation was slowly re-established 3 weeks later. CONCLUSIONS: We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching. This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described. Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

Genome-wide identification and transcript profile of the whole cathepsin superfamily in the intertidal copepod Tigriopus japonicus.

Modulation of expression of cathepsins in innate immune response has previously been reported in mollusks and large crustaceans including crabs, lobsters, and shrimps in response to immune challenges. However, similar responses in copepods and the related cathepsin members remain under-investigated. To understand molecular and innate immune responses in the intertidal copepod Tigriopus japonicus, we identified the full spectra of cathepsin members (2 aspartyl proteases, 18 cysteine proteases, and 4 serine proteases) and also analyzed transcriptional expression of cathepsin (Tj-cathepsin) genes in developmental stages, lipopolysaccharide (LPS)- and two Vibrio species-exposed T. japonicus. The transcriptional levels of most Tj-cathepsin genes were highly increased during the molting transition from the nauplius to the copepodid stages. LPS treatment induced innate immune response via significant transcriptional increase of serine cathepsin (e.g., cathepsin As) members with induction of several cysteine cathepsin genes. However, Tj-aspartyl cathepsin E-like and a novel cysteine cathepsin were slightly reduced in response to LPS exposure. Interestingly, Vibrio species showed very low transcriptional sensitivity in the expression of entire cathepsins, while LPS induced several cathepsin gene-involved primitive immune responses in T. japonicus. In this paper, we discuss how whole cathepsin expression profiling can be linked to host defense mechanism to better understand and uncover the underlying mechanism of copepods' innate immunity.

Effects of maternal education on diet, anemia, and iron deficiency in Korean school-aged children.

BACKGROUND: We investigated the relationship among socioeconomic status factors, the risk of anemia, and iron deficiency among school-aged children in Korea. METHODS: The sample consisted of fourth-grade students aged 10 y recruited from nine elementary schools in Korean urban areas in 2008 (n = 717). Anthropometric and blood biochemistry data were obtained for this cross-sectional observational study. Anemia was defined as hemoglobin levels lower than 11.5 g/dl. Iron deficiency was defined as serum iron levels lower than 40 ug/dl. We also obtained data on parental education from questionnaires and on children's diets from 3-day food diaries. Parental education was categorized as low or high, with the latter representing an educational level beyond high school. RESULTS: Children with more educated mothers were less likely to develop anemia (P = 0.0324) and iron deficiency (P = 0.0577) than were those with less educated mothers. This group consumed more protein (P = 0.0004) and iron (P = 0.0012) from animal sources than did the children of less educated mothers, as reflected by their greater consumption of meat, poultry, and derivatives (P < 0.0001). Logistic regression analysis revealed a significant inverse relationship between maternal education and the prevalence of anemia (odds ratio: 0.52; 95% confidence interval: 0.32, 0.85). CONCLUSIONS: As a contributor to socioeconomic status, maternal education is important in reducing the risk of anemia and iron deficiency and in increasing children's consumption of animal food sources.

The influence of dietary patterns on the nutritional profile in a korean child cohort study.

OBJECTIVES: The aim of the present study was to identify the major dietary patterns of seven- and eight-year-old Korean children and to examine the relationship between dietary patterns and obesity, nutrient intake, and diet quality. METHODS: The subjects were 284 seven- and eight-year-old children who participated in the Gwacheon child cohort study. Three dietary patterns emerged from the factor analysis: Korean, modified Western, and Western. Cluster analysis was used to classify the subjects into two dietary groups: Korean and Western diet patterns. RESULTS: The two different dietary patterns were closely related to dietary quality which in turn was related to health risks. The Western diet group had a lower fiber intake, a higher intake of energy, fat and calcium and a higher dietary diversity score (DDS) than the Korean diet group. The number of days when fruit, milk and dairy products were omitted from the diet was higher for the Korean diet group than for the Western group. CONCLUSIONS: Dietary patterns and related diet quality should be considered when designing nutrition policy and intervention programs for children.

Dining-out behaviors of residents in Chuncheon city, Korea, in comparison to the Korean National Health and Nutrition Survey 2001.

Dining-out behavior is associated not only with socio-demographic characteristics such as gender, education, occupation, residence, and marital status, but also with individual preferences, such as eating-out activities, interests, and opinions. We investigated dining-out behaviors and their associated factors. Announcements by health practioners and the Chief of Dong Office were used to recruit 739 residents (217 males and 522 females) in Chuncheon, Korea. Information on the frequency and reasons for eating out, the standards for meal selection, and the overall satisfaction with restaurants, based on taste, nutrition, amount, price, service, sanitation, and subsidiary facilities of restaurants, was obtained through personal interviews with a structured questionnaire. Among all respondents, 46.3% of subjects ate outside of the home once or twice a month, and 33.8% reported that they ate out only a few times a year, or never. This was much higher than the national average of 52.0% as reported by the Korean National Health and Nutrition Survey (KNHNS) in 2001. The frequency of eating out differed significantly according to age (p=0.001), family income (p<0.001), residential area (p<0.001), and educational level (p<0.001). The most common reasons for dining out were meetings (46.7%), followed by special celebrations (15.4%), and enjoyment (11.2%). Korean food (55.3%) was the most frequently selected type of meal when eating out, and food was most often selected based on personal preferences (41.4%) and taste (29.8%); only 5.5% and 7.7% of subjects considered nutrition or other factors (e.g., sanitation), respectively. The results showed that the frequency of eating out for Chuncheon residents was much lower than the national average; in addition, eating-out behaviors depended on the residents' socio-demographic and personal characteristics.