A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos

In embryogenesis, a series of developmental events begins immediately after fertilization. During the early embryo development, the expression of many genes is spatiotemporally triggered or suppressed, under tight transcriptional control. However, the intricate changes in gene expression in the early embryo have yet to be investigated in detail in mammals or birds.

As avian species are oviparous, the embryo is readily accessible even at the earliest stages and can effectively be manipulated for purposes including profiling the genes expressed in embryogenesis. Recently, the chicken genome was completely sequenced [1], and furthermore, Burt et al [2] reported that the organization of the human genome is actually closer to that of the chicken than the mouse. Thus, the chicken is an interesting and relevant experimental animal model.

In avian species, the embryonic developmental process is quite different from that in mammals. The first cleavage begins 4 h after fertilization, as the embryos enter the magnum of the reproductive duct [3], but the first differentiation does not occur until the egg is laid. Cells in the embryo continue to proliferate until the Eyal-Giladi and Kochav stage X and the laid egg consists of 40,000 to 60,000 undifferentiated embryonic cells [4]. After 12 to 13 h of incubation at Hamburger and Hamilton (HH) stage 3, the primitive streak extends to about the center of the area pellucida [5]. The groove in the primitive streak is gradually established as the cells of surrounding epiblast rapidly divide and migrate to the lower regions of the embryo, where they spread laterally across the surface of the yolk into two layers, the endoderm and mesoderm.

Together with when the three germ layers begin to form, the first appearance of germ cells is an important event during the early embryo development. Primordial germ cells, the progenitors of sperm or egg cells after sexual maturity, first appear from the epiblast in the blastoderm at stage X and translocate to the hypoblast of the area pellucida [6–8]. During gastrulation, they circulate through the vascular system and finally settle in the gonadal anlagen [9].

Understanding the molecular mechanisms that underlie germ cell segregation during early embryogenesis is important not only from the perspective of fundamental research in embryology but also from that of practical use of genetic methods. Additionally, discovering developmental stage-specific genes will aid in uncovering the molecular mechanism involved in the early stages of chicken embryo development.

In this study, we investigated the linkage of gene expression with morphological events, including germ cell segregation and identified gene transcripts from microarray GeneChip technology (stage X versus HH stage 3). We further analyzed our gene expression data using quantitative RT-PCR.

Recently, with chicken genome data and advances in DNA microarray technology, integration of these genomic data with the wealth of existing descriptive embryology will strengthen the position of the chicken embryo as a useful experimental model [10–13]. The chicken embryo provides an excellent model system for studying the developmental biology of vertebrates because of the accessibility of the chick embryo, allowing embryonic manipulations that are not possible in a mammalian system. But, there are few reports on microarray GeneChip technology and functional genomics for profiling gene transcripts expressed during early embryo development in avian species.

To investigate the linkage of gene expression with morphological events, including germ cell segregation, we examined differentially expressed genes between undifferentiated Eyal-Giladi and Kochav (EK) stage X and differentiated Hamburger and Hamilton (HH) stage 3 chicken embryos. Specifically, we used microarray GeneChip technology to identify a set of genes with stage-specific expression, and its results were confirmed by the relative gene expression profiling using quantitative RT-PCR. From our results based on both of microarray and quantitative RT-PCR, we identified a set of stage-specific genes in the developing chick embryo: 21 genes were relatively up-regulated in stage X embryo and 12 genes were relatively up-regulated in the HH stage 3 embryo.

Confirming GeneChip results with quantitative RT-PCR is important in determining whether a specific transcript is truly up- or down-regulated. Our quantitative RT-PCR results demonstrated a higher than 90% concordance with the GeneChip data, consistent with the findings of Rajeevan et al. [14]. Their results showed a similar the degree of agreement between GeneChip and QRT-PCR and that the agreement was related to hybridization intensity, with the highest level of concordance for those genes with the highest signal intensity on the GeneChip. From the quantitative RT-PCR in this study, most (21/23) of genes up-regulated in stage X embryo gradually decreased according to developmental process, while almost (12/13) of genes up-regulated in HH stage 3 embryo were relatively higher than in stage X and increased according to ages of incubation time until HH stage 9 (30 h). However, three gene transcripts (2 at stage X and 1 at HH stage 3) showed the discrepancy between microarray data and the quantitative RT-PCR analysis. Thus, further intensive experiments such as Northern blot and in situ hybridization as well as functional study would be conducted.

The freshly laid egg, at stage X, consists of 40,000 to 60,000 undifferentiated embryonic cells. At this stage, notably, anti-oxidation-related transcripts such as Gga.12503.2.S1_at (Nucleoredoxin) and Gga.4401.1.S1_a_at (prostaglandin-endoperoxide synthase 2) were expressed higher than at HH stage 3. Oxidative stress is one of the main damage sources in living organisms, and the anti-oxidant defence mechanisms of biological systems play major roles in preventing oxidative stress and the consequent cell damage [15]. Thus, higher expression of anti-oxidation-related transcripts could be one of the critical mechanisms for survival at stage X embryo.

Signal transduction pathways are known to play key roles in cell fate commitments during early embryogenesis. FGF13, highly expressed in stage X embryo, would be involved in the signalling transduction during the early chick embryogenesis. FGF signaling not only mediates cell fate specification in the early embryo but also maintains an undifferentiated cell state in many cellular contexts [16]. Streit et al [17] suggested that FGF secreted from a population of organizer precursors initiates induction of neural system before the beginning of gastrulation and Sato et al [18] reported that in human embryonic stem cells, FGF2/13 expression is enriched and important to understand the molecular mechanisms underlying stemness.

At HH stage 3 (12 h of incubation), three germ layers and germline segregation with the appearance of primordial germ cells (PGCs) begin to form in chick embryogenesis. Since the number of these PGCs is rare at the early developmental stags, it would be difficult to investigate the germline-specific genes. In this study, 12 gene transcripts which were relatively up-regulated in HH stage 3 embryos might be important in germ cell segregation as well as morphological events. Interestingly, Gga.12446.1.S1_s_at, transcript up-regulated at HH stage 3, was gallus gallus staufen homolog to Drosophila STAU1 and gradually increased during developmental process. Staufen, a double-stranded RNA binding protein, was first identified in Drosophila as an essential factor required for germline specification and the anterior-posterior axis formation [19]. Ramasamy et al. [20] recently reported that Zebra fish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells.

Septin2 (GgaAffx.11516.1.S1_s_at), GTP-binding protein, is involved in cytokinesis in various species from yeast and vertebrates [21]. Expression level of Septin2 was significantly found in mouse embryonic heart in a developmentally regulated fashion and gradually down-regulated around birth [21]. In our GeneChip data, Septin2 were expressed higher at HH stage 3 than at stage X and it might be that embryonic cells actively proliferate and organogenesis begins during gastrulation. However, the results from quantitative RT-PCR showed septin2 transcript consistently expressed from stage X to HH stage 9. Thus, spatiotemporal regulation(s) of septin2 transcript expression remain to be further investigated.

Although the number of genes analyzed in this study was limited, our data would be contributed to understanding how developmentally important genes are integrated with developmental processes and germline segregation during early embryogenesis in the chicken. Further experiments examining in situ cellular expression and functional properties of the identified genes would be needed to elucidate the transcriptional control mechanisms of gene expression associated with early embryogenesis in chickens.

In this study, we identified a set of genes with stage-specific expression using microarray Genechip and quantitative RT-PCR during the early embryo development in avian species. Discovering stage-specific genes will aid in uncovering the molecular mechanisms involved the formation of the three germ layers and germ cell segregation in the early chick embryos.