척삭동물문에 속하는 붉은멍게(Halocynthia aurantium)는 우렁쉥이와 같이 유용한 양식 품종으 로 사료되지만, 발생과 생태 등 생물학적 특성에 대해 잘 알려지지 않았다. 본 연구에서는 붉 은멍게 양식을 위한 기초자료를 얻기 위해 강원도 동해 연안에 서식하는 붉은멍게의 배발생을 조사하여 근연종인 우렁쉥이와 비교하였다. 그 결과, 붉은멍게의 수정부터 난할기, 낭배기, 신 경배기의 배아 및 올챙이형 유생의 발달 단계 및 형태가 우렁쉥이와 매우 유사하였다. 붉은멍 게의 수정란은 수온 11℃에서 부화까지 약 42.1시간이 소요되어 우렁쉥이의 40.9시간과 거의 유사하였다. 부화 후 어린개체로 변태하는 데 소요되는 시간도 두 종 사이에서 매우 유사하였 다. 수온 11℃에서 부화한 두 종의 유생은 모두 약 23일이 경과해서 입수공과 출수공이 명확 하게 구분되는 어린개체로 발생하였다. 수온 변화에 따른 발생 속도는 저온에서 느렸고 고온 에서 빠른 결과를 나타냈다. 붉은멍게의 경우는 9℃에서 부화까지 평균 62.3시간, 11℃에서 42.1시간, 13℃에서 36.3시간이 소요되었다. 우렁쉥이의 경우는 평균 60.4시간, 40.9시간, 35.2시 간이 소요되었다. 붉은멍게 배아의 대부분은 수온 15℃ 이상에서 정상적으로 발생이 이루어 지지 않아 종묘생산 과정에 주의가 필요한 것으로 사료된다.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. It is, however, still unclear what signaling and molecular event control polarized distribution of mitochondria in the early ascidian embryonic development. To obtain molecular markers for studying mechanisms involved in polarized distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondria-rich cytoplasm in all cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like a mesh structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. These antibodies showed that mitochondria were distributed evenly in the animal hemisphere blastomeres at cleavage stages, whereas did not in the vegetal hemisphere blastomeres. Mitochondria were transferred more into cells of the marginal zone, such as muscle and nerve cord lineage cells, than into cells of the central zone, such as mesenchyme, notochord and endoderm lineage, in the vegetal hemisphere. Therefore, it is suggested that these antibodies may be useful as markers for analysing mechanisms involved in polarized distribution of mitochondria during ascidian embryogenesis.

Asymmetric cell divisions play crucial roles during ascidian embryogenesis. In these processes, an FGF signaling is an essential inductive signal for establishing cell fate polarization, such as mesenchyme and notochord. It was well reported that the FGF signaling cascade is composed of FGF, FGF receptor, Ras, MEK, Erk and Ets. However, mechanisms of communication between the FGF and other signaling pathways and of integrated regulation of signaling pathways have remained largely unknown. In this study, we isolated HrS6K, a homologue of the S6K gene that belongs to the S6-H1 kinase of the ribosomal S6 kinase family, and HrNck1, a homologue of Nck1 gene that encodes an adaptor protein containing Src homology 2 and 3 domains, from the ascidian Halocynthia roretzi, to elucidate the mechanisms. Zygotic expression of HrS6K was initiated as early as the 16-cell stage. In the 64-cell stage embryos, expression of HrS6K was seen in mesenchyme precursor cells. The signal was detected in dorsal midline cells and mesenchyme clusters of the early tailbud embryos, and then down-regulated by the late tailbud stage. In adults, HrS6K mRNA was highly detected in muscle and stomach by QPCR method. On the other hand, HrNck1 transcripts are detected maternally. Zygotic HrNck1 mRNA was strongly expressed in mesenchyme clusters of the neurula, and in tail tip cells of the early tailbud embryos. These results suggest that HrS6K and HrNck1 are involved in formation of mesenchyme cells, which are specified by the FGF signaling.

In animal development, the mechanisms by which localized factors and organelles in egg cytoplasm were exactly distributed into each daughter cell are essential for formation of various cell types. During ascidian Halocynthia roretzi embryogenesis, ooplasmic mitochondria were mainly segregated into muscle and neural precursor cells. At the 32-cell stage, localized mitochondria in the B6.2 blastomeres were preferentially distributed into the B7.4 muscle precursors compared with the B7.3 mesenchyme/ notochord precursors. When the B6.2 blastomeres were isolated from the early 32-cell stage embryos and then allowed to divide 2 times of cell division, the resultant partial embryos showed symmetric distribution of mitochondria, and the partial embryos were composed of equal size cells. In normal development, cell fates of the B7.3 blastomere were correlated with the unequal cleavage of B6.2 lineage cells that normally occurs in the next two-cell division stages to produce a large B8.5 mesenchyme and a small B8.6 notochord cell. Mitochondria are distributed asymmetrically in both cells. When embryos were treated with FGF receptor inhibitor SU5402 and MEK inhibitor U0126 between the 32-cell and the early 64-cell stages, the resultant embryos showed equal cleavage pattern and symmetric distribution of mitochondria in daughter cells of the B6.2 blastomeres. However, blocking of Nodal and Notch signaling did not affect the cell division pattern and mitochondrial distribution in the B6.2 lineage blastomeres between the 32-cell and 110-cell stages. Therefore, it is likely that FGF/MEK signaling is involved in asymmetric distribution of mitochondria and unequal cleavage of the B6.2 lineage blastomeres in ascidian embryo.

FGF9/16/20 signaling pathway specify the developmental fates of notochord, mesenchyme, and neural cells in ascidian embryos. Although a conserved Ras/MEK/Erk/Ets pathway is known to be involved in this signaling, the detailed mechanisms of regulation of FGF signaling pathway have remained largely elusive. In this study, we have isolated Hr-Erf, an ascidian orthologue of vertebrate Erf, to elucidate interactions of transcription factors involved in FGF signaling of the ascidian embryo. The Hr-Erf cDNA encompassed 3110 nucleotides including sequence encoded a predicted polypeptide of 760 amino acids. The polypeptide had the Ets DNA-binding domain in its N-terminal region. In adult animals, Hr-Erf mRNA was predominantly detected in muscle, and at lower levels in ganglion, gills, gonad, hepatopancreas, and stomach by quantitative real-time PCR (QPCR) method. During embryogenesis, Hr-Erf mRNA was detected from eggs to early developmental stage embryos, whereas the transcript levels were decreased after neurula stage. Similar to the QPCR results, maternal transcripts of Hr-Erf was detected in the fertilized eggs by whole-mount in situ hybridization. Maternal mRNA of Hr-Erf was gradually lost from the neurula stage. Zygotic expression of Hr-Erf started in most blastomeres at the 8-cell stage. At gastrula stage, Hr-Erf was specifically expressed in the precursor cells of brain and mesenchyme. When MEK inhibitor was treated, embryos resulted in loss of Hr-Erf expression in mesenchyme cells, and in excess of Hr-Erf in a-line neural cells. These results suggest that zygotic Hr-Erf products are involved in specification of mesenchyme and neural cells.

The mechanisms by which embryo exactly distributes mitochondria into the blastomeres during embryogenesis are one of the important issues in developmental biology. Although the mechanisms has been thought to be important for the proper embryonic development, our understanding has remained limited. In the present study, the distribution of mitochondria was examined in embryos of the ascidian, Halocynthia roretzi, by immunohistochemical staining with three-types of the mitochondria-specific antibodies and vital staining of mitochondria with a fluorescent probe, DiOC2(3). Results of the immunohistochemical staining coincided with that of vital staining, which is able to detect the distribution of mitochondria in cytoplasm of the embryo. Mitochondria was mainly segregated into the B4.1 posterior-vegetal blastomeres at the 8-cell stage. During the next stages, mitochondria was preferentially partitioned into cells of the B-line muscle and the A-line nerve cord precursor compared with each sister cell, endoderm in the 5th cleavage stage, and mesenchyme and notochord in the 6th cleavage stage. However, the mitochondria-rich cytoplasm is divided equally among the blastomeres of the animal hemisphere between the 8-cell and the 64-cell stages. When B6.2 blastomeres were isolated at the early 32-cell stage embryo and cultured in seawater, until control embryos reached the 64-cell stage, pattern of mitochondria distribution was similar to results of the coisolated B7.3 and B7.4 blastomeres from the 64-cell stage embryos. Therefore, it is likely that mitochondria are asymmetrically segregated into the marginal cells in the vegetal hemisphere of the ascidian embryo without cell-cell interaction.

Mechanisms that regulate the number of cells that constitute the body have remained largely elusive. We approached this issue in the ascidian, Halocynthia roretzi, which develops into tadpole larva with small number of cells. Embryonic cells divide 11 times on average from fertilization to hatching. The number of cell division rounds varies between tissue types. For example, notochord cells divide 9 times and give rise to large postmitotic cells in the tadpole. The number of cell division rounds in the partial embryos that were derived from tissue-precursor blastomeres isolated at the 64-cell stage also varied between tissues, and coincided with their counterparts in the intact whole embryos to some extent, suggesting tissueautonomous regulation of cell division. Manipulation of cell fates in notochord, nerve cord, muscle, and mesenchyme lineage cells by inhibition or ectopic activation of the inductive FGF signal changed the number of cell division according to the altered fate. Knockdown and missexpression of Brachyury (Bra), an FGF-induced notochord-specific key transcription factor for notochord differentiation, indicated that Bra is responsible not only for notochord differentiation but also regulates the number of cell division rounds in the notochord lineage cells, suggesting that Bra activates a putative machinery to stop cell division at the specific stage. Results of precocious expression of Bra suggested that the machinery refers the developmental clock that is likely shared in other blastomeres than notochord, and functions to terminate cell division at three rounds after the 64-cell stage. Bra does nothing about the progression of developmental clock itself.