Ontogeny of primary lymphoid organs and lymphoid stem cells

Cells of the immune system go through a series of important developmental steps that begin early in embryonic life and include, first, the various waves of hemopoietic-cell production in the embryo and, second, the homing of these cells to the hemopoietic organs, which are the sites of hemopoiesis and lymphopoiesis in embryonic and adult life. The avian embryo is an important model for investigating these early steps; and this paper presents a comprehensive review of the work done on the early ontogeny of the avian immune system.     

Retinoic acid is a negative physiological regulator of N‐cadherin during early avian heart morphogenesis

The vitamin A‐deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A‐active form, retinoic acid (RA). Here we examine the role of N‐cadherin (N‐cad) in RA‐regulated early cardiovascular morphogenesis. N‐cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N‐cad overproducing VAD embryos reveal N‐cad involvement in the RA‐regulated cardiovascular development and suggest that N‐cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N‐cad. We hypothesize that a critical endogenous level of N‐cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage‐specific, developmentally regulated RA signaling.     

Characterization of c-lil, a chicken cellular sequence associated with a stock of B77 avian sarcoma virus.

Using biochemical methods, we have shown that a new specific sequence, v-lil, is associated with a given stock of B77 avian sarcoma virus (clone 9). We prepared a DNA complementary to v-lil sequences, using substractive hybridizations, and investigated the properties of this sequence. v-lil has a genetic complexity of ca. 2,000 nucleotides and is not present in various stocks of avian sarcoma virus, avian leukosis virus, or defective leukemia virus. v-lil is not associated with B77 avian sarcoma virus isolated from the original tumor and thus has been acquired by in vitro passage of the virus on chicken embryo fibroblasts. A search for the origin of the v-lil sequence among the DNAs of different avian species has shown that a similar sequence, c-lil, is present in normal chicken DNA (1 to 2 copies per haploid genome). c-lil is not highly conserved but is present in the DNA of all chickens from the genus Gallus. The c-lil sequence is transcribed at a low level (1 to 3 copies per cell) in normal chicken embryo fibroblasts. The biological function, if any, of v-lil or its cellular equivalent has yet to be determined.     

Developmental imaging: Insights into the avian embryo

The study of embryonic events using different animal model systems is crucial for gaining insights into human development and birth defects. Biological imaging plays a major role in this effort by providing a spatiotemporal framework to link complex cell movements with molecular data. However, depending on the age of the embryo and the location of a morphogenetic event, visualization often requires the design of novel culture and imaging techniques. One of the primary model systems for biological imaging is the avian embryo, due to its accessibility to manipulation, relatively two-dimensional morphogenesis early on, and viability when grown in culture. Significant work in avian embryo culture and cell labeling, together with advances in imaging technology, now make it possible to monitor many developmental events within the period from egg laying to hatching. Here, we present the latest in avian developmental imaging, focusing on cell labeling, embryo culture, and imaging technologies.     

Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase.

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.     

Controlled release of particle-associated RNA-dependent DNA polymerase by primary chick embryo cell cultures

Chick embryo cell cultures release a particle-associated RNA-dependent DNA polymerase into the culture medium. The release shows a characteristic time course with a maximum on the 3rd or 4th day in culture. The release of enzyme decreases when the cells are further cultivated and passaged. The enzyme was characterized as an RNA-dependent DNA polymerase by its ability to transcribe heteropolymeric RNA into DNA. It is different from the polymerase of the avian leukosis/sarcoma virus group and indistinguishable from an RNA-dependent DNA polymerase from normal embryonated chicken eggs described previously [1, 2]. The release of enzyme is independent of the genetic systems regulating the complete or partial expression of the endogenous avian leukosis virus genome. The amount of enzyme released is dependent on the age of the embryo from which the cell cultures are prepared. Cells prepared from 6-day-old embryos release maximal enzyme activity.     

Why Eberl is wrong. Reflections on the beginning of personhood

In a paper published in Bioethics, Jason Eberl has argued that early embryos are not persons and should not be granted the status possessed by them. Eberl bases this position upon the following claims: (1) The early embryo has a passive potentiality for development into a person. (2) The early embryo has not established both 'unique genetic identity' and 'ongoing ontological identity', which are necessary conditions for ensoulment. (3) The early embryo has a low probability of developing into a more developed embryo. This paper examines these claims. I argue against (1) that a plausible view is that the early embryo has an active potentiality to grow into a more developed embryo. Against (2), I argue that neither 'unique genetic identity' nor 'ongoing ontological identity' are necessary conditions for ensoulment, and that 'ongoing ontological identity' is established between early embryos and more developed embryos. Against (3), I argue that the fact that the early embryo has a low probability of developing into a more developed embryo, if true, does not warrant the conclusion that the early embryo is not a person. If Eberl is right that the human soul is that which organises the activities of a human being and that ensouled humans are persons, embryos are persons from conception.     

禽流感病毒H5N1全基因组克隆与分析

为明确广东地区分离的一株禽流感病毒H5N1的遗传背景,建立流感病毒反向遗传的平台。对该株禽流感病毒进行了空斑纯化与组织细胞培养,检测其在MDCK细胞中的增殖特性;利用H5N1病毒通用引物,通过RT-PCR对该病毒全基因组的8条片段进行全长克隆及测序分析;将H5N1的8条全长基因组片段分别插入反向遗传通用载体中,构建禽流感病毒H5N1的感染性克隆。结果表明,该H5N1毒株在MDCK细胞中可不依赖胰酶进行有效增殖与复制,可使MDCK细胞出现典型细胞病变,具有高致病性禽流感病毒的细胞增殖特征。RT-PCR克隆得到该H5N1毒株的PB2、PB1、PA、HA、NP、NA、M和NS八条全长片段,经测序分析确认该毒株的基因序列,其内部编码序列出现多处突变,其中HA连接肽为多个连续碱性氨基酸,表明该毒株可不依赖胰酶进行有效复制,与细胞培养结果一致,未出现抗药性的遗传突变。PCR与测序证明,插入H5N1八个全长基因组片段的载体序列完全正确,表明成功构建了该毒株的感染性克隆。为明确病毒遗传信息,建立流感病毒反向遗传的平台,为进一步研究禽流感病毒相关疫苗提供了研究基础。     

The importance of the posterior midline region for axis initiation at early stages of the avian embryo

The avian blastoderm acts during its early stages of development as an integrative system programmed to form a single embryonic axis. Here, I report the results of a variety of transplantation experiments of the midline region at stages X-XII, which were carried out to study their relevance for axis initiation. The results of the experimental series discussed herein emphasizes the importance of the posterior midline region (including the marginal zone and Koller's sickle) for axis initiation. This ability resides mainly at stage X in the posterior side of a narrow midline region, while at stages XI-XII it is exhibited at the region which is located more anterior and lateral to the posterior midline region. This posterior midline region has developmental abilities which allow it to initiate a single embryonic axis and at the same time to prevent other regions that also have such abilities to do so. Therefore, in normal development only one embryonic axis develops in the avian blastoderm. It is proposed that the cells which are important to initiate the avian embryonic axis are concentrated mainly at the region of the posterior midline region. These cells may have organizer properties which determine the initiation site of the axis in the avian embryo.     

Gastrulating chick embryo as a model for evaluating teratogenicity: a comparison of three approaches

BACKGROUND: Teratology studies must be carefully designed to minimize potential secondary effects of vehicle and delivery routes. A systematic method to evaluate chick models of early embryogenesis is lacking. METHODS: We investigated 3 experimental approaches that are popular for studies of early avian development, in terms of their utility for teratogen assessment starting at gastrulation. These included in vitro embryo culture, egg windowing followed by direct application of a carrier vehicle to the embryo, and injection of a carrier vehicle into the egg yolk. We also developed a morphologically based scoring system to assess development of the early embryo. RESULTS: The in vitro culture and egg windowing approaches both caused an unacceptably high incidence of central nervous system and cardiac abnormalities in vehicle-treated embryos, which made it difficult to identify teratogen-specific defects. In contrast, exposing chick embryos to vehicle via direct egg yolk injection did not induce developmental anomalies. CONCLUSIONS: Optimization of the exposure route of potential toxicants to the embryo is critical because control treatments can cause developmental anomalies. In ovo yolk injection minimizes perturbation of young embryos and may be appropriate for teratogen delivery.     

Culture system for embryos of blue-breasted quail from the blastoderm stage to hatching.

The blue-breasted quail (Coturnix chinensis), the smallest species in the order Galliforms, is a candidate model animal for avian developmental engineering because it is precocious and prolific. This species requires 17 days to hatch and 8 to 9 weeks to mature to an adult body weight of about 50 g, whereas the Japanese quail (Coturnix japonica) requires 16 days to hatch and 6 to 8 weeks to mature to an adult body weight of 100 to 150 g. The early embryo is the most challenging embryonic stage in terms of culture and manipulation for avian biotechnology. We have evaluated various conditions for the culture of blue-breasted quail embryos from the blastoderm stage to hatching. A hatchability rate of 26% (10/39) is among the best of the various culture conditions examined in the present study and the embryo culture system should facilitate advances in avian biotechnology.     

H5亚型禽流感重组鸡痘病毒活载体疫苗的构建及其遗传稳定性与免疫效力

以鹅源H5亚型禽流感病毒(AIV)基因组为模板,用RTPCR扩增血凝素（Hemagglutinin, HA）基因,克隆入鸡痘病毒表达载体pFG1175,转染鸡痘病毒感染的鸡胚成纤维细胞,通过蓝斑筛选和间接免疫荧光检测,获得表达HA基因的重组鸡痘病毒（Recombinant fowlpox virus, rFPVHA）。rFPVHA经鸡胚成纤维细胞连续传15代后,报告基因LacZ和HA基因可稳定表达。用103PFU和105PFU的rFPVHA免疫无特定病原体的(Specific pathogen free, SPF)鸡,免疫后22d 血凝抑制(Hemagglutinin inhibition,HI)抗体监测阳性率分别为0%和20%,但均抵御了H5亚型毒株的致死性攻击,保护率为100%。结果表明,构建了表达HA基因的重组鸡痘病毒,该重组病毒具有良好遗传稳定性,免疫鸡可提供完全保护,显示出了一定的应用前景。     

Commitment of hematopoietic stem cells in avian and mammalian embryos: an ongoing story

During ontogeny, hematopoietic stem cells (HSC) become committed outside of hematopoietic organs. Once held to emerge from the yolk sac, they are currently thought to originate from the early aorta. However we now show that the allantois in the avian embryo and the placenta in the mouse embryo produce HSC in very large numbers. These sites thus appear to have a central role in the process of HSC emergence.     

Avian reoviruses cause apoptosis in cultured cells: viral uncoating,but not viral gene expression,is required for apoptosis induction

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.     

Early arterial differentiation and patterning in the avian embryo model

Of the many models to study vascular biology the avian embryo remains an informative and powerful model system that has provided important insights into endothelial cell recruitment, assembly and remodeling during development of the circulatory system. This review highlights several discoveries in the avian system that show how arterial patterning is regulated using the model of dorsal aortae development along the embryo midline during gastrulation and neurulation. These discoveries were made possible through spatially and temporally controlled gain-of-function experiments that provided direct evidence that BMP signaling plays a pivotal role in vascular recruitment, patterning and remodeling and that Notch-signaling recruits vascular precursor cells to the dorsal aortae. Importantly, BMP ligands are broadly expressed throughout embryos but BMP signaling activation region is spatially defined by precisely regulated expression of BMP antagonists. These discoveries provide insight into how signaling, both positive and negative, regulate vascular patterning. This review also illustrates similarities of early arterial patterning along the embryonic midline in amniotes both avian and mammalians including human, evolutionarily specialized from non-amniotes such as fish and frog.