鹌鹑、红嘴鸥、黑翅长脚鹬胚胎期 小肠黏膜表面形态结构

应用扫描电镜观察了鹌鹑（Coturnix coturnix）、红嘴鸥（Larus ridibundus）、黑翅长脚鹬（Himantopus himantopus）胚胎期小肠黏膜的形态变化。鹌鹑与红嘴鸥胚胎发育过程中,小肠黏膜形态结构可分为3个阶段。第一阶段为鹌鹑卵孵育第10 ~ 11天、红嘴鸥卵孵育第13 ~ 14天（相当于胚胎发育60%的阶段）,小肠黏膜为山脊状纵行皱襞;第二阶段为鹌鹑卵孵育第12 ~ 13天、红嘴鸥卵孵育第15 ~ 16天（相当于胚胎发育70%的阶段）,小肠黏膜为“W”形板状皱襞;第三阶段为鹌鹑卵孵育第14 ~ 17天、红嘴鸥卵孵育第17 ~ 22天（相当于胚胎发育到80% ~ 100%阶段）,小肠黏膜为指状绒毛。黑翅长脚鹬卵孵育第10天（相当于胚胎发育到60%的阶段）,小肠黏膜为山脊状纵行皱襞;从卵孵育第12天（相当于胚胎发育到70%的阶段）一直到孵出小肠黏膜均为“W”形板状皱襞。初步判断3种鸟小肠黏膜形态发生这种有规律的变化可能是鸟类对其祖先系统发育的重演。     

Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail

Studies in our laboratory have focused on endocrine, neuroendocrine, and behavioral components of reproduction in the Japanese quail. These studies considered various stages in the life cycle, including embryonic development, sexual maturation, adult reproductive function, and aging. A major focus of our research has been the role of neuroendocrine systems that appear to synchronize both endocrine and behavioral responses. These studies provide the basis for our more recent research on the impact of endocrine disrupting chemicals (EDCs) on reproductive function in the Japanese quail. These endocrine active chemicals include pesticides, herbicides, industrial products, and plant phytoestrogens. Many of these chemicals appear to mimic vertebrate steroids, often by interacting with steroid receptors. However, most EDCs have relatively weak biological activity compared to native steroid hormones. Therefore, it becomes important to understand the mode and mechanism of action of classes of these chemicals and sensitive stages in the life history of various species. Precocial birds, such as the Japanese quail, are likely to be sensitive to EDC effects during embryonic development, because sexual differentiation occurs during this period. Accordingly, adult quail may be less impacted by EDC exposure. Because there are a great many data available on normal development and reproductive function in this species, the Japanese quail provides an excellent model for examining the effects of EDCs. Thus, we have begun studies using a Japanese quail model system to study the effects of EDCs on reproductive endocrine and behavioral responses. In this review, we have two goals: first, to provide a summary of reproductive development and sexual differentiation in intact Japanese quail embryos, including ontogenetic patterns in steroid hormones in the embryonic and maturing quail. Second, we discuss some recent data from experiments in our laboratory in which EDCs have been tested in Japanese quail. The Japanese quail provides an excellent avian model for testing EDCs because this species has well-characterized reproductive endocrine and behavioral responses. Considerable research has been conducted in quail in which the effects of embryonic steroid exposure have been studied relative to reproductive behavior. Moreover, developmental processes have been studied extensively and include investigations of the reproductive axis, thyroid system, and stress and immune responses. We have conducted a number of studies, which have considered long-term neuroendocrine consequences as well as behavioral responses to steroids. Some of these studies have specifically tested the effects of embryonic steroid exposure on later reproductive function in a multigenerational context. A multigenerational exposure provides a basis for understanding potential exposure scenarios in the field. In addition, potential routes of exposure to EDCs for avian species are being considered, as well as differential effects due to stage of the life cycle at exposure to an EDC. The studies in our laboratory have used both diet and egg injection as modes of exposure for Japanese quail. In this way, birds were exposed to a specific dose of an EDC at a selected stage in development by injection. Alternatively, dietary exposure appears to be a primary route of exposure; therefore experimental exposure through the diet mimics potential field situations. Thus, experiments should consider a number of aspects of exposure when attempting to replicate field exposures to EDCs.     

Bigger brains cycle faster before neurogenesis begins: a comparison of brain development between chickens and bobwhite quail

The chicken brain is more than twice as big as the bobwhite quail brain in adulthood. To determine how this species difference in brain size emerges during development, we examined whether differences in neurogenesis timing or cell cycle rates account for the disparity in brain size between chickens and quail. Specifically, we examined the timing of neural events (e.g. neurogenesis onset) from Nissl-stained sections of chicken and quail embryos. We estimated brain cell cycle rates using cumulative bromodeoxyuridine labelling in chickens and quail at embryonic day (ED) 2 and at ED5. We report that the timing of neural events is highly conserved between chickens and quail, once time is expressed as a percentage of overall incubation period. In absolute time, neurogenesis begins earlier in chickens than in quail. Therefore, neural event timing cannot account for the expansion of the chicken brain relative to the quail brain. Cell cycle rates are also similar between the two species at ED5. However, at ED2, before neurogenesis onset, brain cells cycle faster in chickens than in quail. These data indicate that chickens have a larger brain than bobwhite quail mainly because of species differences in cell cycle rates during early stages of embryonic development.     

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.     

Temporal and tissue-specific expression of myosin heavy chain isoforms in developing and adult avian muscle

We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.     

Genetic regulation of CFA expression in interspecific avian hybrids and somatic cell hybrids

Chicken fetal-leukemic antigen (CFA) is an oncodevelopmental antigen present on embryonic and neonatal chicken peripheral red blood cells (RBCs) but is not restricted to fetal stages of development in other avian species. Crosses between white Leghorn chickens and Japanese quail resulted in adult hybrids whose peripheral RBCs were positive for CFA. Of the four CFA determinants normally found in adult quail RBCs, only two were present on quail-chicken hybrid RBCs. Adult quail--chicken hybrid RBCs also possessed on CFA determinant associated with early development in both quail and chicken and one chicken-specific CFA determinant. Evidence is presented for the possible association of CFA-positive adult peripheral RBCs and the level of circulating reticulocytes. Crosses between pheasant and turkey (both with CFA-positive adult RBCs) resulted in hybrid adult RBCs expressing only a portion of the parental CFA determinants. Through the formation of somatic cell hybrids between adult chicken and embryonic Japanese quail RBCs, it was possible to induce the appearance of CFA determinants normally restricted to embryonic chicken RBCs. Approximately 50% of the hybrid cells showed reexpression of CFA, and this induction was both time and temperature dependent. Hybridization between RBCs of adult chicken and those of either adult Japanese quail or adult turkey failed to elicit the reexpression of chicken-specific CFA.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Immunomagnetic purification of viable primordial germ cells of Japanese quail (Coturnix japonica).

Immunomagnetic cell sorting (MACS) with the monoclonal antibody (mAb) QCR1 was compared with the Ficoll density-gradient centrifugation system (FICS) in terms of the efficiency of enrichment of quail (Coturnix japonica) primordial germ cells (PGCs) from blood. The purified PGCs were tested for their ability to settle in the chick (Gallus domesticus) embryonic gonad. Blood containing 60-100 PGCs microliter-1 was taken from the dorsal aorta of quail embryos at Hamburger and Hamilton's stages 14-16. The amount and concentration of PGCs in the PGC-rich fraction purified by MACS were greater than in the fraction purified by FICS. Purified quail PGCs were transfused into chick embryos at stages 14-16 and immunohistochemically stained with mAb QCRI on day 8 of chick development. Transfused PGCs purified by either MACS or FICS were positively stained in the chick embryonic gonads.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.     

Xenogenic oogenesis of chicken (Gallus domesticus) female primordial germ cells in germline chimeric quail (Coturnix japonica) ovary

In present study, chicken primordial germ cells (PGCs) were transferred into quail embryos to investigate the development of these germ cells in quail ovary. Briefly, 2 microl of chicken embryonic blood (stage 14) or about 100 purified circulating PGCs were transferred into quail embryo. Contribution of chicken PGCs were detected in gonads of chimeric quail embryos (stage 28) by immunocytochemical staining of cell surface antigen SSEA-1, and by in situ hybridization (ISH) with female chicken specific DNA probe. As a result, 52.0+/-43.2 (n=18) and 42.7+/-27.3 (n=17) chicken PGCs were found in the gonads of chimeric quail embryo that was injected with chicken embryonic blood (stage 14) and about 100 purified circulating PGCs, respectively. Furthermore, the ovaries of 81.8% (9/11) 12 days post incubation (dpi) chimeric quail embryos were observed with a mean of 457.6+/-237.1 female chicken PGCs-derived oogonia scattered in ovarian cortex area. In 9 out of 12 newly hatched and one week old chimeric quail chicks, on average of 2883.0+/-1924.1 primary oocytes and 3 follicles derived from chicken PGCs were found, respectively. The present results suggest that chicken female PGCs are able to migrate, colonize, proliferate and differentiate into oogonia, primary oocytes in chimeric quail ovary.     

Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation.

During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation.     

Role of tissue interaction between pineal primordium and neighboring tissues in avian pineal morphogenesis studied by intraocular transplantation

Tissue interactions play an essential role in organogenesis during embryonic development. However, virtually no attempts have been made to study the role of tissue interaction in pineal development. In the present study we examined the inductive role of the epidermis and mesenchyme in the morphogenesis of quail pineal glands. The pineal rudiment is first observed at embryonic day 2 (E2: 2 days of incubation) at the dorsal midline of the diencephalon as a short semi-spherical protrusion. Electron microscopic observations revealed that no mesenchymal cells are found between the epidermis and the distal end of the E2 pineal primordium but that a thin layer of mesenchymal cells separate the epidermis from the pineal primordium at E3. Small pieces containing pineal rudiment were cut off from E2 or E3 embryos. They were treated with enzymes to eliminate the epidermis and/or mesenchyme, grafted into E5 chicken eyes, and cultured there for 1 week. When E3 pineal rudiment was treated with Dispase to remove the epidermis, the pineal gland developed normally. When the rudiment was further treated with collagenase to remove the surrounding mesenchymal cells, a multi-follicular structure was still formed, but to a lesser extent than when rudiments were treated with Dispase alone. When E2 quail pineal rudiment with the epidermis was grafted without any treatment, a multi-follicular structure developed which morphologically resembled embryonic pineal organs. When the epidermis was removed from E2 rudiments by Dispase, a single large vesicular structure was formed. These results suggest that the overlying epidermis and/or mesenchymal cells play some inductive role in the initial pineal development, while the mesenchymal tissue plays an important role in pineal follicular formation later during development. Since only a few experimental studies have been done to examine pineal morphogenesis, the present study provides fundamental insights into avian pineal development.     

Effects of egg yolk testosterone on growth and immunity in a precocial bird

In oviparous vertebrates, maternal steroid allocation to eggs can have important fitness consequences for the offspring. However, elevated testosterone levels are not only associated with beneficial postnatal effects, such as enhanced growth and high social status, but may also entail costs by suppressing the immune system. In this study, testosterone levels in eggs of Chinese painted quail (Coturnix chinensis) were experimentally manipulated to evaluate its effects on growth and immunocompetence. Testosterone did not affect embryonic development, body size or growth during the first 20 days. However, elevated testosterone levels during embryonic development were immunosuppressive for chicks with inherently higher growth rate. Adaptive scenarios where only beneficial effects of increased testosterone levels are considered may therefore need to be re-evaluated.     

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.     

In vivo and in vitro studies on the segregation of autonomic and sensory cell lineages

Analysis of interspecific quail/chick chimaeras (made by grafting neural primordium from one species to the other) has demonstrated that the neural crest cell population, which gives rises to a large number of derivatives, including the great majority of peripheral ganglion cells, is pluripotential. When peripheral ganglia themselves are transplanted, it can be shown that many of the developmental potentialities of the parent structure are retained, their ultimate expression depending on the microenvironment in which they become located. One of the conclusions obtained from these in vivo studies, that sensory ganglia contain dormant precursors with autonomic potentialities, has been confirmed and extended by the results of in vitro investigations with dissociated 9- to 15-day embryonic quail dorsal root ganglia. Undetectable during normal embryonic development, adrenergic properties (tyrosine hydroxylase immunoreactivity, radio- and cytochemically demonstrable catecholamine production) develop in a population of small, multipolar cells after four days in culture. This differentiation is strongly dependent on the presence of chick embryo extract in the medium. Unlike the postmitotic primary sensory neurons of the ganglia, many of the adrenergic cells were found to incorporate 3H-thymidine during the culture period. These results support the contention that the latent autonomic percursors belong to the non-neuronal compartment of sensory ganglia.     

The Claude Bernard lecture, 1987. Embryonic chimeras: a tool for studying the development of the nervous and immune systems

Chimeras have been constructed in the avian embryo following the observation of the particular structure of the interphase nucleus in the Japanese quail (Coturnix coturnix japonica). In all embryonic and adult cell types of this species a large amount of heterochromatin is associated with the nucleolus, making quail cells readily distinguishable from those of the chick where the constitutive heterochromatin is evenly dispersed in the nucleus. These structural differences have been used to devise a cell-marking technique through which cell migrations and cell interactions during embryogenesis can be followed in the embryo in ovo by grafting quail cells into chick embryos or vice versa. This method was applied to the ontogeny of the neural crest and of the immune system. Recently quail-chick chimeras have been allowed to hatch and the immunological status of the embryonic grafts after birth scrutinized. Xenogeneic tissue grafts made in the embryo are rejected after birth with a more or less prolonged delay according to the nature of the graft. However, rejection can be prevented and a permanent state of tolerance induced for the embryonic tissue grafts by isotopically implanting the thymic epithelium from the same quail donor.     

Sensitivity of expression of perivitelline membrane glycoprotein ZP1 mRNA in the liver of Japanese quail (Coturnix japonica) to estrogenic compounds

Avian perivitelline membrane protein, ZP1, is synthesized and secreted by the liver with the stimulation of estrogens. In the present study, we measured the expression of ZP1 gene in the liver of immature male quail treated with various estrogenic compounds and in the liver of male quail embryos that were developed in the fertilized eggs laid by mother quail injected with various estrogenic compounds during vitellogenesis. Total RNA extracted from the liver was reverse-transcribed and cDNA was subjected to real-time PCR. Both diethylstilbestrol and ethinyl estradiol caused significant effect on the increase in mRNA in immature male quail. In contrast, diethylstilbestrol administered via the route of maternal injection was not effective for induction of embryonic mRNA, although the effect of ethinyl estradiol administered via the same route was prominent. These results showed that direct administration of estrogenic compounds, diethylstilbestrol and ethinyl estradiol, stimulates the induction of ZP1 gene, but the rate of accumulation of these compounds in the yolk is different during vitellogenesis. The present studies suggest that although ZP1 gene is a sensitive biomarker to evaluate the effects of endocrine disruptors, the route of administration is an important factor to compare the effectiveness.