The Developing Chicken Yolk Sac Acquires Nutrient Transport Competence by an Orchestrated Differentiation Process of Its Endodermal Epithelial Cells

During chicken yolk sac (YS) growth, mesodermal cells in the area vasculosa follow the migrating endodermal epithelial cell (EEC) layer in the area vitellina. Ultimately, these cells form the vascularized YS that functions in nutrient transfer to the embryo. How and when EECs, with their apical aspect directly contacting the oocytic yolk, acquire the ability to take up yolk macromolecules during the vitellina-to-vasculosa transition has not been investigated. In addressing these questions, we found that with progressive vascularization, the expression level in EECs of the nutrient receptor triad, LRP2-cubilin-amnionless, changes significantly. The receptor complex, competent for uptake of yolk proteins, is produced by EECs in the area vasculosa but not in the area vitellina. Yolk components endocytosed by LRP2-cubilin-amnionless, preformed and newly formed lipid droplets, and yolk-derived very low density lipoprotein, shown to be efficiently endocytosed and lysosomally processed by EECs, probably provide substrates for resynthesis and secretion of nutrients, such as lipoproteins. In fact, as directly demonstrated by pulse-chase experiments, EECs in the vascularized, but not in the avascular, region efficiently produce and secrete lipoproteins containing apolipoprotein A-I (apoA-I), apoB, and/or apoA-V. In contrast, perilipin 2, a lipid droplet-stabilizing protein, is produced exclusively by the EECs of the area vitellina. These data suggest a differentiation process that orchestrates the vascularization of the developing YS with the induction of yolk uptake and lipoprotein secretion by EECs to ensure embryo nutrition.     

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.     

Differentiation of pinopsin-immunoreactive cells in the developing quail pineal organ: an in-vivo and in-vitro immunohistochemical study

The avian pineal organ contains several types of photoreceptors with different photopigments: rhodopsin, iodopsin, and pinopsin. We have previously examined the differentiation of both rhodopsin-like and iodopsin-like immunoreactive cells during pineal development in quail embryos to determine the onset of synthesis of specific proteins and their cellular localization. In the present study, we have performed pinopsin immunohistochemistry on in-vivo developing and in-vitro cultured pineal organs of quail embryos. The results were compared with those obtained with rhodopsin and iodopsin immunohistochemistry. In the developing pineal organs, pinopsin immunoreactivity was detected at embryonic day 8, i.e. five days earlier than rhodopsin-like and iodopsin-like immunoreactivities. It was localized exclusively in the protrusions extending into the lumen throughout development, whereas rhodopsin-like and iodopsin-like immunoreactivities were usually found both in cell bodies and processes. These differences were also observed under two different types of culture conditions (dissociated cell culture and organ culture) indicating that, in the avian pineal organ, the expression pattern of the pinopsin gene is basically different from those of the other two pineal photopigments. The present study suggests that pineal cells have a mechanism for the polarized transport of pinopsin molecules.     

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.     

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.     

Antioxidant systems of the developing quail embryo are compromised by mycotoxin aurofusarin

The effects of aurofusarin in the quail diet on the antioxidant systems of the developing embryo are investigated. Thirty eight 45-day-old Japanese quails (Coturnix japonica) were divided into two groups and were fed on a corn-soya diet or the same diet supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. Eggs obtained after 7 weeks of feeding were incubated. Samples of quail tissues were collected at day 17 of embryonic development and from day old hatchlings. Antioxidants and malondialdehyde were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased concentrations of alpha- and gamma-tocopherols, alpha- and gamma-tocotrienols, retinol, lutein and zeaxanthin in egg yolk. The vitamin E (tocopherols and tocotrienols) concentration in the liver and yolk sac membrane (YSM) of the day 17 embryos and the hatchlings from aurofusarin-fed group was significantly decreased. Alpha-tocopherol concentration was also reduced in kidney, lung, heart, muscle and brain of day-old quails. In the liver of day-old quails, concentrations of lutein, zeaxanthin, retinol, retinyl linoleate, retinyl oleate, retinyl palmitate and retinyl stearate were also reduced. As a result of these diminished antioxidant concentrations, tissue susceptibility to lipid peroxidation was significantly increased. It is suggested that a compromised antioxidant system of the egg yolk and embryonic tissues could predispose quails to increased mortality at late stages of their embryonic development.     

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.     

A new marker for identifying quail cells in embryonic avian chimeras: a quail-specific antiserum

The characterization of cell behavior in quail chick chimeras has greatly increased our knowledge of the ontogeny of embryonic cell populations and the role of cell-cell interactions in development. We sought to extend the value of avian chimeras by producing a marker that would recognize cell surface components and that could be used instead of the traditional nuclear marker to identify quail cells within chimeras. We describe here a quail-specific antiserum produced by injecting chickens with a membrane fraction of 6-10-day quail embryos. By use of peroxidase coupling of a second antibody, serum reactivity was tested in tissue sections of normal quail and chick embryos and of somitic mesoderm and neural tube chimeras. The primary time period examined was 6-10 days of development. At these stages, the antiserum recognizes only quail cells and stains both plasma membrane-associated and cytoplasmic cell components. The latter characteristics allow the identification of quail axons in chimeras and facilitate visualization of quail cells at low magnification. We show that antiserum staining can also be used to identify quail cells in culture and can be combined with orthograde HRP labeling of neurons.     

Differential incorporation of docosahexaenoic and arachidonic acids by the yolk sac membrane of the avian embryo

During avian development, lipoproteins derived from yolk lipid are assembled in the yolk sac membrane (YSM) for secretion into the embryonic circulation. To investigate how yolk polyunsaturated fatty acids, essential for the development of certain tissues, are distributed among the lipid classes of the lipoproteins, pieces of YSM were incubated in vitro with [14C]arachidonic and [14C]docosahexaenoic acids (DHA). There was a marked difference in the partitioning of these two precursors among the lipid classes of the tissue. Of the radioactivity incorporated into total lipid from [14C]-arachidonic acid during 1 h of incubation, 67.3% was esterified as phospholipid and 29.5% as triacylglycerol. In contrast, only 14.6% of the label incorporated from [14C]-DHA was esterified as phospholipid, whereas 73.2% was recovered in triacylglycerol. This pattern of differential partitioning was observed at all time points and across a 20-fold range of fatty acid concentrations. There was no evidence for conversion of the radioactive arachidonic and DHAs to other fatty acids prior to incorporation into tissue lipids. It is suggested that the selective incorporation of yolk-derived DHA into the triacylglycerol of secreted lipoproteins represents part of a mechanism for directing this polyunsaturate to particular embryonic tissues.     

Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail

Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts.     

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.     

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.     

Towards the Conservation of Endangered Avian Species: A Recombinant West Nile Virus Vaccine Results in Increased Humoral and Cellular Immune Responses in Japanese Quail (Coturnix japonica)

West Nile Virus (WNV) arrived in North America in 1999 and is now endemic. Many families of birds, especially corvids, are highly susceptible to WNV and infection often results in fatality. Avian species susceptible to WNV infection also include endangered species, such as the Greater Sage-Grouse (Centrocercus uropbasianuts) and the Eastern Loggerhead Shrike (Lanius ludovicianus migrans). The virus has been shown to contribute towards the likelihood of their extinction. Although a clear and present threat, there exists no avian WNV vaccine available to combat this lethal menace. As a first step in establishing an avian model for testing candidate WNV vaccines, avian antibody based reagents were assessed for cross-reactivity with Japanese quail (Coturnix japonica) T cell markers CD4 and CD8; the most reactive were found to be the anti-duck CD8 antibody, clone Du-CD8-1, and the anti-chicken/turkey CD4 antibody, clone CT4. These reagents were then used to assess vaccine performance as well as to establish T cell populations in quail, with a novel population of CD4/CD8 double positive T cells being identified in Japanese quail. Concurrently, non-replicating recombinant adenoviruses, expressing either the WNV envelope or NS3 ‘genes’ were constructed and assessed for effectiveness as avian vaccines. Japanese Quail were selected for testing the vaccines, as they provide an avian model that parallels the population diversity of bird species in the wild. Both the level of WNV specific antibodies and the number of T cells in vaccinated birds were increased compared to unvaccinated controls. The results indicate the vaccines to be effective in increasing both humoral and cellular immune responses. These recombinant vaccines therefore may find utility as tools to protect and maintain domestic and wild avian populations. Their implementation may also arrest the progression towards extinction of endangered avian species and reduce the viral reservoir that potentiates infection in humans.     

The roles of ERAS during cell lineage specification of mouse early embryonic development

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.     

Skeletal Muscle Characterization of Japanese Quail Line Selectively Bred for Lower Body Weight as an Avian Model of Delayed Muscle Growth with Hypoplasia

This study was designed to extensively characterize the skeletal muscle development in the low weight (LW) quail selected from random bred control (RBC) Japanese quail in order to provide a new avian model of impaired and delayed growth in physically normal animals. The LW line had smaller embryo and body weights than the RBC line in all age groups (P<0.05). During 3 to 42 d post-hatch, the LW line exhibited approximately 60% smaller weight of pectoralis major muscle (PM), mainly resulting from lower fiber numbers compared to the RBC line (P<0.05). During early post-hatch period when myotubes are still actively forming, the LW line showed impaired PM growth with prolonged expression of Pax7 and lower expression levels of MyoD, Myf-5, and myogenin (P<0.05), likely leading to impairment of myogenic differentiation and consequently, reduced muscle fiber formation. Additionally, the LW line had delayed transition of neonatal to adult myosin heavy chain isoform, suggesting delayed muscle maturation. This is further supported by the finding that the LW line continued to grow unlike the RBC line; difference in the percentages of PMW to body weights between both quail lines diminished with increasing age from 42 to 75 d post-hatch. This delayed muscle growth in the LW line is accompanied by higher levels of myogenin expression at 42 d (P<0.05), higher percentage of centered nuclei at 42 d (P<0.01), and greater rate of increase in fiber size between 42 and 75 d post-hatch (P<0.001) compared to the RBC line. Analysis of physiological, morphological, and developmental parameters during muscle development of the LW quail line provided a well-characterized avian model for future identification of the responsible genes and for studying mechanisms of hypoplasia and delayed muscle growth.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail-chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-alpha are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2alpha, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.     

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.