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Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.

Increases in the size of the neuronal structures that mediate specific behaviors are believed to be related to enhanced computational performance. It is not clear, however, what developmental and evolutionary mechanisms mediate these changes, nor whether an increase in the size of a given neuronal population is a general mechanism to achieve enhanced computational ability. We addressed the issue of size by analyzing the variation in the relative number of cells of auditory structures in auditory specialists and generalists. We show that bird species with different auditory specializations exhibit variation in the relative size of their hindbrain auditory nuclei. In the barn owl, an auditory specialist, the hindbrain auditory nuclei involved in the computation of sound location show hyperplasia. This hyperplasia was also found in songbirds, but not in non-auditory specialists. The hyperplasia of auditory nuclei was also not seen in birds with large body weight suggesting that the total number of cells is selected for in auditory specialists. In barn owls, differences observed in the relative size of the auditory nuclei might be attributed to modifications in neurogenesis and cell death. Thus, hyperplasia of circuits used for auditory computation accompanies auditory specialization in different orders of birds., (Copyright 2004 S. Karger AG, Basel)

(AMH/MIS) was first suggested by Jost, more than Four decades before this gonadal glycoprotein was purified and its gene and promoter sequenced. In mammals, AMH expression is triggered by SOX9 in Sertoli cells at the onset of testicular differentiation, and regulated by SF1, GATA factors, WT1, DAX1 and FSH. Ovarian granulosa cells also secrete AMH from late foetal life. In males, AMH is secreted into the bloodstream at high levels until puberty when it is down-regulated by androgens and meiotic germ cells and its directional secretion switches from the basal compartment to the seminiferous tubule lumen. In birds and reptiles, AMH expression shows particular features. Serum AMH determination is useful to study testicular function in boys and in patients with gonadal tumours. AMH levels in seminal and follicular fluid may also be of clinical use.

This study examines the metabolic fate of total and individual yolk fatty acids (FA) during the embryonic development of the king penguin, a seabird characterized by prolonged incubation (53 days) and hatching (3 days) periods, and a high n-3/n-6 polyunsaturated FA ratio in the egg. Of the approximately 15 g of total FA initially present in the egg lipid, 87% was transferred to the embryo by the time of hatching, the remaining 13% being present in the internalized yolk sac of the chick. During the whole incubation, 83% of the transferred FA was oxidized for energy, with only 17% incorporated into embryo lipids. Prehatching (days 0-49), the fat stores (triacylglycerol) accounted for 58% of the total FA incorporated into embryo lipid. During hatching (days 49-53), 40% of the FA of the fat stores was mobilized, the mobilization of individual FA being nonselective. At hatch, 53% of the arachidonic acid (20:4n-6) of the initial yolk had been incorporated into embryo lipid compared with only 15% of the total FA and 17-24% of the various n-3 polyunsaturated FA. Similarly, only 32% of the yolk's initial content of 20:4n-6 was oxidized for energy during development compared with 72% of the total FA and 58-66% of the n-3 polyunsaturated FA. The high partitioning of yolk FA toward oxidization and the intense mobilization of fat store FA during hatching most likely reflect the high energy cost of the long incubation and hatching periods of the king penguin. The preferential partitioning of 20:4n-6 into the structural lipid of the embryo in the face of its low content in the yolk may reflect the important roles of this FA in tissue function.

Accessory organs of the integument are locally modified parts of the potentially feather-bearing skin in birds (e.g., the rhamphotheca, claws, or scales), and of the potentially hairy skin in mammals (e.g., the rhinarium, nails, claws, or hooves). These special parts of the integument are characterised by a modified structure of their epidermal, dermal and subcutaneous layers. The developmental processes of these various integumentary structures in birds and mammals show both similarities and differences. For example, the development of the specialised epidermal structures of both feathers and the hoof capsule is influenced by the local three-dimensional configuration of the dermis. However, in feathers, in contrast to hooves, the arrangement of the corneous cells is only partially a direct result of the particular arrangement and shape of the dermal surface of the papillary body. Whereas the diameter of the feather papilla, as well as the number, length, and width of dermal ridges on the surface of the feather papilla influence the three-dimensional architecture of the feather rami, there is no apparent direct correlation between the dermo-epidermal interface and the development of the highly ordered architecture of the radii and hamuli in the feather vane. In order to elucidate this morphogenic problem and the problem of locally different processes of keratinisation and cornification, the structure and development of feathers in birds are compared to those of the hoof capsule in horses. The equine hoof is the most complex mammalian integumentary structure, which is determined directly by the dermal surface of the papillary body. Perspectives for further research on the development of modified integumentary structures, such as the role of the dermal microangioarchitecture and the selective adhesion and various differentiation pathways of epidermal cells, are discussed., (Copyright 2003 Wiley-Liss, Inc.)

Plumage is a complex component of the avian phenotype. The plumage of an individual is composed of numerous hierarchically arranged developmental and morphological modules. We present a hierarchical model of plumage that provides an intellectual framework for understanding the development and evolution of feathers. Independence, covariation, and interaction among plumage modules create numerous opportunities for developmental and evolutionary diversification of feather complexity and function. The hierarchical relationships among plumage modules are characterized by both top-down and bottom-up effects in which properties of modules at one level of the hierarchy determine or influence the properties of modules at lower or higher levels of the hierarchy. Plumage metamodules are created by covariation or interaction among modules at different levels of the hierarchy., (Copyright 2003 Wiley-Liss, Inc.)

The discovery of several dinosaurs with filamentous integumentary appendages of different morphologies has stimulated models for the evolutionary origin of feathers. In order to understand these models, knowledge of the development of the avian integument must be put into an evolutionary context. Thus, we present a review of avian scale and feather development, which summarizes the morphogenetic events involved, as well as the expression of the beta (beta) keratin multigene family that characterizes the epidermal appendages of reptiles and birds. First we review information on the evolution of the ectodermal epidermis and its beta (beta) keratins. Then we examine the morphogenesis of scutate scales and feathers including studies in which the extraembryonic ectoderm of the chorion is used to examine dermal induction. We also present studies on the scaleless (sc) mutant, and, because of the recent discovery of "four-winged" dinosaurs, we review earlier studies of a chicken strain, Silkie, that expresses ptilopody (pti), "feathered feet." We conclude that the ability of the ectodermal epidermis to generate discrete cell populations capable of forming functional structural elements consisting of specific members of the beta keratin multigene family was a plesiomorphic feature of the archosaurian ancestor of crocodilians and birds. Evidence suggests that the discrete epidermal lineages that make up the embryonic feather filament of extant birds are homologous with similar embryonic lineages of the developing scutate scales of birds and the scales of alligators. We believe that the early expression of conserved signaling modules in the embryonic skin of the avian ancestor led to the early morphogenesis of the embryonic feather filament, with its periderm, sheath, and barb ridge lineages forming the first protofeather. Invagination of the epidermis of the protofeather led to formation of the follicle providing for feather renewal and diversification. The observations that scale formation in birds involves an inhibition of feather formation coupled with observations on the feathered feet of the scaleless (High-line) and Silkie strains support the view that the ancestor of modern birds may have had feathered hind limbs similar to those recently discovered in nonavian dromaeosaurids. And finally, our recent observation on the bristles of the wild turkey beard raises the possibility that similar integumentary appendages may have adorned nonavian dinosaurs, and thus all filamentous integumentary appendages may not be homologous to modern feathers., (Copyright 2003 Wiley-Liss, Inc.)

The auditory nerve of birds and mammals exhibits differences and similarities, but given the millions of years since the two classes diverged from a common ancestor, the similarities are much more impressive than the differences. The avian nerve is simpler than that of mammals, but share many fundamental features including principles of development, structure, and physiological properties. Moreover, the available evidence shows that the human auditory nerve follows this same general organizational plan. Equally impressive are reports that homologous genes in worms, flies, and mice exert the same heredity influences in man. The clear implication is that animal studies will produce knowledge that has a direct bearing on the human condition.

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar Type II cells. It reduces alveolar surface tension and hence the work of breathing. Despite the tremendous diversity of lung structures amongst the vertebrates, the composition of surfactant is highly conserved. Conserved elements of the surfactant system amongst distantly related species are likely to be crucial factors for successful lung development. Understanding the mechanisms by which the surfactant system becomes operational in animals with dramatically different birthing strategies and in distantly related species will provide important information about the role of the surfactant system in the commencement of air breathing and the processes regulating surfactant maturation and secretion. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones, and autonomic neurotransmitters. Here we review the mechanisms controlling the maturation of surfactant production, including birthing strategy, phylogeny, lung structure, and posthatching environment. Using four species of egg-laying amniote (chicken, dragon lizard, sea turtle, and crocodile) previously described in detail and the large amount of information available for mammals, we examine the hypothesis that the control of surfactant production is dependent on glucocorticoids (dexamethasone [Dex]), thyroid hormones (T3), and autonomic neurotransmitters (epinephrine and carbachol). We also examine whether the overall intrinsic pattern of the control of surfactant maturation is conserved throughout the vertebrate radiation and then how the environment (extrinsic factors) may account for the observed differences in the patterns of development. We also discuss the utility of a coculture system of embryonic Type II cells and fibroblasts to determine the evolutionary pattern behind the control of surfactant and to demonstrate that the surfactant system matures under multihormonal control. We demonstrate that Dex and T3 are stimulators of surfactant production during embryonic development, but they lose their efficacy closer to hatching or birth. Epinephrine stimulates surfactant secretion beyond 75% of development and also after hatching or birth. Carbachol stimulates surfactant secretion in the bearded dragon and saltwater crocodile but not in the sea turtle, chicken, or mammals. It is likely that the differences in control of surfactant development are likely to be primarily related to metabolic activity and the duration of incubation (i.e., the "speed" of development). Moreover, the hormones examined appear important in promoting development and therefore appear conserved within the amniotes. However, the autonomic neurotransmitters induced different responses in different species. Hence, some factors are crucial for the proper maturation of the surfactant system, whereas others vary throughout evolution without being detrimental to the overall function of the system.

Sibling competition has been proposed as an important evolutionary pressure driving interspecific variation in developmental rates. We tested this hypothesis using rates of extra-pair paternity and brood parasitism, as well as the degree of hatching asynchrony, as indices of sibling competition in a comparative analysis of 70 species of bird. We found mixed support for the influence of sibling competition on prenatal development. Brood parasitism was negatively correlated with the length of incubation, and hatching asynchrony was positively correlated with the length of incubation, but both correlations disappeared when phylogeny was controlled for. Extra-pair paternity, however, was negatively correlated with incubation length even when phylogeny was controlled for. The latter could represent support for the influence of sibling competition on prenatal development or indirect effects of correlated selection on both traits by adult mortality. The existence of these correlations demonstrates that life-history strategies include linkages among a larger suite of traits than previously recognized.

Because the yolk lipids of the king penguin (Aptenodytes patagonicus) contain the highest concentrations of long-chain n-3 polyunsaturated fatty acids yet reported for an avian species, the consequences for the establishment of the brain's fatty acid profile in the embryo were investigated. To place the results in context, the fatty acid compositions of yolk lipid and brain phospholipid of the king penguin were compared with those from three other species of free-living birds. The proportions of docosahexaenoic acid (22:6n-3; DHA) in the total lipid of the initial yolks for the Canada goose (Branta canadensis), mallard (Anas platyrhynchos), moorhen (Gallinula chloropus), and king penguin were (% w/w of fatty acids) 1.0+/-0.1, 1.9+/-0.2, 3.3+/-0.1, and 5.9+/-0.2, respectively. The respective concentrations of DHA (% w/w of phospholipid fatty acids) in brains of the newly hatched chicks of these same species were 18.5+/-0.2, 19.6+/-0.7, 16.9+/-0.4, and 17.6+/-0.1. Thus, the natural interspecies diversity in yolk fatty acid profiles does not necessarily produce major differences in the DHA content of the developing brain. Only about 1% of the amount of DHA initially present in the yolk was recovered in the brain of the penguin at hatch. There was no preferential uptake of DHA from the yolk during development of the king penguin.

It was shown on the example of chick embryo that the number tau 0 (tau n/tau 0) can be recommended as a measure of biological time and, for this purpose, the duration of the minimal mitotic cycle during synchronous cleavage divisions should be determined (in minutes) in various avian species. Based on the preliminary data, one can propose the comparability and similarity of the temporal programs of gastrulation and somitogenesis in the chick embryo and embryos of some fish and amphibians.

A toxicokinetic model was developed to describe polychlorinated biphenyl (PCB) accumulation by herring gull (Larus argentatus) embryos during development. The model consists of two components, a bioenergetics model that predicts the lipid mass balance of embryo and yolk compartments with time and an empirical toxicokinetic model that describes PCB partitioning between lipid compartments in the egg. The model was calibrated using data on PCB and lipid partitioning between embryo and yolk + albumen at four time points during incubation in herring gull eggs injected with a PCB mixture, combined with data sets on herring gull embryo growth rates and bioenergetic demands with time. The model was validated using independent data consisting of maternally exposed, field-incubated Lake Superior herring gull eggs that varied in incubation ages over the range of 8.5 d to pipping age (26-28 days). PCB concentrations in 6-9 d embryos were nearly an order of magnitude less than predicted by equilibrium lipid partitioning between the embryo and yolk + albumen compartments of the eggs. PCB concentrations in embryos were adequately predicted by equilibrium partitioning, however, for eggs incubated for 23-28 d. An empirical relationship was developed to account for the apparent nonequilibrium behaviour of PCBs during early development. The model was sensitive to the mass of yolk lipids and the mass of PCBs deposited to fresh eggs and much of the variability in embryo PCB concentrations could by explained by accounting for variability in these input parameters. Consistent with experimental data for other avian species, the model predicts that the highest PCB concentrations in the embryo/chick occur during pipping or soon after when yolk lipids have been completely resorbed by the embryo.

Patterning events along the anterior-posterior (AP) axis of vertebrate embryos result in the distribution of muscle and bone forming a highly effective functional system. A key aspect of regionalized AP patterning results from variation in the migratory pattern of somite cells along the dorsal-ventral (DV) axis of the body. This occurs as somite cell populations expand around the axis or migrate away from the dorsal midline and cross into the lateral plate. The fate of somitic cells has been intensely studied and many details have been reported about inductive signaling from other tissues that influence somite cell fate and behavior. We are interested in understanding the specific differences between somites in particular AP regions and how these differences contribute to the global pattern of the organism. Using orthotopic transplants of segmental plate between quail and chick embryos, we have mapped the interface of the somitic and lateral plate mesoderm during the formation of the body wall in cervical and thoracic regions. This interface does not change dramatically in the mid-cervical region, but undergoes extensive changes in the thoracic region. Based on this regional mapping and consistent with the extensive literature, we suggest a revised method of classifying regions of the body wall that relies on embryonic cell lineages rather than adult functional criteria.

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.

Common terns (Sterna hirundo) and roseate terns (Sterna dougallii) breed on Bird Island, Massachusetts, USA, near a Superfund site highly contaminated with polychlorinated biphenyls (PCBs). Observations of skewed sex ratios and female-female pairings among endangered roseate terns (Nisbet and Hatch (1999) Ibis 141, 307) suggested the possibility of contaminant-related endocrine disruption in these birds and prompted investigation of common terns as a surrogate species. In 1993 and 1994, 60-90% of pipping male common tern embryos sampled exhibited ovarian cortical tissue in their testes (ovotestes) (Nisbet et al. (1996) Bull. Environ. Contam. Toxicol. 57, 895; Hart et al. (1998) Mar. Environ. Res. 46, 174). To examine the possible impact of ovotestes on the reproductive capabilities of common terns, we examined gonadal histology in common tern prefledglings (approximately 21 days old) collected from Bird Island in 1995. As a measure of embryonic contaminant exposure, contaminants were measured in a subset of eggs collected from the same nests as the prefledglings. Concentrations of total PCBs in these eggs ranged from 14.4 to 546 microg/g lipid. No evidence of ovotesticular development was observed in any of the 19 male prefledglings examined. Some gonadal irregularities were observed, including small nodules of testicular tissue within the epithelial capsule of the testes, but these were judged not likely to affect testicular function. There was no relationship between any observed irregularities and levels of contaminants present in the matched eggs. The results suggest that the ovotestes that occur in 60-90% of pipping common tern embryos from this site become fully regressed by approximately 21 days posthatch. Our data from this and previous studies are consistent with the idea that ovotestes occur naturally in some individual common terns at hatching, although the frequency of occurrence may be enhanced by exposure to chlorinated organic contaminants such as PCBs. In either case, we suggest that the presence of ovotestes in common tern embryos from PCB-contaminated sites such as Bird Island does not lead to permanent alterations in gonadal histology that would be expected to impair reproductive function.

Cu-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied in pheasants after administration as single compounds or in combination. The test materials were injected directly into the air-chamber of eggs on day 12 of the hatching period and evaulation was carried out on day 23 of incubation. The pheasant embryos were examined for the following: rate of embryo mortality, body mass, type of developmental anomalies, light microscopic examination. After the administration of copper-sulphate on day 12 of incubation, the average body weight of pheasant embryos significantly did not decrease as compared to the control. The embryonic mortality was 68%. After the administration of a mancozeb containing fungicide formulation (Dithane M-45) on day 12 of incubation, the average body weight of embryos did not decrease as compared to the control. The embryonic mortality was 50%. After the individual administration of pesticide, the incidence of developmental anomalies was very high. After the combined administration of copper-sulphate and the 80% mancozeb containing fungicide formulation (Dithane M-45) on day 12 of incubation the embryonic mortality markedly increased. The rate of embryo mortality was 93%. We did not find any degenerative change in the liver tissue in either the control group or the treated groups by light microscopic examination. In summary, it can be established that the simultaneous administration of Cu-sulphate and mancozeb containing fungicide formulation caused high toxicity compared with the individual toxicity of test materials.

The differentiation and patterning of tendon fibroblasts is at present a poorly understood aspect of musculoskeletal development in the vertebrate limb. Precursors of tendon fibroblasts originate in the somatic mesoderm adjacent to the early limb bud and gradually become incorporated into the limb mesenchyme as development proceeds. It is unclear whether these progenitor cells are committed to the tendon lineage at this early stage, or whether cells become committed only as they are incorporated into a developing tendon. Following a review of our current knowledge of early tendon development, we present recent results from our preliminary studies looking at tendon cell commitment. Using a lacZ encoding replication-deficient retrovirus, we have mapped regions of the early limb bud that contain presumptive tendon progenitor cells, and later used these sites for implanting labelled fetal tendon fibroblasts into developing limbs. Following implantation, we found that these cells successfully re-incorporated into developing proximal and distal tendons, but also surprisingly contributed to other tissue lineages within the limb. Our results suggest that fetal tendon fibroblasts may not be irreversibly committed to a tendon cell fate in the limb and may be somewhat plastic in their ability to integrate into other tissue lineages during development.

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate that is used in the United States and other countries as a larvicide for mosquito suppression. As part of a multi-species evaluation of the potential effects of GB-1111 on birds, red-winged blackbird eggs were collected, artificially incubated, and treated with one of five amounts of GB-1111 varying from 0 to 10 times the expected exposure from a spray application of the maximum recommended amount (X=47 l/ha, 5 gal/ac). The application of 10 X caused a significant reduction in hatching success. A dose-related reduction of hepatic microsomal monooxygenase activity (EROD) was detected. Among body weights, skeletal measurements, and age at death, only crownrump length was different among experimental groups. Overall, the potential hazard to embryos of a representative wetland passerine appears minimal until the application rate exceeds 3 X.

Hematopoietic stem cells (HSCs) are responsible for generating all the lineages of the blood. During vertebrate development, waves of hematopoietic activity can be found in distinct anatomical sites, and they contribute to both embryonic and adult hematopoiesis. The origin of the HSCs that ultimately give rise to all the adult blood lineages has been a controversial issue in the field of hematopoiesis. Studies of amniotes have linked HSC activity to the aorta-gonad-mesonephros (AGM) region, whereas others suggest that the yolk sac is the true source of HSCs. This review describes both primitive and definitive hematopoiesis in mice, humans, chicks, frogs, and zebrafish and examines the current debate over the embryonic origins of HSCs.

The anterior avian cornea possesses several distinct cellular and extracellular regions including the epithelial basal lamina, Bowman's layer and the interfacial matrix that separates Bowman's layer from the stroma. These unique regions differ biochemically, physically and morphologically but all contain type XII collagen. Previously, the collagen fibrils of several of these interfacial regions were shown to be stable to thermal and enzymatic denaturation. We reasoned that type XII collagen, a fibril-associated collagen, would be a good candidate to confer such stabilizing properties. The studies described herein were performed to localize type XII collagen and to assess its role in the interfacial matrices (IM). Using antibodies that react with both the short and long type XII collagen isoforms and that react specifically with the long isoform, we demonstrate that it is the short isoform that is present in Bowman's layer and the associated interfacial matrix lying between Bowman's and the stroma proper. In situ hybridization analyses demonstrate that both the epithelial and endothelial cells synthesize type XII collagen. In vitro cell culture analyses, however, demonstrate that in addition to epithelial cell synthesis, the stromal fibroblasts are capable of synthesizing type XII collagen as well. Immunofluorescence analyses performed at elevated temperature demonstrate that type XII collagen is thermally stable in Bowman's layer, but not in the anterior interfacial matrix or Descemet's layer. In addition, we observed that the distribution of type XII collagen during the development of the anterior extracellular matrices correlates precisely with an elevated density of keratocytes populating the interfacial matrix just deep to Bowman's layer. We show that this cellular density is developmentally regulated and does not arise from a localized increase in cell proliferation. These data demonstrate that Bowman's layer and the anterior interfacial matrix have unique biochemical and morphologic properties. Type XII collagen is thermally stable in Bowman's layer and, as a surface component of type I collagen fibrils, may contribute to the stability of the fibrils in this region. Neither type XII nor type I collagen is stable in the adjacent interfacial matrix, suggesting that differences in the type I-XII collagen fibril organization may exist between Bowman's layer and IM.

The Baltic Sea is a heavily polluted area. To assess the current contaminant pressure on the common guillemot (Uria aalge) living there, whole-body extracts of guillemots from the Baltic Sea were prepared and subdivided over six fractions, which differed in composition due to lipophilicity and polarity of the contaminants. The fractions were tested in the chicken embryo assay and compared to fractions of Atlantic guillemot extracts. Fertilized chicken eggs were injected with 0.03, 0.3, or 3 bird egg equivalents (BEQ) of the contaminants present in the fractions and then incubated for 19 d. Endpoints were selected to cover several mechanisms that may play a role in reproductive failures of fish-eating birds. Fractions I and IV from the Baltic guillemots induced ethoxyresorufin-O-deethylase (EROD) activity up to 15-fold in embryos exposed to 0.3 BEQ and up to 17-fold in embryos exposed to 3 BEQ. Corresponding Atlantic fractions induced EROD activity only at the higher dose of 3 BEQ. Morphological alterations were observed in the bursa of Fabricius in embryos exposed to the fractions that induced EROD, and for the Baltic fractions, this was apparent at the dose of 0.3 BEQ. The higher toxic potency of fractions I and IV was confirmed by higher mortality and occurrence of malformations among embryos exposed to these fractions. No other effects were observed; morphometry, hepatic porphyrin levels, thiamine-dependent enzymes, and acetylcholinesterase activity were not affected by any fraction. During interpretation of the results, concentrations in the whole-body guillemot extracts were compared to concentrations reported in field studies. In general, concentrations in the guillemot extract were lower than those associated with biomarker responses in other wild-bird species. However, because the relative sensitivity of guillemot toward immunotoxic effects remains to be resolved, effects on the immunocompetence of guillemot could not be excluded.

Avian communal breeding systems generate alternative behavioural strategies for females, resulting in differences in reproductive success. Identifying eggs of different females in such systems is problematic, however, due to egg destruction before incubation, difficulty of capturing adults, and/or inaccuracy of egg identification based on egg morphometry. Here, we describe a technique that uses electrophoresis of yolk proteins to determine egg ownership, which we applied to communally breeding guira cuckoos (Guira guira). Validation of the method included identical yolk protein banding patterns in all eggs of the same female, but different patterns in eggs of different females in budgerigars (Melopsittacus undulatus), and identical patterns in yolk follicles of the same females in guira cuckoos. We applied the protocol to 195 guira cuckoo eggs from 34 joint nests in 2 years. All multiple guira cuckoo eggs laid on the same day in single nests had distinct banding patterns of yolk proteins, practically eliminating the possibility of more than one female being represented by the same pattern. Some identical banding patterns were repeated in different days within a nesting bout, indicating that some females laid several eggs in shared nests. Identical patterns occasionally occurred in renestings of groups, indicating that some females lay eggs in consecutive nestings. Yolk protein electrophoresis is a useful tool to identify egg maternity in other circumstances, such as polygynous mating systems with joint nests and intraspecific parasitism. Additionally, it is an alternative method for species where electrophoresis of egg white proteins does not show sufficient polymorphism.

We examined whether the previously reported low cost of embryonic development in pelicans could be attributed to a more efficient conversion of egg energy to hatchling tissues as a result of high initial egg water content, low embryonic metabolic rate and growth later in incubation than in more precocious species. We therefore determined egg and hatchling composition and the development of embryonic respiration in the Australian pelican Pelecanus conspicillatus, which lays one of the largest eggs (140-210 g) with an altricial developmental mode. The small yolk fraction (21%) is typical of all pelecaniforms; however, we found that intraspecific variability in fresh egg mass was related to water content (principally in the albumen), but independent of yolk mass (mean 13 g dry mass). P. conspicillatus eggs have, on average, 635 kJ of energy, irrespective of egg mass across the whole range of egg mass. The embryonic developmental pattern of O(2) consumption and CO(2) production showed clear plateaus lasting 2-3 days immediately prior to internal pipping, resembling the typical precocial pattern. However, the rate of pre-internal pipping O(2) consumption was low in comparison with that of precocial species of similar egg mass. There is no evidence to support the hypothesis that the observed plateau in rates of O(2) uptake is due to a diffusion limitation of the eggshell gas conductance in this species. Embryonic metabolic rate nearly doubled during the pipping period, but the mass-independent metabolic rate of the hatchling was low in comparison with that of the resting adult. The total O(2) consumed (11 063 ml) is equivalent to 217.3 kJ (or 34% of egg energy) based on indirect calorimetry and the observed respiratory exchange ratio of 0.71. Thus, the cost of development (direct calorimetry) was 0.29 kJ J(-1) in the egg (mean egg mass 168 g), which is one of lowest reported values. As a result, the production efficiency of pelican embryonic development was 61.6%, higher than the average for birds in general (56.9%) and, in particular, of seabirds that have prolonged incubation periods on the basis of egg mass. High efficiency in embryonic development in this species was attained as a result of rapid embryonic growth later in incubation, low hatchling energy density (23.6 kJ g(-1) dry matter) and dry matter content, low embryonic metabolic rate throughout incubation and a shorter than expected incubation period of 33 days (predicted 36 days).

Necropsy records for 102 Attwater's prairie chickens (Tympanuchus cupido attwateri) that had been captive-reared and had died at the San Antonio Zoo over a 4-yr period were evaluated to determine the causes of mortality. Gross necropsy, histopathology, and bacterial culture findings are summarized. Mortality was highest in young birds (<6 mo) and embryos. Husbandry- and gastrointestinal-related diseases were the most common causes of mortality.

To examine the role of development in the origin of evolutionary novelties, we investigated the developmental mechanisms involved in the formation of a complex morphological novelty-branched feathers. We demonstrate that the anterior-posterior expression polarity of Sonic hedgehog (Shh) and Bone morphogenetic protein 2 (Bmp2) in the primordia of feathers, avian scales, and alligator scales is conserved and phylogenetically primitive to archosaurian integumentary appendages. In feather development, derived patterns of Shh-Bmp2 signaling are associated with the development of evolutionarily novel feather structures. Longitudinal Shh-Bmp2 expression domains in the marginal plate epithelium between barb ridges provide a prepattern of the barbs and rachis. Thus, control of Shh-Bmp2 signaling is a fundamental component of the mechanism determining feather form (i.e., plumulaceous vs. pennaceous structure). We show that Shh signaling is necessary for the formation and proper differentiation of a barb ridge and that it is mediated by Bmp signaling. BMP signaling is necessary and sufficient to negatively regulate Shh expression within forming feather germs and this epistatic relationship is conserved in scale morphogenesis. Ectopic SHH and BMP2 signaling leads to opposing effects on proliferation and differentiation within the feather germ, suggesting that the integrative signaling between Shh and Bmp2 is a means to regulate controlled growth and differentiation of forming skin appendages. We conclude that Shh and Bmp signaling is necessary for the formation of barb ridges in feathers and that Shh and Bmp2 signaling constitutes a functionally conserved developmental signaling module in archosaur epidermal appendage development. We propose a model in which branched feather form evolved by repeated, evolutionary re-utilization of a Shh-Bmp2 signaling module in new developmental contexts. Feather animation Quicktime movies can be viewed at http://fallon.anatomy.wisc.edu/feather.html., (Copyright 2002 Wiley-Liss, Inc.)

Heart rate (HR) in avian embryos developing inside an eggshell has been measured by various means while maintaining adequate gas exchange through the eggshell. This is an important requirement in order to avoid adverse effects of impeding gas exchange on the cardiac rhythms of developing embryos. The present report is a review of our ontogenetic study on embryonic HR, which was measured with fulfillment of the above requirement and also hatchling HR measured non-invasively. Firstly, we reviewed measurements of daily changes (developmental patterns) in embryonic mean heart rate (MHR), which were determined from a short-term measurement of HR once a day, in 34 species of altricial and precocial birds. The allometric relationship between the MHR during pipping in altricial birds and their fresh egg masses was the same as that between the MHR at 80% of incubation duration and fresh egg masses in pre-cocial birds. Secondly, we presented the developmental patterns of MHR in chick embryos and hatchlings, which were determined from long-term, continuous measurement of HR before, during and after hatching. The ultradian and circadian rhythms of HR were clearly shown in embryos and hatchlings, respectively. Thirdly, we summarized instantaneous HR fluctuations: HR variability and HR irregularities, in chick embryos and hatchlings. The distinctive patterns were shown in pre-pipped and pipped embryos and newly hatched chicks, individually, which were partly related to autonomic nervous functions and physiological functions.

Using the quail-chick chimera technique, we followed the fate of Rauber's sickle cells in older whole blastoderms (cultured for approximately 2 days): after removal of the autochthonous Rauber's sickle from an unincubated chicken blastoderm, a quail Rauber's sickle was grafted isotopically and isochronically in its place. In transverse sections through these chimeras, the grafted quail Rauber's sickle cells were seen to have transformed into a broad row or ridge of quail junctional endoblast cells extending at the inner border of the area containing blood islands. After unilateral removal of the junctional endoblast from an intermediate streak chicken blastoderm (Stage 3; Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed during further in vitro culture that at the operated side, in the area previously occupied by this junctional endoblast, blood islands no longer developed. If after such a unilateral removal of the chicken junctional endoblast quail junctional endoblast was apposed in its place, then blood islands reappeared in the operated area. The intimate contact between the apposed quail junctional endoblast and the recently formed blood islands, derived from peripherally migrating mesoderm, was very obvious on sections through such chimeras. We further demonstrate that Rauber's sickle vs. junctional endoblast is indispensable for the anlage of blood islands in avian blastoderms. Indeed, in the absence of Rauber's sickle material no blood islands develop (even when mesoderm is present after ingression of the upper layer via a primitive streak) in the isolated central region of the area centralis of unincubated chicken blastoderms after culture in vitro. Also, no junctional endoblast and no sickle canal appear in these explants. By contrast, if a Rauber's sickle fragment is placed on such an isolated central blastoderm region, then blood islands develop. These blood islands start to develop from peripherally migrating mesoderm in the neighborhood of the Rauber's sickle-derived junctional endoblast., (Copyright 2002 Wiley-Liss, Inc.)

A computer-controlled microscopy system was devised to allow the observation of avian embryo development over an extended time period. Parallel experiments, as well as extended specimen volumes, can be recorded at cellular resolution using a three-dimensional scanning procedure. The resulting large set of data is processed automatically into registered, focal- and positional-drift corrected mosaic images, assembled as montages of adjacent microscopic fields. The configuration of the incubator and a sterile embryo chamber prevents condensation of the humidified culturing atmosphere in the optical path and is compatible with both differential interference contrast and epifluorescence optics. As a demonstration, recordings are presented showing the large-scale remodelling of the embryonic primordial vascular structure.

Body size and temperature are the two most important variables affecting nearly all biological rates and times. The relationship of size and temperature to development is of particular interest, because during ontogeny size changes and temperature often varies. Here we derive a general model, based on first principles of allometry and biochemical kinetics, that predicts the time of ontogenetic development as a function of body mass and temperature. The model fits embryonic development times spanning a wide range of egg sizes and incubation temperatures for birds and aquatic ectotherms (fish, amphibians, aquatic insects and zooplankton). The model also describes nearly 75% of the variation in post-embryonic development among a diverse sample of zooplankton. The remaining variation is partially explained by stoichiometry, specifically the whole-body carbon to phosphorus ratio. Development in other animals at other life stages is also described by this model. These results suggest a general definition of biological time that is approximately invariant and common to all organisms.

Water, soil, vegetation, grasshoppers, bird eggs, and bird livers collected at a 23.5-ha (58 acres) grassland irrigated with wastewater from an in situ uranium mine (study area) and a reference site in 1998 were analyzed for selenium and other trace elements. Selenium concentrations in the uranium mine wastewater applied onto the grassland ranged from 340 to 450 microg/L. Selenium in the upper 15 cm (6 in) of soil from the irrigated grassland at the mine ranged from 2.6 to 4.2 microg/g dry weight (DW). Mean selenium concentrations in soil and water were 5 and 15 times higher at the study area than at the reference site. Selenium concentrations in grasses and grasshoppers ranged from 6.8 to 24 microg/g and 11 to 20 microg/g DW, respectively. Selenium in red-winged blackbird eggs and livers collected from the study area ranged from 13.2 to 22 microg/g and 33 to 53 microg/g DW, respectively, and concentrations were well in excess of toxic thresholds. Mean selenium concentrations in grasses, grasshoppers, and bird eggs and livers were 5.8 to 30 times higher at the study area than at the reference site. Elevated selenium concentrations in water, soil, grasshoppers, and red-winged blackbird eggs and livers collected from the study area demonstrate that selenium is being mobilized and is bioaccumulating in the food chain.

The aim of this paper is to summarise the results of earlier experiments on thermoregulation and heat balance in birds, to present new results concerning thermoregulation during the perinatal period in precocial embryos and to develop a model of the ontogeny of thermoregulation over the whole lifespan of birds. The ontogeny of thermoregulation in precocial birds is characterised by three phases with different efficiency of the system. In the prenatal phase, all control elements of the thermoregulatory system can function, but the efficiency of the system is low. It is postulated that endothermic reactions during the prenatal period do not have a proximate (immediate), but rather an ultimate influence on the efficiency of thermoregulation. They may support adaptivity to expected environmental conditions and may be involved in epigenetic adaptation processes. During the early postnatal phase, the thermoregulatory system develops and matures. Summit metabolism and resting metabolic rate and their thermoregulatory set points increase. Preferred temperature is significantly different during different behavioural activities. The phase of full-blown homeothermy starts at approximately the 10th day of life. It is characterised by an activation order of thermoregulatory control elements and by secondary chemical thermoregulation. The influence of thermal and non-thermal climatic factors on heat production and heat loss may be described by mathematical models.

Excess selenium (Se) in the aquatic food chain is embryotoxic and teratogenic to avocets, stilts, and other waterbirds. American avocet (Recurvirostra americana) and black-necked stilt (Himantopus mexicanus) eggs were collected from three sites in the Tulare Lake Basin of California, USA, and hatched in the laboratory. These sites included the Tulare Lake Drainage District-north (TLDD-N, water 2.5 ppb Se), TLDD-south (TLDD-S, water 8.6 ppb Se), and Westfarmers (WF, water 190 ppb Se). Highest egg Se concentrations occurred at WF (geometric mean 31.4 ppm dry wt for avocets and 20.5 ppm dry wt for stilts). Mean egg Se concentrations were 6.7 ppm for avocets and 8.4 ppm for stilts at TLDD-S, and 3.3 ppm for avocets and 2.3 ppm for stilts at TLDD-N. Hatching success and incidence of malformations did not differ among sites, but yolk sac-free hatching weights and bone lengths were less for avocets at the WF site, whereas liver weights and liver to body weight ratios were greater at that site. With increasing Se concentration, oxidative stress was most apparent in avocet hatchlings from WF: hepatic glutathione (GSH) peroxidase activity increased, glucose-6-phosphate dehydrogenase activity decreased, and oxidized glutathione (GSSG) concentration as well as the ratio of GSSG to reduced GSH concentration increased. In stilts, hepatic GSH concentration was lower in WF hatchlings. In conclusion, our findings of Se-impaired embryo growth and hepatotoxicity in avocet hatchlings suggest that oxidative stress observed in hatchlings may be related to these biological effects and may serve as a potential bioindicator of subsequent impaired functions.

Viewing life-history evolution in birds based on an age-specific mortality framework can explain broad life-history patterns, including the long incubation periods in southern latitudes documented here. I show that incubation periods of species that are matched phylogenetically and ecologically between Argentina and Arizona are longer in Argentina. Long incubation periods have mystified scientists because they increase the accumulated risk of time-dependent mortality to young without providing a clear benefit. I hypothesize that parents of species with low adult mortality accept increased risk of mortality to their young from longer incubation if this allows reduced risk of mortality to themselves. During incubation, songbird parents can reduce risk of mortality to themselves by reducing nest attentiveness (percentage of time on the nest). Here I show that parents of species with lower adult mortality exhibit reduced nest attentiveness and that lower attentiveness is associated with longer incubation periods. However, the incubation period is also modified by juvenile mortality. Clutch size variation is also strongly correlated with age-specific mortality. Ultimately, adult and juvenile mortality explain variation in incubation and other life-history traits better than the historical paradigm.

Can developmental processes account for vertebrate limb homology, the overall similarity of definitive limb structure despite differences in different taxa which often relate to evolutionary adaptations? Relevant evidence is from molecular studies, from 'cut & paste' experimental embryology and from classical descriptive accounts of embryology and structure. There is striking evidence of a similar pattern of homologous regulatory gene expression (eg Shh, and Hox A & D genes) in tetrapod limb buds, and both similarity and differences when these are compared with expression patterns in a teleost fish paired fin bud. But these findings are as yet from too few tetrapod species (chick and mouse) to permit a 'molecular bauplan' for the limb to be proposed with any certainty. Further, the identification of similar networks of regulatory genes common to non-homologous developmental systems limits possibilities for finding a basis for classical structural homology in terms of expression of system-specific genes or gene networks. An integrated approach is needed, combining evidence from the fin-limb transition, and from study of the patterns and processes of amphibian and avian limb embryology, and this points towards a conserved developmental bauplan for the pentadactyl skeleton of the type earlier proposed by Alberch. Key features include the digital arch, restriction of digit number to a maximum of 5 and stereotyped connections between prechondrogenic condensations. But this is a dynamic and not rigidly fixed bauplan. It has no single set of skeletal elements (except proximally), since the position of joint formation in the prechondrogenic condensations is not stereotyped. Urodele amphibians in particular demonstrate heterochronic differences in the timing of events. Heterochrony may underlie some of the important changes in the pentadactyl pattern during evolution.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.

1. Embryonic mortality of domestic pheasant (Phasianus colchinus) eggs was recorded for a 10-week laying season for two groups of laying hens maintained under mating ratios of 8:1 and 12:1 (females:males). 2. Mating ratio had no significant effect on embryonic mortality between 1 and 4 or 5 and 20 d but values for these categories doubled as the laying season progressed, both rising to 8% to 9% of fertile eggs. 3. Higher embryonic mortality was observed between 21 and 25 d for the eggs from the 8:1 mating ratio (14.5% of fertile eggs compared with 12.1%) but this was unaffected by week of setting. 4. Reasons for these patterns of embryonic mortality were not clear and further research is needed to determine the factors affecting embryonic mortality in pheasants.

A bioassay based on the measurement of vitellogenin (VTG) mRNA in avian embryo hepatocyte cultures by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was developed. To allow sequence comparison and design of suitable PCR primers, a short region of VTG cDNA was cloned and sequenced for seven species of birds. Cell cultures were prepared from both chicken and herring gull embryos and treated with the estradiol analogue moxestrol or the organochlorine insecticide o,p'-DDT. Using primers based on an area of the VTG gene that was identical for herring gull and chicken, in vitro VTG mRNA induction was observed for both moxestrol- and o,p'-DDT-treated cultures. Herring gull embryo hepatocyte cultures responded with VTG mRNA induction at moxestrol concentrations of 1 nM compared with 10 nM in chicken embryo hepatocyte cultures. Both herring gull and chicken embryo hepatocyte cultures responded with substantial VTG mRNA induction when treated with 10,000 nM o,p'-DDT. These results suggest that the bioassay will be useful for comparing avian embryo hepatocyte culture concentration-response data in terms of intra- and interspecies sensitivities to pharmacological estrogens or environmental contaminants., (Copyright 2001 Academic Press.)

A review of one of the key problems of experimental hematology: the origin of hemopoietic stem cells in the development of vertebrates (amphibians, birds, and mammals). The appearance and functioning of two independent sources of hemopoietic stem cells (extra- and intraembryonic) were considered in amphibians, birds, and mammals. The contribution of each source to the formation of definitive hemopoietic tissue was analyzed. It was shown for amphibians and birds that intraembryonic organs such as the dorsolateral plate and the mesenchyme of dorsal aorta are involved in the formation of adult hemopoietic tissue, while the extraembryonic organs such as ventral islets and the yolk sac are devoid of true stem cells and provide only for the primary, transient hemopoiesis. New data have been considered concerning the previously unknown intraembryonic hemopoietic organ in mammals, a region of aorta-gonad-mesonephros arising in embryogenesis simultaneously with the yolk sac. Two extreme views on the involvement of stem cells of all these organs in the formation of definitive hemopoiesis have been considered. The data are provided on the interaction of the embryonic hemopoietic stem cells and the hemopoietic microenvironment of adult recipients.

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1., (Copyright 2001 Wiley-Liss, Inc.)

During the development of the cerebral cortex, radial glia serve as a scaffold to support and direct neurons during their migration. This view is now changing in the light of emerging evidence showing that these cells have a much more dynamic and diverse role. A recent series of studies has provided strong support for their role as precursor cells in the ventricular zone that generate cortical neurons and glia, in addition to providing migration guidance.

Turning biases have been reported in some mammalian species, but less is known about such biases in nonmammalians. This study investigated turning biases in domestic chicks, bobwhite and Japanese quail, leopard geckos, and snapping turtles. Domestic chicks (white leghorn and bantam) and bobwhite quail demonstrate strong group laterality. Japanese quail chicks, snapping turtles, and leopard geckos demonstrate no significant group bias. Results are discussed with regard to differences in embryonic experience, hatching behavior, and postnatal environment., (Copyright 2001 John Wiley & Sons, Inc.)

It has been suggested that female birds put more resources into eggs fathered by attractive males by laying larger eggs or by adding more testosterone, but this inference could be undermined if eggs of different sex are provisioned differently, as these studies did not control for sex differences. Here we compare hormone concentrations in the yolks of male and female eggs and find that these are significantly different. Our results indicate that it is premature to conclude that female birds invest more in eggs sired by a preferred male, and raise the possibility that yolk sex steroids may be part of the sex-determining process in birds.

1. The histochemistry and ultrastructure of pheasant eggs were compared on the basis of blue or brown shell colour. 2. Differences in lectin histochemistry of the outer shell membrane calcification surface indicate a biochemical disruption of the calcification sites in blue eggs. 3. Significant differences were observed in all aspects of eggshell ultrastructure with blue eggs having thinner shells with structural defects. 4. Poor hatchability of blue eggs may reflect high rates of weight loss associated with a defective eggshell.

The author presents for the first time empirical allometrical equations corresponding the mass of dinosaurs with the mass of their eggs, clutch size and its total mass. Comparison of these equations with those that were proposed for modern taxa of reptiles and birds shows that dinosaurs can be characterized by intermediate value of allometry index.

The concentrations (microg/g wet yolk) of total carotenoids in eggs of the common moorhen (Gallinula chloropus), American coot (Fulica americana) and lesser black-backed gull (Larus fuscus), collected in the wild, were 47.5, 131.0 and 71.6, respectively. In contrast to data for eggs of the domestic chicken, beta-carotene was a significant component in the yolks of these three wild species, forming 25-29% by wt. of the total carotenoids present. The concentration of total carotenoids in the livers of the newly-hatched chicks was 5-10 times higher than in the other tissues and beta-carotene was again a major component, forming 37-58% of the hepatic carotenoids. In the newly-hatched gull, the proportions of both lutein and zeaxanthin were very low in the liver but high in the heart and muscle when compared with the yolk. By contrast canthaxanthin, echinenone and beta-carotene were very minor constituents of heart and muscle when compared with their proportions in the yolk of the gull. The proportions of lutein and zeaxanthin in the liver of the newly-hatched coot and moorhen were also far lower than in the yolk whereas the liver was relatively enriched with beta-cryptoxanthin, beta-carotene and (in the moorhen) echinenone. The results indicate that avian embryos discriminate between different carotenoids during their distribution from the yolk to the various tissues.

Experimental embryology performed on avian embryos combines tissue manipulations and cell-labeling methods with increasing opportunities and demands for critical assays of the results. These approaches continue to reveal unexpected complexities in the normal patterns of cell movement and tissue origins, documentation of which is critical to unraveling the intricacies of cell and tissue interactions during embryogenesis. Viktor Hamburger's many pioneering contributions helped launch and promote the philosophical as well as technical elements of avian experimental embryology. Furthermore, his scholarship and profoundly positive presence influenced not just those of us fortunate to have trained with him, but several generations of developmental biologists. The first part of this article presents examples of the opportunities and rewards that have occurred due to his influences. Surgical manipulation of avian embryonic tissues always introduces a greater number of variables than the experimenter can control for or, often, readily identify. We present the results of dorsal and ventral lesions of hindbrain segments, which include defects in structures within, beside, and also at a considerable distance from the site of lesion. Extramedullary loops of longitudinal tract axons exit and re-enter the neural tube, and intra-medullary proliferation of blood vessels is expanded. Peripherally, the coalescence of neural crest- and placode-derived neuroblasts is disrupted. As expected, motor neurons and their projections close to the sites of lesion are compromised. However, an unexpected finding is that the normal projections of cranial nerves located distant to the lesion site were also disrupted. Following brainstem lesions in the region of rhombomeres 3, 4 or 5, trigeminal or oculomotor axons penetrated the lateral rectus muscle. Surprisingly, the ability of VIth nerve axons to reach the lateral rectus muscle was not destroyed in most cases, even though the terrain through which they needed to pass was disrupted. These axons typically followed a more ventral course than normal, and usually, the axons emerging from individual roots failed to fasciculate into a common VIth nerve, which suggests that each rootlet contains pathfinder-competent axons. The lesson from these lesions is that surgical intervention in avian embryos may have substantial effects upon tissues within, adjacent to, and distant to those that are being manipulated.