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The review contains data on functional shifts in fishes, amphibians and birds caused by changes in the otolith system operation after stay under weightlessness conditions. These data are of theoretical and practical significance and are important to resolve some fundamental problems of vestibulogy. The analysis of the results of space experiments has shown that weightlessness conditions do not exert a substantial impact on formation and functional state of the otolith system in embryonic fishes, amphibians and birds developed during space flight. Weightlessness conditions do pot inhibit embryonic development of lower vertebrates but even have rather beneficial effect on it. This is consistent with conclusions concerning development of mammalian fetuses. The experimental results show that weightlessness can cause similar functional and behavioral vestibular shifts both in lower vertebrates and in mammals. For example, immediately after an orbital flight the vestibuloocular reflex in fish larvae and tadpoles (without lordosis) was stronger than in control individuals. A similar shift of the otolith reflex was observed in the majority of cosmonauts after short-term orbital flights. Immediately after landing adult terrestrial vertebrates, as well as human beings, exhibit lower activity levels, worse equilibrium and coordination of movements. Another interesting finding observed after landing of the cosmic apparatus was an unusual looping character of tadpole swimming. It is supposed that the unusual motor activity of animals as well as appearance of illusions in cosmonauts and astronauts after switching from 1 to 0 g have the same nature and are related to the change in character of otolith organs stimulation. Considering this similarity of vestibular reactions, using animals seems rather perspective. Besides it allows applying in experiments various invasive techniques.

The anklebone (astragalus) of dinosaurs presents a characteristic upward projection, the 'ascending process' (ASC). The ASC is present in modern birds, but develops a separate ossification centre, and projects from the calcaneum in most species. These differences have been argued to make it non-comparable to dinosaurs. We studied ASC development in six different orders of birds using traditional techniques and spin-disc microscopy for whole-mount immunofluorescence. Unexpectedly, we found the ASC derives from the embryonic intermedium, an ancient element of the tetrapod ankle. In some birds it comes in contact with the astragalus, and, in others, with the calcaneum. The fact that the intermedium fails to fuse early with the tibiale and develops an ossification centre is unlike any other amniotes, yet resembles basal, amphibian-grade tetrapods. The ASC originated in early dinosaurs along changes to upright posture and locomotion, revealing an intriguing combination of functional innovation and reversion in its evolution.

Polyspermy is a major puzzle in reproductive biology. In some taxa, multiple sperm enter the ovum as part of the normal fertilization process, whereas in others, penetration of the ovum by more than one sperm is lethal. In birds, several sperm typically enter the germinal disc, yet only one fuses with the female pronucleus. It is unclear whether supernumerary sperm play an essential role in the avian fertilization process and, if they do, how females regulate the progression of sperm through the oviduct to ensure an appropriate number reach the ovum. Here, we show that when very few sperm penetrate the avian ovum, embryos are unlikely to survive beyond the earliest stages of development. We also show that when the number of inseminated sperm is limited, a greater proportion than expected reach and penetrate the ovum, indicating that females compensate for low sperm numbers in the oviduct. Our results suggest a functional role for supernumerary sperm in the processes of fertilization and early embryogenesis, providing an exciting expansion of our understanding of sperm function in birds., (© 2015 The Authors.)

Although eggshell thinning has been described mainly in the context of environmental pollution, it can also be the effect of reproductive changes induced by a developing embryo. On the basis of a literature survey of 25 bird species (26 published papers) we reviewed data on embryo-induced eggshell thinning (EET) in three groups of birds: precocials, semi-precocials and altricials. The average EET at the equator of the eggs was 6.4% (median = 4.7%). Our review did not confirm a general prediction of elevated EET at the egg equator in precocial species: altricial birds exhibited the highest EET (average = 12.0%), followed by precocials (7.6%) and semi-precocials (4.2%). We make certain critical recommendations based on the results of this study. Studies aiming to assess variation in eggshell thickness should examine intrinsic factors affecting shell properties of avian eggs, like thickness, which are the result of anatomical or reproductive changes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The avian beak is a key evolutionary innovation whose flexibility has permitted birds to diversify into a range of disparate ecological niches. We approached the problem of the mechanism behind this innovation using an approach bridging paleontology, comparative anatomy, and experimental developmental biology. First, we used fossil and extant data to show the beak is distinctive in consisting of fused premaxillae that are geometrically distinct from those of ancestral archosaurs. To elucidate underlying developmental mechanisms, we examined candidate gene expression domains in the embryonic face: the earlier frontonasal ectodermal zone (FEZ) and the later midfacial WNT-responsive region, in birds and several reptiles. This permitted the identification of an autapomorphic median gene expression region in Aves. To test the mechanism, we used inhibitors of both pathways to replicate in chicken the ancestral amniote expression. Altering the FEZ altered later WNT responsiveness to the ancestral pattern. Skeletal phenotypes from both types of experiments had premaxillae that clustered geometrically with ancestral fossil forms instead of beaked birds. The palatal region was also altered to a more ancestral phenotype. This is consistent with the fossil record and with the tight functional association of avian premaxillae and palate in forming a kinetic beak., (© 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.)

Embryonic remains within a small (4.75 by 2.23 cm) egg from the Late Cretaceous, Mongolia are here re-described. High-resolution X-ray computed tomography (HRCT) was used to digitally prepare and describe the enclosed embryonic bones. The egg, IGM (Mongolian Institute for Geology, Ulaanbaatar) 100/2010, with a three-part shell microstructure, was originally assigned to Neoceratopsia implying extensive homoplasy among eggshell characters across Dinosauria. Re-examination finds the forelimb significantly longer than the hindlimbs, proportions suggesting an avian identification. Additional, postcranial apomorphies (strut-like coracoid, cranially located humeral condyles, olecranon fossa, slender radius relative to the ulna, trochanteric crest on the femur, and ulna longer than the humerus) identify the embryo as avian. Presence of a dorsal coracoid fossa and a craniocaudally compressed distal humerus with a strongly angled distal margin support a diagnosis of IGM 100/2010 as an enantiornithine. Re-identification eliminates the implied homoplasy of this tri-laminate eggshell structure, and instead associates enantiornithine birds with eggshell microstructure composed of a mammillary, squamatic, and external zones. Posture of the embryo follows that of other theropods with fore- and hindlimbs folded parallel to the vertebral column and the elbow pointing caudally just dorsal to the knees. The size of the egg and embryo of IGM 100/2010 is similar to the two other Mongolian enantiornithine eggs. Well-ossified skeletons, as in this specimen, characterize all known enantiornithine embryos suggesting precocial hatchlings, comparing closely to late stage embryos of modern precocial birds that are both flight- and run-capable upon hatching. Extensive ossification in enantiornithine embryos may contribute to their relatively abundant representation in the fossil record. Neoceratopsian eggs remain unrecognized in the fossil record.

Feathers are an evolutionary novelty found in all extant birds. Despite recent progress investigating feather development and a revolution in dinosaur paleontology, the relationship of feathers to other amniote skin appendages, particularly reptile scales, remains unclear. Disagreement arises primarily from the observation that feathers and avian scutate scales exhibit an anatomical placode-defined as an epidermal thickening-in early development, whereas alligator and other avian scales do not. To investigate the homology of feathers and archosaur scales we examined patterns of nuclear β-catenin localization during early development of feathers and different bird and alligator scales. In birds, nuclear β-catenin is first localized to the feather placode, and then exhibits a dynamic pattern of localization in both epidermis and dermis of the feather bud. We found that asymmetric avian scutate scales and alligator scales share similar patterns of nuclear β-catenin localization with feathers. This supports the hypothesis that feathers, scutate scales, and alligator scales are homologous during early developmental stages, and are derived from early developmental stages of an asymmetric scale present in the archosaur ancestor. Furthermore, given that the earliest stage of β-catenin localization in feathers and archosaur scales is also found in placodes of several mammalian skin appendages, including hair and mammary glands, we hypothesize that a common skin appendage placode originated in the common ancestor of all amniotes. We suggest a skin placode should not be defined by anatomical features, but as a local, organized molecular signaling center from which an epidermal appendage develops., (© 2015 Wiley Periodicals, Inc.)

Temperature profoundly affects the rate and trajectory of embryonic development, and thermal extremes can be fatal. In viviparous species, maternal behaviour and physiology can buffer the embryo from thermal fluctuations; but in oviparous animals (like most reptiles and all birds), an embryo is likely to encounter unpredictable periods when incubation temperatures are unfavourable. Thus, we might expect natural selection to have favoured traits that enable embryos to maintain development despite those fluctuations. Our review of recent research identifies three main routes that embryos use in this way. Extreme temperatures (i) can be avoided (e.g. by accelerating hatching, by moving within the egg, by cooling the egg by enhanced rates of evaporation, or by hysteresis in rates of heating versus cooling); (ii) can be tolerated (e.g. by entering diapause, by producing heat-shock proteins, or by changing oxygen use); or (iii) the embryo can adjust its physiology and/or developmental trajectory in ways that reduce the fitness penalties of unfavourable thermal conditions (e.g. by acclimating, by exploiting brief windows of favourable conditions, or by producing the hatchling phenotype best suited to those incubation conditions). Embryos are not simply passive victims of ambient conditions. Like free-living stages of the life cycle, embryos exhibit behavioural and physiological plasticity that enables them to deal with unpredictable abiotic challenges., (© 2014 The Authors. Biological Reviews © 2014 Cambridge Philosophical Society.)

Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Avian chemical communication is a rapidly emerging field, but has been hampered by a critical lack of information on volatile chemicals that communicate ecologically relevant information (semiochemicals). A possible, but as yet unexplored, function of olfaction and chemical communication in birds is in parent-embryo and embryo-embryo communication. Communication between parents and developing embryos may act to mediate parental behaviour, while communication between embryos can control the synchronicity of hatching. Embryonic vocalisations and vibrations have been implicated as a means of communication during the later stages of development but in the early stages, before embryos are capable of independent movement and vocalisation, this is not possible. Here we show that volatiles emitted from developing eggs of Japanese quail (Coturnix japonica) convey information on egg fertility, along with the sex and developmental status of the embryo. Specifically, egg volatiles changed over the course of incubation, differed between fertile and infertile eggs, and were predictive of embryo sex as early as day 1 of incubation. Egg odours therefore have the potential to facilitate parent-embryo and embryo-embryo interactions by allowing the assessment of key measures of embryonic development long before this is possible through other modalities. It also opens up the intriguing possibility that parents may be able to glean further relevant information from egg volatiles, such as the health, viability and heritage of embryos. By determining information conveyed by egg-derived volatiles, we hope to stimulate further investigation into the ecological role of egg odours.

We show-in contrast to the traditional textbook contention-that the first amniote lungs were complex, multichambered organs and that the single-chambered lungs of lizards and snakes represent a secondarily simplified rather than the plesiomorphic condition. We combine comparative anatomical and embryological data and show that shared structural principles of multichamberedness are recognizable in amniotes including all lepidosaurian taxa. Sequential intrapulmonary branching observed during early organogenesis becomes obscured during subsequent growth, resulting in a secondarily simplified, functionally single-chambered lung in lepidosaurian adults. Simplification of pulmonary structure maximized the size of the smallest air spaces and eliminated biophysically compelling surface tension problems that were associated with miniaturization evident among stem lepidosaurmorphs. The remaining amniotes, however, retained the multichambered lungs, which allowed both large surface area and high pulmonary compliance, thus initially providing a strong selective advantage for efficient respiration in terrestrial environments. Branched, multichambered lungs instead of simple, sac-like organs were part and parcel of the respiratory apparatus of the first amniotes and pivotal for their success on dry land, with the sky literally as the limit., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

The feather is a complex ectodermal organ with hierarchical branching patterns. It provides functions in endothermy, communication, and flight. Studies of feather growth, cycling, and health are of fundamental importance to avian biology and poultry science. In addition, feathers are an excellent model for morphogenesis studies because of their accessibility, and their distinct patterns can be used to assay the roles of specific molecular pathways. Here we review the progress in aspects of development, regeneration, and evolution during the past three decades. We cover the development of feather buds in chicken embryos, regenerative cycling of feather follicle stem cells, formation of barb branching patterns, emergence of intrafeather pigmentation patterns, interplay of hormones and feather growth, and the genetic identification of several feather variants. The discovery of feathered dinosaurs redefines the relationship between feathers and birds. Inspiration from biomaterials and flight research further fuels biomimetic potential of feathers as a multidisciplinary research focal point.

With the flood of published research encountered today, it is important to occasionally reflect upon how we arrived at our current understanding in a particular scientific discipline, thereby positioning new discoveries into proper context with long-established models. This historical review highlights some of the important scientific contributions in the field of neurogenic placode development. By viewing cumulatively the rich historical data, we can more fully appreciate and apply what has been accomplished. Early descriptive work in fish and experimental approaches in amphibians and chick yielded important conceptual models of placode induction and cellular differentiation. Current efforts to discover genes and their molecular functions continue to expand our understanding of the placodes. Carefully considering the body of work may improve current models and help focus modern experimental design., (Copyright © 2014 Wiley Periodicals, Inc.)

The cardiac neural crest cells (CNCCs) have played an important role in the evolution and development of the vertebrate cardiovascular system: from reinforcement of the developing aortic arch arteries early in vertebrate evolution, to later orchestration of aortic arch artery remodeling into the great arteries of the heart, and finally outflow tract septation in amniotes. A critical element necessary for the evolutionary advent of outflow tract septation was the co-evolution of the cardiac neural crest cells with the second heart field. This review highlights the major transitions in vertebrate circulatory evolution, explores the evolutionary developmental origins of the CNCCs from the third stream cranial neural crest, and explores candidate signaling pathways in CNCC and outflow tract evolution drawn from our knowledge of DiGeorge Syndrome., (© 2014 Wiley Periodicals, Inc.)

The New and Early Chick (EC) methods, two commonly used techniques for ex ovo culture of early-stage avian embryos, are limited by poor survivability after initiation of circulation. This limitation is circumvented with two recent technical advancements: the modified Cornish pasty culture and whole-embryo transplantation. The former supports optimal ex ovo growth till stage HH18, and the latter allows ex-ovo-manipulated embryos to have long-term in ovo-survivability. Here we provide step-by-step instructions for both methods. These two new techniques can also be combined to achieve targeted labeling, imaging and electroporation in early-stage embryos ex ovo, and phenotypic and functional analyses at more advanced developmental stages in ovo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent studies have demonstrated that thermoregulatory behavior occurs not only in posthatching turtles but also in turtles prior to hatching. Does thermoregulatory behavior also occur in the embryos of other reptile and bird species? Our experiments show that such behavior is widespread but not universal in reptile and bird embryos. We recorded repositioning within the egg, in response to thermal gradients, in the embryos of three species of snakes (Xenochrophis piscator, Elaphe bimaculata, and Zaocys dhumnades), two turtles (Chelydra serpentina and Ocadia sinensis), one crocodile (Alligator sinensis), and four birds (Coturnix coturnix, Gallus gallus domesticus, Columba livia domestica, and Anas platyrhynchos domestica). However, we detected no significant thermoregulation by the embryos of two lizard species (Takydromus septentrionalis and Phrynocephalus frontalis). Overall, embryonic thermoregulatory behavior is widespread in reptile as well as bird species but may be unimportant in the small eggs laid by most lizards.

Nonylphenol (NP) is an endocrine disruptor with harmful effects including feminization and carcinogenesis on various organisms. This substance is a degradation product of nonylphenol ethoxylates (NPEO) that is used in several industrial and agricultural processes. In this paper, we examined the assessment of NP exposure on chick embryo development, using a concentration consistent with the environmental concentrations of NP. With this aim, NP (between 0.1 and 50 μg/egg) was injected into the yolk of egg through a small needle hole in the shell. We report the effect of NP on chick reproductive system development although the effect we observed is lower than those observed by exposition to other endocrine disruptors. However, histological analysis highlighted a decrease of intraluminal seminiferous surface area in 64.12% of case (P=0.0086) and an heterogeneous organization of the renal tubules when 10 μg/egg were injected. Moreover, an impairment of liver development with an abnormal bile spillage was observed when higher concentration of NP was injected (50 μg/egg)., (© 2013.)

Although vertebrates display a superficial bilateral symmetry, most internal organs develop and locate with a consistent left:right asymmetry. There is still considerable debate as to when this process actually begins, but it seems that, at least for some species, the initial steps occur at a very early stage of development. In recent years, a number of model systems, including the chick embryo, have been utilised to increase our understanding of the molecular basis of this complex developmental process. While the basic elements of asymmetry are clearly conserved in chick development, the chick embryo also exhibits an additional unusual asymmetry in terms of the development of the gonads. In the female chick embryo, only 1 gonad and accessory structures fully develop, with the result that the adult hen has only 1 ovary and a single oviduct - both on the left side. With a small number of exceptions, this is a consistent feature of avian development. Here, we describe the morphological development and molecular basis of this unusual asymmetry, consider the implications for avian sex determination, and discuss the possible biological reasons why many birds have adopted a single-ovary system.

Unlike the mammalian fetus, development of the avian embryo is independent of the maternal uterus and is potentially vulnerable to physiological and environmental stresses close to hatch. In contrast to the fetus of late gestation in mammals, skeletal muscle in avian embryos during final incubation shows differential developmental characteristics: 1) muscle mobilization (also called atrophy) is selectively enhanced in the type II fibers (pectoral muscle) but not in the type I fibers (biceps femoris and semimembranosus muscle), involving activation of ubiquitin-mediated protein degradation and suppression of S6K1-mediated protein translation; 2) the proliferative activity of satellite cells is decreased in the atrophied muscle of late-term embryos but enhanced at the day of hatch, probably preparing for the postnatal growth. The mobilization of muscle may represent an adaptive response of avian embryos to external (environmental) or internal (physiological) changes, considering there are developmental transitions both in hormones and requirements for glycolytic substrates from middle-term to late-term incubation. Although the exact mechanism triggering muscle fiber atrophy is still unknown, nutritional and endocrine changes may be of importance. The atrophied muscle fiber recovers as soon as feed and water are available to the hatchling. In ovo feeding of late-term embryos has been applied to improve the nutritional status and therein enhances muscle development. Similarly, in ovo exposure to higher temperature or green light during the critical period of muscle development are also demonstrated to be potential strategies to promote pre- and posthatch muscle growth.

Evolution involves interplay between natural selection and developmental constraints. This is seen, for example, when digits are lost from the limbs during evolution. Extant archosaurs (crocodiles and birds) show several instances of digit loss under different selective regimes, and show limbs with one, two, three, four or the ancestral number of five digits. The 'lost' digits sometimes persist for millions of years as developmental vestiges. Here we examine digit loss in the Nile crocodile and five birds, using markers of three successive stages of digit development. In two independent lineages under different selection, wing digit I and all its markers disappear. In contrast, hindlimb digit V persists in all species sampled, both as cartilage, and as Sox9- expressing precartilage domains, 250 million years after the adult digit disappeared. There is therefore a mismatch between evolution of the embryonic and adult phenotypes. All limbs, regardless of digit number, showed similar expression of sonic hedgehog (Shh). Even in the one-fingered emu wing, expression of posterior genes Hoxd11 and Hoxd12 was conserved, whereas expression of anterior genes Gli3 and Alx4 was not. We suggest that the persistence of digit V in the embryo may reflect constraints, particularly the conserved posterior gene networks associated with the zone of polarizing activity (ZPA). The more rapid and complete disappearance of digit I may reflect its ZPA-independent specification, and hence, weaker developmental constraints. Interacting with these constraints are selection pressures for limb functions such as flying and perching. This model may help to explain the diverse patterns of digit loss in tetrapods. Our study may also help to understand how selection on adults leads to changes in development.

Methylmercury (MeHg) is a toxic metal that has been frequently linked to neurochemical alterations, brain lesions, neurobehavioral changes, and reproductive impairments in wild and captive birds. Much less is known about the effects of MeHg on the developing avian brain and resulting effects on hatchling behavior. The objective of this work was to use air cell injection studies to investigate the effect of in ovo MeHg exposure on brain pathology and four neurochemical biomarkers (N-methyl-d-aspartate (NMDA) receptor, γ-aminobutyric acid (GABA) receptor, glutamine synthetase (GS) and glutamic acid decarboxylase (GAD)) that have previously been studied in wild birds, and on hatchling righting response, balance, and startle response. In a series of six studies, we exposed white leghorn chicken and Japanese quail embryos to methylmercury chloride (MeHgCl) (range: 0-6.4μg/g egg) via egg injection on embryonic day (ED) 0 and measured receptor levels and enzyme activity at different stages of embryonic (days 11, 14, and 19 in chicken; day 15 in quail) and hatchling (day 1 and day 7) development, and in whole brain or discrete brain regions (cerebrum, cerebellum, optic lobe). We assessed neurobehaviors on post hatch (PH) days 1 and 7. Despite accumulating relatively high levels of Hg in the brain, embryos and hatchlings did not consistently display neurochemical changes consistent with those seen in wild birds and laboratory mammals. Hatchlings also did not demonstrate behavioral alterations. Pathology did not indicate a difference in occurrence and types of lesions between control and dosed birds. These findings suggest that in ovo MeHg exposure alone may not be responsible for neurological impacts in bird. This work draws attention to factors, such as age and species, that may influence responses to MeHg in birds., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Methylmercury (MeHg) is transferred by female birds into their eggs thus leaving developing embryos exposed to MeHg from the time of fertilization through hatching. Although Hg is a developmental toxicant, little is known about how it distributes among embryonic tissues and subsequently affects neurodevelopment in birds. The main objective of the present study (Part 1 of 2) was to evaluate the distribution of Hg in tissues during different developmental stages in order to better understand potential targets of Hg in the embryo and hatchling. Eight independent, yet related, egg injection studies were conducted. In five studies, white leghorn chicken embryos were air cell injected with methylmercury chloride (MeHgCl; range injected: 0.17-6.4μg/g egg) and Hg concentrations were assessed in seven tissues. We found that soft tissue distribution in embryos and hatchlings was similar to that seen in older birds, with higher total Hg concentrations in liver and kidney than in heart, muscle, and brain (e.g., 5.1, 3.8, 1.9, 2.3, and 1.9μg/g wet weight, respectively, in day 19 embryos after injection with 6.4μg/g MeHgCl). Concentrations were highest in feathers and unabsorbed yolk (e.g., 24.1 and 13.0μg/g in day 19 embryos after injection with 6.4μg/g MeHgCl). Tissue concentrations rose through embryonic days 11, 14, 16, and 19 but generally leveled off at days 1 and 7 post-hatch. We also report on pilot studies that demonstrated that tissue Hg accumulation after MeHgCl injection is similar in chicken and Japanese quail embryos, and that tissue Hg accumulation in chicken embryos after methylmercury cysteine, but not mercury (2) chloride, injection is similar to accumulation after MeHgCl injection. These findings suggest that embryos may accumulate kidney and brain Hg concentrations known to cause renal and neurotoxicity seen in older birds, but that sequestration of Hg into liver and excretion into rapidly growing feathers may offer some protection. This work also demonstrates that air cell injection studies are potentially a useful tool for studies of Hg toxicity in the laboratory., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Incubation conditions for eggs influence offspring quality and reproductive success. One way in which parents regulate brooding conditions is by balancing the thermal requirements of embryos with time spent away from the nest for self-maintenance. Age related changes in embryo thermal tolerance would thus be expected to shape parental incubation behavior. We use data from unmanipulated Black-capped Chickadee (Poecile atricapillus) nests to examine the temporal dynamics of incubation, testing the prediction that increased heat flux from eggs as embryos age influences female incubation behavior and/or physiology to minimize temperature fluctuations. We found that the rate of heat loss from eggs increased with embryo age. Females responded to increased egg cooling rates by altering incubation rhythms (more frequent, shorter on- and off- bouts), but not brood patch temperature. Consequently, as embryos aged, females were able to increase mean egg temperature and decrease variation in temperature. Our findings highlight the need to view full incubation as more than a static rhythm; rather, it is a temporally dynamic and finely adjustable parental behavior. Furthermore, from a methodological perspective, intra- and inter-specific comparisons of incubation rhythms and average egg temperatures should control for the stage of incubation.

It is widely accepted that birds are a subgroup of dinosaurs, but there is an apparent conflict: modern birds have been thought to possess only the middle three fingers (digits II-III-IV) of an idealized five-digit tetrapod hand based on embryological data, but their Mesozoic tetanuran dinosaur ancestors are considered to have the first three digits (I-II-III) based on fossil evidence. How could such an evolutionary quirk arise? Various hypotheses have been proposed to resolve this paradox. Adding to the confusion, some recent developmental studies support a I-II-III designation for avian wing digits whereas some recent paleontological data are consistent with a II-III-IV identification of the Mesozoic tetanuran digits. A comprehensive analysis of both paleontological and developmental data suggests that the evolution of the avian wing digits may have been driven by homeotic transformations of digit identity, which are more likely to have occurred in a partial and piecemeal manner. Additionally, recent genetic studies in mouse models showing plausible mechanisms for central digit loss invite consideration of new alternative possibilities (I-II-IV or I-III-IV) for the homologies of avian wing digits. While much progress has been made, some advances point to the complexity of the problem and a final resolution to this ongoing debate demands additional work from both paleontological and developmental perspectives, which will surely yield new insights on mechanisms of evolutionary adaptation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Sex determination in vertebrate embryos has long been equated with gonadal differentiation into testes or ovaries. This view has been challenged over the years by reports of somatic sexual dimorphisms pre-dating gonadal sex differentiation. The recent finding that sex determination in birds is likely to be partly cell autonomous has again called for a broader definition of sex determination. Inherent sexual differentiation in each and every cell may apply widely among vertebrates, and may involve more than one "master sex gene" on a sex chromosome. At the gonadal level, key genes required for proper sexual differentiation are conserved among vertebrates, but their relative positions in the ovarian and testicular cascades differ., Results: We illustrate these differences by comparing key sex genes in fishes versus birds and mammals, with emphasis on DM domain genes and the SOX9-AMH pathway in the testis and the FOXL2-Aromatase pathway in the ovary. Such comparisons facilitate the identification of ancient versus derived genes involved in gonadal sex determination., Conclusions: The data indicate that vertebrate sex-determining cascades are not as conserved as once thought., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The classic model of sex determination in mammals states that the sex of the individual is determined by the type of gonad that develops, which in turn determines the gonadal hormonal milieu that creates sex differences outside of the gonads. However, XX and XY cells are intrinsically different because of the cell-autonomous sex-biasing action of X and Y genes., Results: Recent studies of mice, in which sex chromosome complement is independent of gonadal sex, reveal that sex chromosome complement has strong effects contributing to sex differences in phenotypes such as metabolism. Adult mice with two X chromosomes (relative to mice with one X chromosome) show dramatically greater increases in body weight and adiposity after gonadectomy, irrespective of their gonadal sex. When fed a high-fat diet, XX mice develop striking hyperinsulinemia and fatty liver, relative to XY mice. The sex chromosome effects are modulated by the presence of gonadal hormones, indicating an interaction of the sex-biasing effects of gonadal hormones and sex chromosome genes., Conclusions: Other cell-autonomous sex chromosome effects are detected in mice in many phenotypes. Birds (relative to eutherian mammals) are expected to show more widespread cell-autonomous sex determination in non-gonadal tissues, because of ineffective sex chromosome dosage compensation mechanisms., (Copyright © 2013 Wiley Periodicals, Inc.)

The skull and jaws of extant birds possess secondary cartilage, a tissue that arises after bone formation during embryonic development at articulations, ligamentous and muscular insertions. Using histological analysis, we discovered secondary cartilage in a non-avian dinosaur embryo, Hypacrosaurus stebingeri (Ornithischia, Lambeosaurinae). This finding extends our previous report of secondary cartilage in post-hatching specimens of the same dinosaur species. It provides the first information on the ontogeny of avian and dinosaurian secondary cartilages, and further stresses their developmental similarities. Secondary cartilage was found in an embryonic dentary within a tooth socket where it is hypothesized to have arisen due to mechanical stresses generated during tooth formation. Two patterns were discerned: secondary cartilage is more restricted in location in this Hypacrosaurus embryo, than it is in Hypacrosaurus post-hatchlings; secondary cartilage occurs at far more sites in bird embryos and nestlings than in Hypacrosaurus. This suggests an increase in the number of sites of secondary cartilage during the evolution of birds. We hypothesize that secondary cartilage provided advantages in the fine manipulation of food and was selected over other types of tissues/articulations during the evolution of the highly specialized avian beak from the jaws of their dinosaurian ancestors.

During vertebrate development, the lung inaugurates as an endodermal bud from the primitive foregut. Dichotomous subdivision of the bud results in arborizing airways that form the prospective gas exchanging chambers, where a thin blood-gas barrier (BGB) is established. In the mammalian lung, this proceeds through conversion of type II cells to type I cells, thinning, and elongation of the cells as well as extrusion of the lamellar bodies. Subsequent diminution of interstitial tissue and apposition of capillaries to the alveolar epithelium establish a thin BGB. In the noncompliant avian lung, attenuation proceeds through cell-cutting processes that result in remarkable thinning of the epithelial layer. A host of morphoregulatory molecules, including transcription factors such as Nkx2.1, GATA, HNF-3, and WNT5a; signaling molecules including FGF, BMP-4, Shh, and TFG- β and extracellular proteins and their receptors have been implicated. During normal physiological function, the BGB may be remodeled in response to alterations in transmural pressures in both blood capillaries and airspaces. Such changes are mitigated through rapid expression of the relevant genes for extracellular matrix proteins and growth factors. While an appreciable amount of information regarding molecular control has been documented in the mammalian lung, very little is available on the avian lung.

The primitive streak is where the mesoderm and definitive endoderm precursor cells ingress from the epiblast during gastrulation. It is often described as an embryological feature common to all amniotes. But such a feature has not been associated with gastrulation in any reptilian species. A parsimonious model would be that the primitive streak evolved independently in the avian and mammalian lineages. Looking beyond the primitive streak, can one find shared features of mesoderm and endoderm formation during amniote gastrulation? Here, we survey the literature on reptilian gastrulation and provide new data on Brachyury RNA and laminin protein expression in gastrula-stage turtle (Pelodiscus sinensis) embryos. We propose a model to reconcile the primitive streak-associated gastrulation in birds and the blastopore-associated gastrulation in extant reptiles., (© 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.)

A concise review of the articles about the origin of primordial germ cells (PGCs) in vertebrates is provided. Differences among various taxa concerning the origin of PGCs, not easily understandable on the base of traditional knowledge, are pointed out. All those differences can be explained taking into consideration the recent “theory of the endoderm as secondary layer”. That theory allows us to understand that those differences are only apparent, being related to modifications of stages of the consequent embryogeny, overall, to a different amount of yolk in the egg. Eggs very rich in yolk became meroblastic, and the portion of primordial ectomesenchyme destined to give rise to a part of the mesoderm and the PGCs separates early from the part destined to give rise to the rest of the mesoderm and to the digestive endoderm in order to form the vitelline hypoblast lamina. To this lamina, in contrast to the traditional interpretation, a mesodermal, not endodermal, origin must be attributed. With the misunderstanding regarding the origin of this lamina clarified, all the differences about the origin of PGCs disappears. Furthermore, in taxa where PGCs were considered to be of endodermal origin, they too have a mesodermal origin. Considering that a mesodermal origin of PGCs has been demonstrated in all sponges and cnidarians, as well, a unique, mesodermal origin of germinal cells in all pluricellular animals results.

Skewed sex ratios in zoo breeding programs may require housing single birds of an overrepresented gender, increasing demands on limited resources that could otherwise be diverted to breeding pairs or other important species. The ability to selectively incubate and hatch eggs of a desired sex represents a significant improvement in the long-term management of avian species. This study describes a successful method for in ovo sexing of embryos from stage 30 through 42 of incubation (Hamburger and Hamilton [1951] J Morphol 88:49-92). A 0.01-1 µl blood sample was collected from either the vitelline vessel (VV) or the blood vessels of the chorio-allantoic membrane (CAM) of embryos at stages 14-18 or 30-42, respectively. DNA was isolated from whole blood using the Chelex method (Walsh et al. [1991] Biotechniques 10:506-513; Jensen et al., [2003] Zoo Biol 22:561-571). Sex was determined by PCR amplification using the previously described P2/P8 (Griffiths et al. [1998] Mol Ecol 7:1071-1075) and 1237L/1272H (Kahn et al. [1998] Auk 115:1074-1078) primers or by commercial vendor. Success rate was calculated as the percent of sampled embryos surviving to hatch. Embryos of the undesired sex were not incubated, thus not included in the calculation. There was a considerable difference in success rate when blood was collected from the stage 14-18 VV (0-25%, average 12%) vs. stage 30-42 CAM (33-100%, average 76%). In conclusion, in ovo sexing of embryos between stages 30 and 42 yields acceptable embryo survival rates while providing enough blood for genetic testing., (© 2011 Wiley Periodicals, Inc.)

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs., (© 2012 Blackwell Publishing Ltd.)

Using gross dissections and computed tomography we studied the lungs of juvenile American alligators (Alligator mississippiensis). Our findings indicate that both the external and internal morphology of the lungs is strikingly similar to the embryonic avian respiratory system (lungs + air sacs). We identified bronchi that we propose are homologous to the avian ventrobronchi (entobronchi), laterobronchi, dorsobronchi (ectobronchi), as well as regions of the lung hypothesized to be homologous to the cervical, interclavicular, anterior thoracic, posterior thoracic, and abdominal air sacs. Furthermore, we suggest that many of the features that alligators and birds share are homologous and that some of these features are important to the aerodynamic valve mechanism and are likely plesiomorphic for Archosauria., (Copyright © 2012 Wiley Periodicals, Inc.)

To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotide-primed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10,000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 40-70% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDD-treated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available.

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters.

All living birds are toothless, constituting by far the most diverse toothless vertebrate clade, and are striking examples of evolutionary success following tooth loss. In recent years, an unprecedented number of Mesozoic birds have been described, illustrating the evolution of dentition reductions. Simultaneously, major advances in experimental embryology have yielded new results concerning avian edentulism. Reviewing these lines of evidence, we propose hypotheses for its causes, with a prominent role for the horny beak during development. A horny beak and a muscular gizzard functionally 'replaced' dentition for food acquisition and processing, respectively. Together with edentulism itself, these features and others contributed to the later success of birds, as a result of their high performance or additional functionality working in concert in these complex organisms., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The subpallial region of the avian telencephalon contains neural systems whose functions are critical to the survival of individual vertebrates and their species. The subpallial neural structures can be grouped into five major functional systems, namely the dorsal somatomotor basal ganglia; ventral viscerolimbic basal ganglia; subpallial extended amygdala including the central and medial extended amygdala and bed nuclei of the stria terminalis; basal telencephalic cholinergic and non-cholinergic corticopetal systems; and septum. The paper provides an overview of the major developmental, neuroanatomical and functional characteristics of the first four of these neural systems, all of which belong to the lateral telencephalic wall. The review particularly focuses on new findings that have emerged since the identity, extent and terminology for the regions were considered by the Avian Brain Nomenclature Forum. New terminology is introduced as appropriate based on the new findings. The paper also addresses regional similarities and differences between birds and mammals, and notes areas where gaps in knowledge occur for birds., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hematopoietic stem cells (HSC) are the source of all blood cell types produced during the entire life of an organism. They appear during embryonic development, where they will transit through different successive hematopoietic organs, before to finally colonize the bone marrow. Nowadays, the precise origin of HSC remains a matter of controversy. Different HSC precursor candidates, located in different anatomical sites, have been proposed. Here, we summarize and discuss the different theories in light of the recent articles, especially those using in vivo confocal microscopy technology., (© 2011 médecine/sciences – Inserm / SRMS.)

The article discusses the establishment of pulmonary ventilation (V˙E) in the avian embryo, the metabolic and V˙E sensitivity to hypoxia and the effects of sustained embryonic hypoxia on the hatchling's V˙E chemosensitivity. Throughout embryogenesis, hypometabolism is the common response to hypoxia, with no compensation by anaerobic energy supply. It originates primarily from the depression in body growth and, later in development, from the depression of thermogenesis. The V˙E responses to acute hypoxia or hypercapnia are clearly detectable during the internal pipping phase; their magnitude rapidly increases in the first postnatal day. Sustained prenatal hypoxia diminishes the V˙E chemosensitivity of the hatchling and reduces the hypometabolic response to an acute hypoxic episode. The former most likely originates from a disturbance in the normal development of the carotid bodies, the latter from the central action of hypoxia on thermogenesis. The avian embryo is a model suitable for the studies of the development of respiratory control and offers an alternative to mammalian models for investigations on the short- and long-term effects of prenatal hypoxia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

During avian embryonic development, the carbon dioxide tension inside the egg increases as the shell restricts gas exchange with the environment. Acid-base regulation of the avian embryo is a complex process, not only due to the non-function of the lungs and limited functionality of the kidneys but also because the embryo is affected by the inflow of bicarbonates from the shell, when calcium is reabsorbed for calcification. Moreover, interaction occurs between the embryo and the extraembryonic compartments. It has been shown that carbonic anhydrase plays a crucial role in the formation of sub-embryonic fluid and in the chorioallantoic membrane and kidney. This review provides a detailed overview of the acid-base status of the extraembryonic compartments inside chicken eggs, and their interaction in keeping the acid-base status of the embryo balanced. Secondly, experimental acid-base disturbances are summarized. The last part of the review briefly compares embryos of birds and other amniotes (mammals and reptiles) with regard to acid-base regulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Respiratory chemoreceptors are neurons that detect PCO(2), PO(2), and/or pH in body fluids and provide sensory feedback for the control of breathing. They play a critical role in coupling pulmonary ventilation to metabolic demand in endothermic vertebrates. During birth in mammals and hatching in birds, the state change from placental or chorioallantoic gas exchange to pulmonary respiration makes acute demands on the neonatal lungs and ventilatory control system, including the respiratory chemoreceptors. Here we review the literature on prenatal development of carotid body chemoreceptors, central chemoreceptors, and airway chemoreceptors, with emphasis on the histology, histochemistry, and neurophysiology of chemosensory cells or their afferents, and their physiological genomics if known. In general, respiratory chemoreceptors develop prenatally and are functional but immature at birth or hatching. Each type of respiratory chemoreceptor has a unique prenatal developmental time course, and all studied to date require a period of postnatal maturation to express the full adult response., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Environmental conditions considerably influence embryogenesis and subsequent postnatal development; however, the roles of individual embryonic physiological systems in these effects remain largely unclear. Data on the effects of some environmental factors (temperature variation and hypoxia) on the development of embryos and extra-embryonic organs in reptiles and birds are summarized, with special emphasis on acute changes in these factors. The involvement of several physiological characteristics related to the functions of the extra-embryonic membranes, cardiovascular system, and embryonic behavior (amnion rhythmic contractions, cardiac activity, and embryonic motility) in the integrated response to temperature fluctuations and acute hypoxia is considered. The changes in this response with embryonic age in reptiles and birds are analyzed. Particular attention is paid to the maturation of the regulatory mechanisms, interactions among the above parameters, and their possible contributions to the changes in oxygen consumption (or its stability) during short-term changes in temperature and oxygenation at different stages of embryonic development., (Copyright © 2011 Elsevier B.V. All rights reserved.)

During embryonic development of homeothermic animals like birds and mammals transition from ectothermy to endothermy occurs especially in precocial species of both taxa. Based on some evolutionary aspects of the development of endothermy the review focuses on the prenatal development of endothermy and of the thermoregulatory system using the precocial bird as a model. During final incubation precocial bird embryos have all the prerequisites to respond to environmental (temperature) influences in a nearly appropriate way. Autonomic, neuronal and molecular mechanisms underlying the development of endothermy are established and the transition of the thermoregulatory system from a control system without feedback into a system with feedback mechanisms occurs. Precocial bird embryos are endothermic, but not homeothermic if incubation temperature decreases below the normal level, which seems to be associated with summit metabolism. At increased incubation temperatures the embryos are able to stabilize their body temperature. Therefore, above normal temperatures, homoeothermy can occur over a limited temperature range., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Embryonic growth and development are impacted by environmental conditions. In avian systems, parents tightly control these environments through provisioning of nutrients to the egg and through incubation. Parents can influence embryonic development through egg size, eggshell conductance, hormones, or other substances deposited in eggs and through the onset and temperature of incubation. In addition to these parental influences, evidence suggests that avian embryos are able to perceive and actively respond to their environment during incubation and adjust their own development. Evolution of embryos' responses to developmental environments in birds can be understood in the context of parent-offspring conflicts. When parental investments favor future reproduction over current reproduction, current offspring pay fitness costs, which result in strong selection for offspring that can respond to developmental environments independent of their parents. Here, we review literature indicating that avian embryos actively respond to maternally derived components of the egg, vocalizations, and differences in day length, and we explore these responses in the context of three situations where the consequence of these environments to the fitnesses of offspring and parents differ: the degree of synchrony in hatching, the deposition of hormones in yolks, and seasonal timing of breeding. However, the adaptive significance of responses of embryos to developmental environments arising from parent-offspring conflict has not been adequately explored in birds.

Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker for the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane and the inner zone of the Area Opaca, is notably absent from the blastoderm edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, β-catenin, and E-cadherin, are present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay strongly suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, as opposed to proliferation. Taken together, our data show that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and that the avian epiboly model, which has been dormant for so many years, may yet again prove to be helpful as a unique developmental model for studying partial EMT in the context of collective epithelial cell migration., (Copyright © 2011 Wiley-Liss, Inc.)

Members of the Meis family of TALE homeobox transcription factors are involved in many processes of vertebrate development and morphogenesis, showing extremely complex transcriptional and spatiotemporal expression patterns. In this work, we performed a comprehensive study of chicken Meis genes using multiple approaches. First, we assessed whether the chicken genome contains a Meis3 ortholog or harbors only two Meis genes; we gathered several lines of evidence pointing to a specific loss of the Meis3 ortholog in an early ancestor of birds. Next, we studied the transcriptional diversity generated from chicken Meis genes through alternative splicing during development. Finally, we performed a detailed analysis of chick Meis1/2 expression patterns during early embryogenesis and organogenesis. We show that the expression of both Meis genes begins at the gastrulation stage in the three embryonic layers, presenting highly dynamic patterns with overlapping as well as distinct expression domains throughout development., (Copyright © 2011 Wiley-Liss, Inc.)

Signaling by the hedgehog (Hh) family of secreted growth factors is essential for development of embryonic blood vessels. Embryos lacking Hh function have abundant endothelial cells but fail to assemble vascular cords or lumenized endothelial tubes. However, the role of Hh signaling during later aspects of vascular patterning and morphogenesis is largely unexplored. We have used small molecule inhibitors and agonists to alter activity of the Hh signaling pathway in the chick embryo. When cyclopamine is added after cord formation, aortal cells form tubes, but these are small and disorganized and the density of the adjacent vascular plexus is reduced. Activation of the Hh pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. The number of endothelial cell filopodia is found to correlate with Hh signaling levels. These studies show that Hh signaling levels must be tightly regulated for normal vascular patterning to be achieved., (Copyright © 2011 Wiley-Liss, Inc.)