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The periodic color motifs such as the spots or stripes that adorn the coat of vertebrates have served as emblematic systems in empirical and theoretical studies of pattern formation, because they vary extensively between taxa but often have conserved orientation and are highly reproducible within species. Two major patterning theories have been proposed, namely instructional signaling, in which positional information is encoded as a program, and self-organization, in which position is spontaneously acquired within the developing tissue. We review here recent empirical evidence that supports both theories in vertebrates: with the advent of new molecular techniques and functional approaches, researchers nowadays take advantage of natural populations of mammals, birds and fish species, closely-related to model organisms and varying in periodic patterns. As a whole, results strongly suggest that instruction and self-organization act in combination in space and time. The orientation and reproducibility of periodic patterns relies on initial foundations provided by early developmental landmarks while their periodicity and natural variation are shaped by late-acting self-organizing processes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Early embryonic cells are capable of acquiring numerous developmental fates until they become irreversibly committed to specific lineages depending on intrinsic determinants and/or regional interactions. From fertilization to gastrulation, such pluripotent cells first increase in number and then turn to undergoing differentiation. Mechanisms regulating pluripotency in each species attract great interest in developmental biology. Also, outlining the evolutionary background of pluripotency can enhance our understanding of mammalian pluripotency and provide a broader view of early development of vertebrates. Here, we introduce integrative models of pluripotent states in amniotes (mammals, birds and reptiles) to offer a comprehensive overview of widely accepted knowledge about mammalian pluripotency and our recent findings in non-mammalian amniotes, such as chicken and gecko. In particular, we describe 1) the IL6/Stat3 signaling pathway as a positive regulator of naive pluripotency, 2) Fgf/Erk signaling as a process that prepares cells for differentiation, 3) the role of the interactions between these two signaling pathways during the transition from pluripotency to differentiation, and 4) functional diversification of two transcription factors, Class V POUs and Nanog. In the last section, we also briefly discuss possible relationships of unique cell cycle properties of early embryonic cells with signaling pathways and developmental potentials in the pluripotent cell states., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The study of oogenesis and early development of frogs belonging to the family Hemiphractidae provide important comparison to the aquatic development of other frogs, such as Xenopus laevis, because reproduction on land characterizes the Hemiphractidae. In this review, the multinucleated oogenesis of the marsupial frog Flectonotus pygmaeus (Hemiphractidae) is analyzed and interpreted. In addition, the adaptations associated with the incubation of embryos in the pouch of the female marsupial frog Gastrotheca riobambae (Hemiphractidae) and the embryonic development of this frog are summarized. Moreover, G. riobambae gastrulation is compared with the gastrulation modes of Engystomops randi and Engystomops coloradorum (Leptodactylidae); Ceratophrys stolzmanni (Ceratophryidae); Hyalinobatrachium fleischmanni and Espadarana callistomma (Centrolenidae); Ameerega bilinguis, Dendrobates auratus, Epipedobates anthonyi, Epipedobates machalilla, Epipedobates tricolor, and Hyloxalus vertebralis (Dendrobatidae); Eleutherodactylus coqui (Terrarana: Eleutherodactylidae), and X. laevis (Pipidae). The comparison indicated two modes of frog gastrulation. In X. laevis and in frogs with aquatic reproduction, convergent extension begins during gastrulation. In contrast, convergent extension occurs in the post-gastrula of frogs with terrestrial reproduction. These two modes of gastrulation resemble the transitions toward meroblastic cleavage found in ray-finned fishes (Actinopterygii). In spite of this difference, the genes that guide early development seem to be highly conserved in frogs. I conclude that the shift of convergent extension to the post-gastrula accompanied the diversification of frog egg size and terrestrial reproductive modes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The orderly formation of the avian feather array is a classic example of periodic pattern formation during embryonic development. Various mathematical models have been developed to describe this process, including Turing/activator-inhibitor type reaction-diffusion systems and chemotaxis/mechanical-based models based on cell movement and tissue interactions. In this paper we formulate a mathematical model founded on experimental findings, a set of interactions between the key cellular (dermal and epidermal cell populations) and molecular (fibroblast growth factor, FGF, and bone morphogenetic protein, BMP) players and a medially progressing priming wave that acts as the trigger to initiate patterning. Linear stability analysis is used to show that FGF-mediated chemotaxis of dermal cells is the crucial driver of pattern formation, while perturbations in the form of ubiquitous high BMP expression suppress patterning, consistent with experiments. Numerical simulations demonstrate the capacity of the model to pattern the skin in a spatial-temporal manner analogous to avian feather development. Further, experimental perturbations in the form of bead-displacement experiments are recapitulated and predictions are proposed in the form of blocking mesenchymal cell proliferation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Variation in regional identity, patterning, and structure of epidermal appendages contributes to skin diversity among many vertebrate groups, and is perhaps most striking in birds. In pioneering work on epidermal appendage patterning, John Saunders and his contemporaries took advantage of epidermal appendage diversity within and among domestic chicken breeds to establish the importance of mesoderm-ectoderm signaling in determining skin patterning. Diversity in chickens and other domestic birds, including pigeons, is driving a new wave of research to dissect the molecular genetic basis of epidermal appendage patterning. Domestic birds are not only outstanding models for embryonic manipulations, as Saunders recognized, but they are also ideal genetic models for discovering the specific genes that control normal development and the mutations that contribute to skin diversity. Here, we review recent genetic and genomic approaches to uncover the basis of epidermal macropatterning, micropatterning, and structural variation. We also present new results that confirm expression changes in two limb identity genes in feather-footed pigeons, a case of variation in appendage structure and identity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.)

Microbial communities present on eggshell surfaces of wild birds are weakly studied, especially their influence on embryo infection and, thus, egg viability. Bacterial density of wild bird eggshells is very low, and most DNA extraction protocols are frequently unsuccessful. We have efficiently adapted a chelex-based DNA isolation method for 16S ribosomal gene amplification from the total communities of eggshell surfaces from six avian species., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

The faces of birds and mammals exhibit remarkable morphologic diversity, but how variation arises is not well-understood. We have previously demonstrated that a region of facial ectoderm, which we named the frontonasal ectodermal zone (FEZ), regulates proximo-distal extension and dorso-ventral polarity of the upper jaw in birds. In this work, we examined the equivalent ectoderm in murine embryos and determined that the FEZ is conserved in mice. However, our results revealed that fundamental differences in the organization and constituents of the FEZ in mice and chicks may underlie the distinct growth characteristics that distinguish mammalian and avian embryos during the earliest stages of development. Finally, current models suggest that neural crest cells regulate size and shape of the upper jaw, and that signaling by Bone morphogenetic proteins (Bmps) within avian neural crest helps direct this process. Here we show that Bmp expression patterns in neural crest cells are regulated in part by signals from the FEZ. The results of our work reconcile how a conserved signaling center that patterns growth of developing face may generate morphologic diversity among different animals. Subtle changes in the organization of gene expression patterns in the FEZ could underlie morphologic variation observed among and within species, and at extremes, variation could produce disease phenotypes.

The effects of heavy metal pollutants on aquatic birds have been widely studied in ecotoxicological investigations; however, the predominant focus has been on the postnatal period of life. Limited information on the adverse effects of metals to bird eggs is available. The possible toxic effects of lead and copper were studied in mallard eggs. After the accidental severe heavy metal pollution of the Tisa river (Hungary) in March 2000, these metals were detected in the highest concentration in both the water and the sediment, reaching far beyond acceptable concentrations. Pb treatment (2.9 mg/L) significantly increased the rate of mortality after a single immersion of the eggs into polluted water for 30 min. The rate of dead embryos significantly increased after the combined exposure to Cu and Pb (0.86 and 2.9 mg/L, respectively) both in the single- (once for 30 min) and in the multiple- (10s daily during first trimester of incubation) immersion groups. It was concluded that elevated metal concentrations similar to those found in the Tisa river after the tailing dam failure may cause toxic effects (mortality and teratogenicity) upon exposure of mallard eggs.

Cryopreservation of avian germplasm is becoming better understood and more commonly practiced. However, one area that would be of great benefit for genome resource banking is the preservation of avian embryos. Little is know about the cryobiology of avian embryos, and they have never been successfully cryopreserved. However, it is likely that they share many of the challenges of other yolk-filled multicompartmental embryos. For example, the fish embryo has 1) a large overall size, resulting in a low surface-to-volume ratio, which retards water and cryoprotectant efflux/influx; 2) large-sized cells, such as the yolk, which could increase the likelihood of membrane disruption by intracellular ice formation; 3) compartments, such as the blastoderm and yolk, with differing permeability properties; and 4) susceptibility to chilling injury. Both the avian and fish systems share many physical and anatomical properties, and it is predicted that some of the same permeability barriers would exist in both as well. Although the systems are similar, some of the goals, and thus the practices, to protect the genome may be quite different. One of these major goals in avian developmental biology is to produce chicken:chicken transgenic animals, especially those with germ line transmission. Producing efficient germ line transmissions and being able to cryopreserve these transmissions would be extremely beneficial to both basic and agricultural science. This could be accomplished through the cryopreservation of embryonic gonadal tissue followed by grafting into a host. The gonadal/tail-graft system would provide an advantage for cryopreservation because it is small (in comparison with the whole embryo), has fairly uniform tissue, and contains the essential primordial germ line cells capable of recreating the genetic line of interest. Moreover, because the chicken is such a robust model for most other avian species, the cryopreservation of the gonadal/tail-graft may potentially open up similar treatments for other commercially important species.

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.

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.

Development of an organ is directed by cell and tissue interactions and these also occur during the formation of functional kidney. During vertebrate development inductive signalling between mesenchyme and epithelium controls the organogenesis of all three kinds of kidneys: pronephros, mesonephros and metanephros. In higher animals the metanephros differentiates into the permanent kidney and in this review we will mainly concentrate on its development. Molecular interactions currently known to function during nephrogenesis have primarily been based on the use of knockout techniques. These studies have highlighted the role for transcription factors, signalling molecules, growth factors and their receptors and also for extracellular matrix components in kidney development. Finally in this review we will represent our own model for kidney development according to the knowledge of the genes involved in the development of the functional excretory organ, kidney.

In birds, sensory innervation of skin is restricted to dermis, with few axons penetrating into the epidermis. This pattern of innervation is maintained in vitro, where sensory neurites avoid explants of epidermis but grow readily on dermis. We have used this coculture paradigm to investigate the mechanisms that impede innervation of avian epidermis. The lack of epidermal innervation in birds has been attributed to diffusible chondroitin sulfate proteoglycans (CSPGs) secreted by the epidermis, although direct experimental evidence is weak. We found that elimination of CSPG function with either chondroitinase or neutralizing antibodies did not promote growth of DRG neurites onto epidermis in vitro, indicating that CSPGs alone are not responsible for preventing epidermal innervation. Moreover, the failure of sensory neurites to invade epidermis is not due exclusively to soluble chemorepulsive factors, since sensory neurites also avoid dead epidermis. This inhibition can be overridden, however, by coating epidermis with the growth-promoting molecule laminin, but only if the tissue is killed first. Epidermal innervation of laminin-coated epidermis is even more robust when CSPGs are also eliminated. Thus, the absence of growth-promoting or permissive molecules, such as laminin, may contribute to the failure of sensory neurites to invade avian epidermis. Together these results show that the inhibitory character of avian epidermis is complex. Cell- or matrix-associated CSPGs clearly contribute to the inhibition, but are not solely responsible., (Copyright 1999 Academic Press.)

The lateral wall of the avian braincase, which is indicative of the primitive amniote condition, is formed from mesoderm. In contrast, mammals have replaced this portion of their head skeleton with a nonhomologous bone of neural crest origin. Features that characterize the local developmental environment may have enabled a neural crest-derived skeletal element to be integrated into a mesodermal region of the braincase during the course of evolution. The lateral wall of the braincase lies along a boundary in the head that separates neural crest from mesoderm, and also, neural crest cells migrate through this region on their way to the first visceral arch. Differences in the availability of one skeletogenic population versus the other may determine the final composition of the lateral wall of the braincase. Using the quail-chick chimeric system, this investigation tests if populations of neural crest, when augmented and expanded within populations of mesoderm, will give rise to the lateral wall of the braincase. Results demonstrate that neural crest can produce cartilages that are morphologically indistinguishable from elements normally generated by mesoderm. These findings (1) indicate that neural crest can respond to the same cues that both promote skeletogenesis and enable proper patterning in mesoderm, (2) challenge hypotheses on the nature of the boundary between neural crest and mesoderm in the head, and (3) suggest that changes in the allocation of migrating cells could have enabled a neural crest-derived skeletal element to replace a mesodermal portion of the braincase during evolution., (Copyright 1999 Academic Press.)

Whereas the origin and migration of endothelial cells (ECs) have been studied primarily in the avian embryo, the molecular mechanisms governing these events in birds and mammals were unraveled following the identification of specific growth factors and of their receptors. In particular, analytic and experimental studies of vascular endothelial growth factor (VEGF) and its receptors have provided significant insights into the developmental biology of the vascular system. VEGFR2 is the earliest marker expressed by EC precursors in chickens and mice. Based on the localization of VEGFR2-positive cells in the avian embryo and on findings from clonal culture experiments, two types of EC precursors can be differentiated as early as the gastrulation stage, namely posterior mesoderm hemangioblasts capable of differentiating into both ECs and hematopoietic cells and anterior angioblasts capable of yielding only ECs.

The avian model provides an experimental approach for dissecting the origin, migrations, and differentiation of cell lineages in early embryos. In this model, the endothelial network was shown to stem from both the somites and the splanchnopleural mesoderm. The somite line age produces only endothelial cells, whereas the splanchnopleural line age also produces hematopoietic stem cells. Potentialities of the mesoderm are determined by a positive influence from the endoderm and a negative influence from the ectoderm. A novel mode of blood-borne angiogenesis is also described.

It has been proposed that the subepicardial mesenchymal cells (SEMC) originate from the primitive epicardium and also from migration of extracardiac mesenchyme from the liver area. We have studied the possibility of an origin of SEMC through transformation of the proepicardial mesothelium, as well as the potential of the early proepicardium to generate epicardium and SEMC in quail-chick chimeras. The study was carried out in quail and chick embryos between HH16 and HH29 stages. Most proepicardial cells, mesothelial as well as mesenchymal, were cytokeratin and vimentin immunoreactive, suggesting a cytoskeletal shift from the epithelial to the mesenchymal type. Furthermore, we immunolocated, in the proepicardial mesothelium, three proteins specifically expressed during the endothelial-mesenchymal transition of the endocardial cushions, namely the JB3/fibrillin-associated antigen, the ES/130 protein and the smooth muscle cell alpha-actin. Grafts of proepicardial tissue from HH16-17 quail embryos into chick embryos of the same age originated large areas of donor-derived epicardium, including mesothelial, mesenchymal, and vascular cells. The donor-derived primitive epicardium showed segment-specific features, being squamous and adhered to the myocardium on the atrial wall and showing morphological signs of ingression in the atrioventricular groove and outflow tract. These morphological traits together with the distribution of vimentin, the ES/130 protein, and the JB3/fibrillin-associated antigen suggested a localized transformation of some epicardial mesothelial cells into mesenchyme. Most of the donor-derived cells, mesothelial and mesenchymal, showed the vascular marker QH1, which frequently colocalized with cytokeratin. Heterotopic grafts of quail splanchnopleura into the pericardial cavity of chick embryos originated a squamous, epicardial-like, cytokeratin-immunoreactive cell layer on the heart surface, as well as a few QH1(+) subepicardial and intramyocardial cells. The results suggest that a substantial part of the subepicardial mesenchyme, including the progenitors of the cardiac vessels, originates from the transformation of proepicardial and epicardial mesothelial cells into mesenchyme, and that the epicardial transition could be driven by a segment-specific myocardial signal., (Copyright 1998 Academic Press.)

The formation of the digits in amniota embryos is accompanied by apoptotic cell death of the interdigital mesoderm triggered through BMP signaling. Differences in the intensity of this apoptotic process account for the establishment of the different morphological types of feet observed in amniota (i.e., free-digits, webbed digits, lobulated digits). The molecular basis accounting for the differential pattern of interdigital cell death remains uncertain since the reduction of cell death in species with webbed digits is not accompanied by a parallel reduction in the pattern of expression of bmp genes in the interdigital regions. In this study we show that the duck interdigital web mesoderm exhibits an attenuated response to both BMP-induced apoptosis and TGFbeta-induced chondrogenesis in comparison with species with free digits. The attenuated response to these signals is accompanied by a reduced pattern of expression of msx-1 and msx-2 genes. Local application of FGF in the duck interdigit expands the domain of msx-2 expression but not the domain of msx-1 expression. This change in the expression of msx-2 is followed by a parallel increase in spontaneous and exogenous BMP-induced interdigital cell death, while the chondrogenic response to TGFbetas is unchanged. The regression of AER, as deduced by the pattern of extinction of fgf-8 expression, takes place in a similar fashion in the chick and duck regardless of the differences in interdigital cell death and msx gene expression. Implantation of BMP-beads in the distal limb mesoderm induces AER regression in both the chick and duck. This finding suggests an additional role for BMPs in the physiological regression of the AER. It is proposed that the formation of webbed vs free-digit feet in amniota results from a premature differentiation of the interdigital mesoderm into connective tissue caused by a reduced expression of msx genes in the developing autopod., (Copyright 1998 Academic Press.)

The avian embryo is uniquely amenable to experimental manipulation. The most widely used models are chimeras resulting from heterotopic or orthotopic exchanges of rudiments between chick and quail embryos, according to Le Douarin's technique (1969). Cell migrations and fates are traced in these chimeras either through the identification of quail cell nuclei stained for DNA or by means of monoclonal antibodies that recognize a particular lineage in only one of the two species. The ontogeny of the hemopoietic and endothelial lineages, as enlightened through appropriately designed chimeras, is reviewed in the present article. Homologies recently disclosed in mouse and human embryo are emphasized. Finally, the possibilities afforded by retroviral somatic transgenesis in the avian embryo will be envisaged.

The origin of the germline is studied in avians by tracing primordial germ cells from the stage of the germinal crescent backwards to earlier developmental stages. It has been demonstrated that during primitive streak formation, the germline has already been segregated. However, during this stage, the cells are seen gradually migrating from the epiblast to the hypoblast. The vertical migration is followed by a horizontal translocation to the extra-embryonic germinal crescent, being carried out by the hypoblast that is pushed anteriorly by the invading endodermal cells. In contrast, it has been shown that in the mouse the germ cells are allocated during gastrulation in a cluster of cells. Our results demonstrate that in avians the allocation takes place according to a different mode. The close association between the germ cells and the extra-embryonic mesoderm indicates that the germline in avians, as in the mouse, develops from a subset of cells that have already segregated from the epiblast as extra-embryonic mesoderm.

We studied the early pattern of neural tube (NT) vascularization in quail embryos and chick-quail chimeras. Angioblasts appeared first in the dorsal third at Hamburger and Hamilton (HH) stage 19 as single, migrating cells. Their distribution did not correspond to a segmental pattern. After this initial dorsal immigration, endothelial sprouts invaded the NT on either side of the floor plate (HH stage 21). These cells remained continuous with their arterial vascular sources, connected to the venous perineural vascular plexus at HH-stage 22, and formed the first perfused vessels of the NT at HH-stage 23. The same pattern of angiotrophic vascularization was observed in a craniocaudal sequence starting caudal to the rhombencephalic NT. Extremely long filopodia were observed on sprouting cells, extending toward the central canal and the mantle layer. The exclusively extraneuroectodermal origin of angioblastic cells was demonstrated with chick-quail chimeras. Following replacement of quail NT by chick NT graft, angioblast and sprout distribution in chimeras was the same as in controls. We conclude that the NT receives its first blood vessels by a combination of two different processes, dorsal immigration of isolated migrating angioblastic cells and ventral sprouting of endothelial cells, which derive from perfused vessels. The dorsal invasive angioblasts contribute to the developing intraneural vascular plexus after having traversed the neural tube. The initial distribution of blood vessels within the neuroepithelium corresponds to intrinsic random motility of angioblastic cells; a more regular pattern is seen later. The floor plate apparently prohibits connections between sprouts in both NT sides, whereas in the dorsal NT, such a separating effect on the migrating angioblasts does not exist.

This paper presents the hypothesis that developmental asynchrony between embryos of both sexes, on the one hand, and offspring development and responsiveness of maternal reproductive organs, on the other, may result in significant sex ratio biases at birth in mammals, and in birds. It is argued that the developmental asynchrony hypothesis may account for a significant proportion of findings on sex ratio skews, including the inconsistency of evidence and the lack of successful selection attempts. This mechanism could be used by the maternal organism adaptively to alter the sex ratio by control of the relative time of insemination within the oestrous cycle or by alteration of reproductive hormone levels around the time of ovulation and early embryonic development. However, the developmental asynchrony hypothesis may also imply that many of the sex ratio biases can be viewed as by-products of constraining selection on control of fertility and fecundity and on sexual dimorphic growth rates.

Polarity and its plasticity in the avian mesencephalon and the retina are reviewed. Rostrocaudal polarity of the tectum (dorsal mesencephalon) is revealed by the gradient of engrailed gene expression, by its cytoarchitecture, and by positional specification of tectal cells which retinal fibers read to find their targets. In the ventral mesencephalon, the polarity is revealed by directional growth of tectofugal axons. To examine the plasticity in rostrocaudal polarity, rostrocaudal orientation of the mesencephalon was surgically reversed on E2. Rostrocaudal polarity of the reversed mesencephalon was all adjusted to the host pattern. This suggests that the mesencephalic polarity is not fixed on E2 and is established under environmental influences. Retinal polarity is revealed by the distribution of certain molecules, by retinal structures, and by the retinotectal projection pattern. Retinal structures and retinotectal projection pattern were kept as in the original if retinal primordium was rotated or partially excised after stages 12-14 of Hamburger and Hamilton. The location of an optic fissure and the retinal fiber pattern were adjusted to those of normal eyes when the rotation was performed before stage 12, therefore the retinal polarity was supposed to be plastic before stage 12 and to be fixed around the stages 12-14 under environmental influences.

The sex determination systems of Drosophila, birds, marsupial mammals, and eutherian mammals are briefly reviewed. Evidence from poultry polyploids, aneuploids, and gynandromorphs suggests that avian sex is determined by Z to autosome ratio and also that avian sex-limited traits may be under both cell-autonomous and hormonal controls. To identify aneuploids in avian populations, a cDNA clone linked to both the Z and W chromosomes of many avian groups was used to screen large numbers of psittacine birds and identified five birds with atypical patterns of restriction fragments. Two of these birds were examined by laparoscopy and found to be intersexes. These preliminary observations indicate that structural changes in the Z and W chromosomes result in alterations of sex determination.

The pattern of pigmentation in bird embryos is determined by the spatial organization of melanocyte differentiation. Some of the results from recent, neural crest transplantation experiments support a model based on a prepattern in the feathers; others could be interpreted in terms of a nonspecific pattern resulting from a failure of the crest cells to read the positional values in another species. To distinguish between these possibilities, the crucial test is to construct chimeras from two species with different pigment patterns. We have examined the wing plumage of quail and guinea fowl embryos. The quail has a characteristic pattern of pigmented and unpigmented feather papillae, whereas the guinea fowl shows uniform pigmentation. Chimeras were constructed by grafting wing buds isotopically between embryos. The wing buds were transplanted before they had become invaded by neural crest cells. Quail wing buds grafted to the guinea fowl developed, in most cases, a pigment pattern resembling that of the quail and not that of the guinea fowl. A few cases became uniformly pigmented and appeared to represent nonspecific patterns. The reciprocal grafts (guinea fowl wing buds grafted to the quail) became pigmented all over. We found evidence that the timing of melanocyte differentiation is controlled by cues in the feather papillae. Some cases developed a severe inflammatory response. The model which best accounts for these findings--and which can account for inconsistencies in previous reports--is the following. A prepattern is present in the feathers and this can control the differentiation of melanoblasts, even if they come from a different species. The local cues which constitute the prepattern are not positional values. In some chimeras melanoblasts fail to respond to the prepattern and so a nonspecific pattern of uniform pigmentation is produced.

Microliter quantities of a Prudhoe Bay crude oil (PBCO) applied to the shell of fertile chick eggs during various stages of development induced cytochrome P-450 levels and mixed-function oxidase activities within the liver of the embryo. PBCO (5 microliter) applied on Day 11 of incubation was found to maximally induce within 24 hr embryo hepatic cytochrome P-450 levels (fourfold), naphthalene hydroxylase (sixfold), benzo[a]pyrene 3-hydroxylase (14-fold), and 7-ethoxyresorufin O-deethylase (24-fold). Glutathione S-transferase was not induced. Crude oils are known to be highly toxic to avian embryos, especially during the early stages of development. The LD50 of PBCO and Hibernia crude oil applied to the egg shell on Day 8 of incubation was found to be 1.3 and 2.2 microliter, respectively. Mixed-function oxidase-dependent metabolism of crude oil components may be required for toxicity since administration of 20 micrograms of disulfiram in dioxane 1 hr prior to application of 1.3 microliter of PBCO reduced embryo mortality from 60 to 20%.

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged varied with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explanation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.

The authors hypothesized that donor primordial germ cells (PGC) are useful as vehicles of gene transfer in birds. The PGC have been identified in the blastula prior to incubation. They have been isolated from the Stage 7 germinal crescents, Stage 17 blood, and Stage 30 gonads. The percentages of PGC in these three embryonic sources were 2, .003 and 1.5%, respectively. The isolated PGC have been used as antigens to produce specific antibodies that have served as major tools in identification, isolation, and forming enriched in vitro cultures of PGC. Sterile hosts for normal donor PGC have been induced by use of ultraviolet irradiation, treatment with the chemical busulfan, and crossing rooster with female quail to form a sterile host termed "quickens." Micromanipulator technology, along with refinement of techniques to avoid trauma, will allow 80% hatchability. Integration of all the above biotechnology has led to successful use of PGC as vehicles of gene transfer and production of autogenic quail.

Eggs from pheasants, Chukar partridge and Japanese quail were stored 1 to 28 days in a cold box where temperature and relative humidity were maintained at approximately 16 degrees C. and 70 percent, respectively. Half the eggs from each species was turned once daily from a 45 degree angle from the verticle to the opposite angle. Turning eggs prior to incubation had no appreciable effect on hatchability of eggs stored up to 28 days. Length of storage for eggs from the pheasant and quail was critical, with significant declines in fertility in eggs stored beyond 14 days. Storage of Chukar eggs up to 28 days had little effect on subsequent hatchability, and their holding quality was comparable to that for commercial strains of chickens and turkeys.

We have isolated molecular clones encoding RNAs which appear to be transcribed predominantly or exclusively during oogenesis in Drosophila melanogaster. These clones were isolated by virtue of their homology to polyadenylated RNAs present in very early embryos and absent from older embryos. An analysis of the developmental expression of three cloned genes is presented. The genomic position of one of these clones coincides closely with the position of the maternal effect lethal gene swallow, providing strong evidence that we have cloned this locus.