Homotopic and heterotopic transplantations of quail tectal primordia in chick embryos: Organization of the retinotectal projections in the chimeric embryos

To study the adaptative capabilities of the retinotectal system in birds, the primordium of one optic tectum from 12-somite embryos of Japanese quail was transplanted either homotopically, to replace the ablated same primordium, or heterotopically, to replace the ablated dorsal diencephalon in White Leghorn chick embryos of the same stage. The quail nucleolar marker was used to recognize the transplants. The cytoarchitecture of the tecta and the retinal projections from the eye contralateral to the graft were studied on the 17th or 18th day of incubation in the chimeric embryos by autoradiographic or horseradish peroxidase tracing methods. Morphometric analysis was applied to evaluate the percentage of the tectal surface receiving optic projections. It was observed that: (i) quail mesencephalic alar plate can develop a fully laminated optic tectum even when transplanted heterotopically; (ii) retinal ganglion cells from the chick not only recognize the tectal neurons of the quail as their specific targets in homotopic grafts, but the optic fibers deviate to innervate the heterotopically grafted tectum; (iii) in the presence of a graft, the chick retina is unable to innervate a tectal surface of similar or larger size than that of the control tectum; (iv) tectal regions devoid of optic projections, whether formed by donor or by host cells, always present an atrophic lamination; (v) the diencephalic supernumerary optic tectum competes with and prevails over the host tectum as a target for optic fiber terminals.     

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.     

Embryonic origin of skeletal muscle cells in the iris of the duck and quail

Summary The origin of skeletal muscle cells in avian iris muscle was investigated by quantitative analysis of heterochromatin profiles at the electron-microscopic level in irides of six types of quail-duck chimeras. Each of the following tissues was transplanted into the head region from quail to duck between stages 9 and 10: cranial neural crest; trunk neural crest; midbrain and adjacent mesoderm; forebrain; forebrain without neural crest; and forebrain without neural crest and mesoderm. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high (quail type) in the dorsal iris, but low (duck type) in the ventral iris of the chimeras resulting from isotopic transplantation of cranial neural crest. Heterotopic transplantation of trunk neural crest to cranial position resulted in failure of development of skeletal muscle cells in the dorsal iris, but not in the appearance of skeletal muscle cells in the ventral iris. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high in the chimeras resulting from transplantation of midbrain region and the chimeras resulting from transplantation of forebrain region, intermediate in the chimeras resulting from transplantation of forebrain region without neural crest, and low in the chimeras resulting from transplantation of forebrain region without neural crest and mesoderm. These results indicate that the skeletal muscle cells in the dorsal iris are of cranial neural crest origin while those in the ventral iris are not, and could possibly arise from cranial mesoderm.     

Epithelia are interchangeable between facial primordia of chick embryos and morphogenesis is controlled by the mesenchyme

The embryonic chick face is composed of a series of facial primordia, epithelium-covered buds of mesenchyme, which surround the presumptive mouth. The protruding adult upper beak containing the prenasal cartilage is formed from the frontonasal mass, the paired maxillary primordia form the sides of the face, while the lower beak is derived from the paired mandibular primordia which contain the two Meckel's cartilages. When grafted to a host wing bud, the frontonasal mass and the mandibular primordia both form elongated outgrowths, whereas the maxillary primordium forms a ball of tissue. Facial epithelium is required for growth and morphogenesis of all primordia. Recombinations between epithelium and mesenchyme from different primordia show that the epithelia are interchangeable and appear to be equivalent. Even the epithelium from the maxillary primordium that does not grow out in a polarized fashion can support outgrowth of the frontonasal mass and mandibular mesenchyme. The form of the recombined graft is determined by the mesenchymal component.     

Concerning variability in the delimitation of the vascular system in root primordia of maize embryos

1. The transfer of immature embryos from maternal plants to artificial media influenced the radial arrangement of vascular bundles in developing root primordia. The variability in the number of poles of the prospective protoxylem and protophloem, observed as a rule during embryogenesis under natural conditions, could not be suppressed even under the conditions ofin vitro cultivation. The possibility is admitted that when using agar medium the nutrient supply need not necessarily be equivalent for all embryos.
2. Using excised embryos of various ages the period of delimination of the vascular system in the root primordium was determined. It is relatively short and occurs in the first half of embryogenesis. The results obtained revealed no relationship between vascular system arrangement in root primordium and mature grain and mature embryo size.
3. Maize ear represents a type of inflorescence of which the apical part is delayed in development. Histogenically this uneven development becomes evident with the formation of a significantly lower mean number of poles in root primordia from the grains originating from the apical region of the cob. This is further evidence of the adaptibility of the vascular system development to environmental conditions.
4. As further causes of the variability in pole number those differences are considered which occur during sex cell formation, pollination and fertilization.

     

Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos.

We report the cloning of two new quail myogenic cDNAs, quail myogenic factor 2 (qmf2) and qmf3, which encode helix-loop-helix proteins homologous to mammalian myogenic factors myogenin and myf-5. In situ hybridization has been used to investigate the developmental expression of qmf2 and qmf3, as well as qmf1, the quail homologue to mammalian MyoD1, during the formation of the brachial somites. These studies show that qmf1 and qmf3 are activated sequentially in medially localized somite cells, immediately following somite formation but prior to myotome formation. qmf1, qmf2, and qmf3 are expressed in the myotome of compartmentalized somites. These findings suggest that determination of the myogenic cell lineage in quail somites is a progressive process controlled by influences of the neural tube on the expression of the qmf regulatory genes in newly forming somites.     

Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development.

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.     

Embryonic development and evolutionary origin of the Orthopteran auditory organs

Two different types of ears characterize the order of Orthopteran insects. The auditory organs of grasshoppers and locusts (Caelifera) are located in the first abdominal segment, those of bushcrickets and crickets (Ensifera) are found in the tibiae of the prothoracic legs. Using neuron-specific antibody labelling, we describe the ontogenetic origin of these two types of auditory organs, use comparative developmental studies to identify their segmental homologs, and on the basis of homology postulate their evolutionary origin. In grasshoppers the auditory receptors develop by epithelial invagination of the body wall ectoderm in the first abdominal segment. Subsequently, at least a part of the receptor cells undergo active migration and project their out-growing axons onto the next anterior intersegmental nerve. During this time the receptor cells and their axons express the cell-cell adhesion molecule, Fasciclin I. Similar cellular and molecular differentiation processes in neighboring segments give rise to serially homologous sensory organs, the pleural chordotonal organs in the pregenital abdominal segments, and the wing-hinge chordotonal organs in the thoracic segments. In more primitive earless grasshoppers pleural chordotonal organs are found in place of auditory organs in the first abdominal segment. In bushcrickets the auditory receptors develop in association with the prothoracic subgenual organ from a common developmental precursor. The auditory receptor neurons in these insects are homologous to identified mechanoreceptors in the meso- and metathoracic legs. The established intra- and interspecies homologies provide insight into the evolution of the auditory organs of Orthopterans.     

Two-step consumption of yolk granules during the development of quail embryos

The mechanism of yolk consumption was studied morphologically and biochemically in Japanese quail Coturnix japonica. The amount of yolk granules in the yolk (or 'yolk cell') decreased in two steps during embryonic development. In the first step, during days 0-4 of incubation, the yolk-granule weight decreased at a rate of 13 mg/day. This decrease was due to segregation by endodermal cells that were newly formed in the developing yolk sac. In the second step after day 6, the decrease was drastic at a rate of 29.8 mg/day during days 6-12 and very slow thereafter. The decrease at the second step was due to the enzymatic digestion of yolk granules by cathepsin D that coexisted in yolk spheres. This digesting reaction was triggered by the solubilization of the granules with high concentrations of salts that were supplied after disruption of the limiting membrane of yolk spheres. The 'yolk cell' seemed to die around day 5 of incubation. Thus the digestion products might be taken up together with yolk lipids by endocytosis into the endodermal cells and transported to blood vessels.     

Preformation of root primordia in shoots and root morphogenesis in Salix viminalis

Root primordia are formed in the stems of Salix viminalis L. during normal growth. Some of these primordia are produced at definite sites in the nodes. The initiation and early structural and ultrastructural development of the nodal primordia were studied in young shoots. In the fourth node below the terminal leaf cluster some parenchyma cells situated at the lateral leaf gaps formed a small group of initial cells. Derivatives of the newly formed interfascicular cambium added cells to that group, in which later on cell divisions in various directions occurred resulting in the formation of a root primordium. Root morphogenesis was studied in cuttings from one-season-old stems. The cells in the dormant primordia contained many lipid bodies but only a small amount of starch. After the cuttings had been 24 hours in water starch was accumulating in the plastids and lipid bodies were seen in the vacuoles. 48 hours after activation cell divisions occurred throughout the primordia and a layered apical mer-istem was organized. After 96 hours a root cap with amyloplasts was formed and the procambium was well developed. The amyloplasts were sedimented in response to gravity. After six days the first roots were ready to emerge from the stems. Their root caps had a well developed columella and endodermal and pericyclic cells were recognizable.     

Mothering during early development influences subsequent emotional and social behaviour in Japanese quail

The aim of the present study was to investigate the influence of mothers on the emotional reactivity and social behaviour in young precocial Japanese quail. We used a classical method of maternal deprivation. Ethological tests and observations analysed and compared the behaviour of young artificially raised quail to that of young raised by adoptive maternal quail. After separation from mothers, brooded young were more fearful (frightened easily) in the presence of humans (human-observer tests) and more neophobic in novel environments (open-field and hole-in-the-wall tests) than young raised artificially. As chicks rarely expressed fear during the brooding period, no differences related to mothering could be observed at that time. In separation tests, brooded chicks jumped significantly more frequently than non-brooded chicks and later, observations of groups revealed that brooded chicks remained closer to one another than non-brooded chicks. Social motivation of brooded chicks appeared to be higher. These results indicate that, during their first days of life, mothers influence the emotional and social behaviour of their young.     

Improved development of rat embryos in culture during the period of craniofacial morphogenesis

To study the mammalian craniofacial development, the culture conditions of rat whole embryo during the period of major craniofacial morphogenesis were examined. The improved rotating apparatus which is gassed continuously was used. Rat embryos explanted at 11.5 days (plug day 0) developed in vitro for up to 72 hr, that is, throughout the period of major craniofacial morphogenesis, and cultured embryos showed normal facial formation. The medium was equilibrated with a gas mixture of 95% 02, 5% CO2. The 100% rat serum improved the protein content of embryos cultured for 48 hr compared with the medium consisting of 50% rat serum and 50% Tyrode solution, although somite number was not altered. Furthermore, 100% rat serum containing 2 mg/ml glucose was the best medium for supporting growth of embryos when it was measured by protein content. Thus, the best culture medium was pure rat serum containing 50 units/ml penicillin, 50 micrograms/ml streptomycin, and 2 mg/ml glucose. Protein content, body weight, craniofacial formation, and somite number of embryos cultured for 48 hr with continuous gassing were much better than those cultured with noncontinuous gassing.     

Lymphatic vascular morphogenesis in development, physiology, and disease

The lymphatic vasculature constitutes a highly specialized part of the vascular system that is essential for the maintenance of interstitial fluid balance, uptake of dietary fat, and immune response. Recently, there has been an increased awareness of the importance of lymphatic vessels in many common pathological conditions, such as tumor cell dissemination and chronic inflammation. Studies of embryonic development and genetically engineered animal models coupled with the discovery of mutations underlying human lymphedema syndromes have contributed to our understanding of mechanisms regulating normal and pathological lymphatic morphogenesis. It is now crucial to use this knowledge for the development of novel therapies for human diseases.     

Development of the skeleton in Japanese quail embryos

In the research fields of experimental embryology, teratological testing, and developmental engineering in avian species, a knowledge of normal embryonic development is necessary so that research may be performed efficiently and precisely. A series of normal stages based on external appearance has been established in both chicken and quail embryos. Those based on skeletal features, however, have not been elucidated. The present study newly established a series of normal stages for the development of the Japanese quail embryo skeleton. This series is composed of 15 stages determined by observing the timing of chondrification and calcification of the skeleton every 24 h, from 3 to 17 days of incubation. Cartilage and ossified bones were stained blue and red with Alcian blue 8GX and alizarin red S, respectively. These skeletogenous stages of the Japanese quail embryo will be useful as a normal control not only in studies of experimental embryology, teratological testing, and developmental engineering, but also in the analysis of mutant embryos with skeletal abnormalities.     

Embryonic development in two species of scleractinian coral embryos: Symbiodinium localization and mode of gastrulation

Reef-building scleractinian corals widely engage in symbiotic relationships with Symbiodinium dinoflagellates (zooxanthellae), which reside inside cells of the gastrodermis. In most cases, sexually produced larvae acquire their symbionts from the environment in the early developmental stages preceding settlement; however, some scleractinian corals maternally "seed" their oocytes with symbionts, and these symbionts are reported to be restricted to the gastrodermis at the time of its formation (gastrulation). A precise mechanism for how Symbiodinium are translocated to endoderm in these seeded species was previously unknown. In order to examine the process of endoderm formation and Symbiodinium localization during gastrulation, we have examined two species of "robust" clade scleractinians: Fungia scutaria (nonseeded) and Pocillopora meandrina (maternally seeded). We determined that both species, independent of whether or not they are seeded, undergo a "nutritive" stage before gastrulation, wherein lipid-rich cells (F. scutaria) or membrane-bound cellular fragments (P. meandrina) are passed to the blastocoel where they are subsequently taken up by the definitive endoderm. This emergent property of anthozoan development has been co-opted to facilitate the movement of Symbiodinium to the blastocoel (future site of endoderm), in the seeded species, where they are later phagocytosed by the newly formed definitive endoderm. Additionally, both species of robust clade scleractinians examined gastrulate by way of invagination, as do the majority of anthozoans. This invagination differs from the prawn chip-type gastrulation seen in the complex clade corals and provides evidence for a possible linkage between gastrulation type and phylogenetic history.     

Embryonic development of pineal gland vesicles: a morphological and morphometrical study in chick embryos.

Pineal cell aggregates in 5, 10 and 15 day-old chick embryos have been studied. Cell aggregates were classified into rosettes or vesicles and spheroid and ellipsoid vesicles distinguished. The number of pineal vesicles per unit of surface (vesicle density) was determined in three pineal portions: apical, anterior and posterior. By day 5, only cellular rosettes were found, mainly in the apical portion. After 10 and 15 days, the presence of rosettes was occasional. The posterior wall showed only small spheroid vesicles, while in the apical and anterior areas ellipsoid vesicles were also observed. Moreover, the spheroid/ellipsoid vesicle ratio increased from the 10th to the 15th day of incubation. The vesicle density decreased between the 10th and 15th day because of the increase in both vesicle and pineal size, without changes in the total number of vesicles. The results suggest that changes in vesicle morphology and density could be related to the functional activity of the pineal gland during embryonic development.     

Magnum morphogenesis during the natural development of the quail oviduct: analysis of egg white proteins and progesterone receptor concentration

The histological development of the quail oviduct and the changes in concentrations of progesterone receptor, ovalbumin, conalbumin, ovomucoid and ovoglycocomponents are analyzed during the period spanning 7-35 days of age. The initiation of luminal epithelial cell proliferation is the first event of magnum growth. The epithelial cells begin to evaginate into subepithelial stroma and form tubular glands. Meanwhile, luminal epithelium starts cellular pleomorphism through ciliogenesis. No egg white proteins are detectable in the developing glands; at the same time, the concentration of the progesterone receptor increases from about 5500 sites/cell to 30,300 sites/cell. Tubular gland cells then begin to synthetize and accumulate egg white proteins, mucous cells differentiate in the luminal epithelium, and the cell proliferation decreases and finally stops. Compared with earlier studies dealing with the blood levels of estrogen and progesterone in developing quails during the same period, and the cellular changes induced in the oviducts of ovariectomized and ovariectomized-hypophysectomized quail by exogenous steroids, these results distinguish between the cellular responses that are physiologically controlled by estradiol and other responses that have multihormonal regulation.