Possible Roles for Cognin in Chick Embryo Neural Retina

In this review, we discuss current information about a cell-cellrecognition protein present in chick embryo neural retina. Thisprotein, retina cognin, has cell adhesion or aggregation promotingpropertiesin vitro. We discuss five questions. First, what isretina cognin (R-cognin)? Second, what do we know about cogninin chick retina? We discuss its histological distribution inretina and how that distribution changes during embryonic andearly post-hatching development. Third, where is cognin withincells? We review light microscopy evidence for its localizationin plasma membranes of somas and neurites of selected retinalneurons as an intrinsic membrane protein. Fourth, how is cognindistributed in membranes? We summarize evidence that cogninmight not be uniformly distributed over cellsurfaces and thatit might bind to specific proteins on the surfaces of otherretina cells. From the available information, we ask what wecan deduce about cognin's biological role in the neural retina.     

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest¹. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest^2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone^6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required¹⁰. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.Download video file.^{(56M, mov)}     

Neural Induction by Previously Induced Epiblast in Avian Embryo in vitro

Pieces of previously neurally induced and competent epiblast of chick and, respectively, quail primitive streak blastoderms were cultured in close contact with each other for 48 h. In several cases, both pieces differentiated into neural direction, which indicates the occurrence of a homoiogenetic induction. There was a considerable mixing of cells of different origin, especially in the undifferentiated controls. In general, the dorsoventral orientation of the previously induced epiblast was retained, but the orientation of the competent epiblast cells was more flexible and could be influenced by the neighbouring neuralised cells.     

Studies on Phosphoprotein Phosphatase in Chick Embryo

Several properties of partially purified phosphoprotein phosphatase from chick embryo are described. The enzyme was active toward casein, phosvitin and phosphopeptone, but not toward low molecular weight phosphate esters including aliphatic and aromatic phos-phomonoesters, a phosphodiester, pyrophosphates and phosphoamides. The enzyme was not activated by reducing compounds. Heavy metal ions and sulfhydryl inhibitors inhibited the enzyme activity, but the inhibited enzyme was partially reactivated with cysteine. Metal chelating agents also inhibited the activity. To the oxalate treated enzyme, Fe⁺⁺ and Co⁺⁺ had a stimulatory effect. Differences in property between phosphoprotein phosphatases of chick embryo and of mammalian tissues are discussed.     

Ontogeny of Cerebral Oxidative Metabolism in the Chick Embryo

Abstract: The low cerebral energy requirements of most mammals at birth reflect an immaturity of the central nervous system, and it has been suggested that energy demands in fetuses are even less well developed than in newborns. Furthermore, fetal cerebral energy requirements are presumed to be met predominantly or exclusively by anaerobic glycolysis. To clarify these issues, we investigated cerebral oxidative metabolism in 9-, 14-, 16-, and 19-day-old chick embryos and in newly hatched peeps. Animals were decapitated and quick-frozen in liquid Freon 0-5 min post-mortem. Forebrain extracts were prepared and assayed for ATP, phosphocreatine, glucose, and lactate. Alterations in these metabolites post-decapitation were used to calculate cerebral metabolic rates (Δ∼P) and rates of maximal anaerobic glycolysis (Δ lactate). Rates of lactate accumulation during cerebral ischemia increased progressively from embryonic day 9 through hatching. Cerebral metabolic rates were not different in 9-, 14-, and 16-day-old embryos, but increased steadily thereafter. The extent to which total cerebral energy utilization could be derived from anaerobic glycolysis (Δ lactate/Δ∼ P) increased from a low at day 9 (0.29) to a maximum at day 16 (0.78). The data suggest that, despite the low cerebral metabolic activity of the chick embryo, at no time during development is anaerobic glycolysis capable of entirely supporting the energy needs of the developing brain.     

Establishment of the Neural Differentiation Pattern in the Prospective Prosencephalic Ectoderm of the Chick Embryo (Gallus domesticus)

Small graft pieces (size: 0.15 × 0.2 mm to 0.1 × 0.4 mm and 0.25 × 0.25 mm) were isolated from the prospective prosencephalic ectoderm of the definitive primitive streak (st. 4) and head process (st. 5) chick blastoderms, and cultured in vivo intracoelomically. In all 1437 graft pieces isolated from 448 donor blastoderms were transplanted into the coelom of 941 host embryos; 216 hosts died before reaching 12 days. The 725 surviving hosts, which carried 1241 implants, yielded 304 analysable grafts. The very small size of the isolates affected the recovery rate. The histological analysis of the recovered grafts reveals that the frequency of neuralization was higher in the grafts isolated from the central region of the prospective prosencephalic ectoderm than in those from its peripheral region. A centrifugal extension of differentiation tendencies of prosencephalic structures was observed from st. 4 to st. 5. At st. 5 frequency of telencephalic structures was high in the grafts of the anterior region, whereas that of diencephalic structures was high in those of the more posterior region. Eye structures (retina as well as tapetum) differentiated in an area measuring ca. 0.4 × 0.1 mm extending transversely about 0.1 mm anterior to the head process at st. 5. Structural elements of the lens were not observed in any graft.     

Pathways of Differentiation in Chick Embryo Neuroretinal Cultures

Markers of neuronal cell differentiation (GABA accumulation, choline acetyltransferase activity) are shown to increase initially and then decline sharply in monolayer cultures of 9 day embryo neuroretinal (NR) cells. A glial marker (glutamine synthetase, GSase) is precociously inducible by hydrocortisone (HC) in dense'monolayer' NR cultures (containing aggregates of neuronal cells overlying the glial sheet) as well as in chick embryo retinal explants. The induced level of GSase activity is not maintained in the continued presence of HC, but rather declines by 20 days in vitro. Choline acetyltransferase (CAT) activity is higher in HC-treated cultures than in controls only during the period when induced GSase activity is detectable. Furthermore, the subsequent transdifferentiation of lens cells (monitored as δ crystallin content) in these cultures is delayed by 10 days and much reduced in extent when HC is present throughout the culture period.
We suggest a simple model to account for these results, on the basis of recent evidence that lens cells are derived mainly from the retinal epithelial cells (immature Müller glia) of 9-day embryonic NR, and that transdifferentiation results from a change in cell determination during the early stages of'monolayer' culture. In outline, our model proposes that early dedetermination of the retinal glia is associated with a decline of neuronal cell markers (dedifferentiation) followed eventually by loss of the neuronal cells. Hydrocortisone, by inducing transient glial cell differentiation (GSase activity), both prolongs the expression of a neuronal marker (CAT) and also reduces later transdifferentiation into lens.     

Biochemical Studies during the Development of the Chick Embryo

The devised method consists of the enzymatic hydrolysis, separation of deoxyribosides in the hydrolysate by paper chromatography and paper electrophoresis, and the estimation of the separated fractions with L. leichmannii. This method permits the determination of deoxyriboside composition of the smaller amounts of DNA or the related compounds with relatively higher accuracy even under the presence of some other compounds when nucleic acids and acid insoluble fractions of the chick embryo were analyzed.

The change of each deoxyriboside composition in acid insoluble fraction prepared from the 3 to 19 day old embryos investigated by the method. Among the major four deoxyribosides, the contents of deoxyguanosine and of deoxycytidine was nearly constant during the development of the embryo, whereas that of thymidine and of deoxyadenosine appeared to undergo the change slightly at the periods from 10 to 15 days incubation. It seems that this periods is also the most active time of the synthesis of DNA and of the changes of deoxyribosyl compounds in acid soluble fraction through the embryo growth.     

Biochemical Studies during the Development of the Chick Embryo

The presence and the change of deoxyribosidic compounds in the acid extract of the embryo with the incubation were examined with an aid of the organism, L. leichmannii. The main deoxyribosidic compounds in the extract prepared from the 18 th day embryo were identified as uracil, cytosine and thymine deoxyribosides and deoxyribotides of cytosine and thymine from the behaviour on paper chromatographic and paper electrophoretic separation. A small amount of purine deoxyribosyl compound which was assumed as hypoxanthine deoxyriboside was detected, and the content of which per 1 g of fresh embryo changed with the lapse of the incubated day; especially, the content was minimum at the period from the 10 th to 15 th day incubation. At this period, the total growth promoting compounds contained 50% of deoxyribotide though deoxyribosides was lower than that of the other days. This period is the most significant stage of the embryo growth and the most active time of synthesis of DNA through the embryo growth.     

Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth

Chick myogenic cells grew in a medium composed of Eagle's minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF. They grew in a medium composed of MEM and CS; they grew much better in a medium composed of MEM, CS, and HS.
In the presence of one of the EFMG, the optimal HS concentration for growth varied depending on its lot. At higher HS concentrations, growth was suppressed. Further, it was suggested that an inhibitory substance(s) for myogenic cell growth was present in HS. The inhibitory effects can usually be minimized by diluting the serum with an artificial medium.     

Biochemical Studies During the Development of the Chick Embryo

The days when the concentration of conjugated- and free- vitamin B₁₂ per wet weight g increased or decreased agreeded with that of nucleic acids in the chick embryo from 3rd through 18th day of incubation. It is intereresting that the day of increasing or decreasing of those compounds concentration corresponds with the important stages through the growth of embryo. The changes of these compounds in the egg contents free of embryo were estimated. Although a considerable large amount of free vitamin B₁₂ was contained in the embryo, the most vitamin B₁₂ in the egg content except the embryo existed in the conjugated state. It seems that the amount of total vitamin B₁₂ in the egg during the incubation is relatively constant.     

Synthetic Orcein as a Stain for Chick Embryo Cartilage Matrix

Chick embryo tissues fixed in Bouin's fluid, in 10% formol saline or in 10% formol saline with subsequent mordanting in saturated picric acid containing 3% HgCl₂, were examined as 5 μ paraffin sections after staining with 1% synthetic orcein in 80% ethanol containing 1% HCl (conc.). Orcein defined the young elastic fibres formed in the truncus arteriosus, aorta and other large arteries after the 5th day of embryonic development but also reacted with the matrix of cartilage in all parts of the skeleton from the 3rd day onward. It is thought that a glycoprotein or proteoglycan shared by these two tissues could account for their mutual affinity for orcein.     

On the Origin of Primordial Germ Cells in the Chick Embryo

An attempt was made to re-examine the location of the primordial germ cells (PGCs) in very young chick embryos. Freshly laid blastoderms, prior to hypoblast formation, of a known anterio-posterior axis, were transversely bisected and each half was separately grown in vitro. Both anterior and posterior halves were shown to be fertile and each was shown to contain roughly the same amount of PGCs as a normal control embryo. It has been concluded that in the chick as well as in the duck there is no concentration of cells containing germinal plasm in the posterior part of the blastoderm.
Two other possibilities should be investigated:
1. A concentric arrangement of cells containing germinal plasm. 2. The absence of a germinal plasm and a relatively late appearance of PGCs as a result of induction.     

Hypothalamic regulation of the Pituitary-Thyroid Unit in the Developing Chick Embryo

SYNOPSIS. In the chick embryo, the period of 10.0 to 13.0 daysof incubation is critical in the maturation of the hypothalamic/adenohypophysealor adenohypophyseal regulation of thyroid function; the establishmentof thyroid regulation at this time is reflected in a markedincrease in thyroid function. There has been general agreementthat the "feed-forward" and the "feed-backward" loops of theadenohypophyseal-thyroid unit are established on or about day11.5, but whether these events were the result of the establishmentof pituitary-thyroid interactions alone or included hypothalamicregulation of the pituitary-thyroid unit has until quite recentlyremained problematical. Data presented in the present reviewindicate that beginning on or about day 11.5 the hypothalamuscan and does regulate the pituitary-thyroid unit. Although thehypothalamus is essential for the maintenance of normal pituitary-thyroidfunction, the data strongly suggest that the immediate eventwhich is responsible for the establishment of a functional hypothalamoadenohypophyseal-thyroid axis in the developing chick embryo is a marked increasein adenohypophyseal thyrotrophs on day 11.5.     

Glycosyltransferases in Different Brain Regions During Chick Embryo Development

Glycosphingolipids, in particular gangliosides, play a crucial role in neuronal development and are known to change dramatically in total content and distribution in different brain areas during embryogenesis. In the present work we analyzed the activity of enzymes involved in the metabolism of gangliosides, at different periods of functional maturation in different regions of chick embryo brain. Our data demonstrate differences in the enzymatic activities in the examined areas; these differences might be correlated with the functional lateralization occurring in the brain during development. Significative differences were found in glycosphingolipid composition between controlateral cerebral hemispheres and optic lobes; these results together with previous data we found, contribute to reinforce our hypothesis on the occurrence of biochemical lateralization during early brain development.     

Early Expression of Hypocretin/Orexin in the Chick Embryo Brain

Hypocretin/Orexin (H/O) neuropeptides are released by a discrete group of neurons in the vertebrate hypothalamus which play a pivotal role in the maintenance of waking behavior and brain state control. Previous studies have indicated that the H/O neuronal development differs between mammals and fish; H/O peptide-expressing cells are detectable during the earliest stages of brain morphogenesis in fish, but only towards the end of brain morphogenesis (by ∼85% of embryonic development) in rats. The developmental emergence of H/O neurons has never been previously described in birds. With the goal of determining whether the chick developmental pattern was more similar to that of mammals or of fish, we investigated the emergence of H/O-expressing cells in the brain of chick embryos of different ages using immunohistochemistry. Post-natal chick brains were included in order to compare the spatial distribution of H/O cells with that of other vertebrates. We found that H/O-expressing cells appear to originate from two separate places in the region of the diencephalic proliferative zone. These developing cells express the H/O neuropeptide at a comparatively early age relative to rodents (already visible at 14% of the way through fetal development), thus bearing a closer resemblance to fish. The H/O-expressing cell population proliferates to a large number of cells by a relatively early embryonic age. As previously suggested, the distribution of H/O neurons is intermediate between that of mammalian and non-mammalian vertebrates. This work suggests that, in addition to its roles in developed brains, the H/O peptide may play an important role in the early embryonic development of non-mammalian vertebrates.     

Development of Immune Tolerance in the Chick Embryo to Borrelia hispanica

Varying degrees of immune tolerance were induced in chick embryos at 10, 14, and 18 days of age by inoculation of living or dead Borrelia hispanica in the yolk sac, allantoic cavity, or intravenously. Relative tolerance was measured by responses to challenge with virulent organisms in relation to altered susceptibility to infection and inability to produce agglutinins for B. hispanica or Proteus strain OXK. Challenges were made 1 week posthatching with the controls and chicks that had received dead organisms embryonically, and all chicks and controls were challenged at 5-week intervals from the 5th through the 30th week posthatching. Infections persisted 7 to 12 days in the tolerant chicks without a recurring parasitemia. Control chicks were never infected, and in almost all instances produced agglutinins. Differences in degree and duration of tolerance were observed in relation to the age of the embryo injected and may, or may not, have been related to differences of antigenic mass. Differences in induced tolerance were also observed in the three inoculation routes (intravenous > allantoic cavity > yolk sac, with the first route as the greatest) with chicks that had received dead organisms embryonically, but not with those that had received living organisms. Tolerance was not transmitted to the progeny of tolerant pullets.     

Influence of Hydrocortisone on Chick Embryo Retina Development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.