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.     

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington ^1,2 and Gallera ^3,4.Open in a separate windowClick here to view.^{(67M, flv)}     

Expression of Transferrin Binding Protein in the Capillaries of the Brain in the Developing Chick Embryo

Transferrin-binding protein (TfBP) has been shown to be a novel protein, structurally related to the chicken heat shock protein 108. The physiological function of this protein, however, has not yet been established. Antiserum to TfBP selectively stains transferrin- and iron-rich oligodendrocytes and choroidal epithelium in the adult and embryonic chick brain, suggesting a role for this protein in transferrin and iron storage in these cells. In this study, we further demonstrate TfBP-immunoreactivity (IR) in the blood vessels of the embryonic chick central nervous system. A strong TfBP-IR was present in blood vessels from E6, declined from E10 and was absent by E18. Thus, the expression of the TfBP in the blood vessels precedes its expression in the oligodendrocytes. At the subcellular level, TfBP-IR was confined to the cytoplasm of capillary pericytes while the Tf-receptor IR was associated with the capillary endothelium of the brain. The up-regulated expression of TfBP, together with the Tf-receptor of the brain capillaries, suggests that pericytes may be associated with the high iron uptake required for the metabolic demands of the developing brain. D. W. Kim and H. N. Lee contributed equally to this work.     

非水介质中辣根过氧化物酶（HRP）荧光活性测定法

建立了一种分析ＨＲＰ催化活力的新方法。该方法基于单体（底物）、聚合物（产物）的荧光发射光谱不重叠,使用荧光光谱仪,通过测量底物荧光淬灭来检测ＨＲＰ在非水介质中（二氧六环－水、乙醇－水、丙酮－水体系）催化酚类、芳香胺类物质聚合的活力。此方法迅速、简便,结果是定量并可重复的,并能定量地计算底物转化率。     

Changes in Soybean Agglutinin (SBA) and Peanut Agglutinin (PNA) Binding Pattern in the Epidermis of the Developing Chick Embryo

Lectin binding pattern in the developing chick embryonic epidermis was studied using peroxidase labeling method. The epidermis of the 13-day-old embryo is in an undifferentiated state. Little binding of soybean agglutinin (SBA), specific for N-acetyl-D-galactosamine, and peanut agglutinin (PNA), specific for β-D-galactose, was seen in such epidermal cells. As the epidermis developed toward keratinization, the cell membrane of the differentiating flattened cells was positively stained with SBA and PNA. The positive staining was also seen in the supranuclear region of the cells located between the flattened cells and the basal cells. The basal cells remained unstained in all the stages of development. Similar staining pattern with SBA and PNA was seen in the cultured skin explants during the epidermal differentiation in vitro. These observations show that the SBA- and PNA-reactive glycoconjugates accumulate during the epidermal cell differentiation, suggesting their important roles in the maintenance of the ordered structure of the epidermis.     

Polymerization of Horseradish Peroxidase in the Presence of Inorganic Adsorbents

Efficiencies of binding between horseradish peroxidase (HRP) and its polymers (HRPp) with inorganic adsorbents (precipitated and coprecipitated) were studied. In aqueous solutions, HRP efficiently adsorbed on aluminum oxide and the coprecipitated sorbent (composed of calcium orthophosphate, magnesium hydroxide, and aluminum hydroxide). HRP readily bound to zinc hydroxide but not to aluminum hydroxide in 25.0 mM bicarbonate buffer (pH 9.0). Several variants of HRP polymerization and HRPp modification with diamines in the presence of Al₂O₃ and Zn(OH)₂ were compared. Synthesis of HRPp according to the scheme comprising HRP activation in solution followed by its polymerization in the presence of Zn(OH)₂ appeared the most efficient. HRP and HRPp bound to Zn(OH)₂ displayed a high catalytic activity in the presence of high H₂O₂ concentrations.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 383–391.Original Russian Text Copyright © 2005 by Eryomin, Makarenko, Budnikova.     

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.     

Regulation of Ganglioside Composition and Synthesis Is Different in Developing Chick Retinal Pigment Epithelium and Neural Retina

Abstract: We examined the immunocytochemical expression of GM3 and QD3 in 3-day-old chick embryo retinal pigment epithelium (RPE) and neural retina (NR). We also compared the composition of gangliosides and the activities of key ganglioside glycosyltransferases of the RPE and NR of 8-, 12-, and 15-day old embryos. The immunocytochemical studies in 3-day-old embryos showed heavy expression of GM3 and GD3 at the inner and outer layers of the optic vesicle that are the precursors of the RPE and NR, respectively. The compositional and enzymatic studies showed pronounced differences between RPE and NR of 8-day and older embryos. HPTLC showed that at 8 days the major species were GM3 and GD3 in RPE and GD3 and GT3 in NR. As development proceeded, GD3 decreased in both tissues, GM3 became the major ganglioside in RPE, and ganglio-series gangliosides (mainly GD1a) became the major species in NR. At 15 days the major species were GD1 a in NR and GM3 in RPE. Enzyme determinations showed that whereas in RPE from 12-day-old embryos GM2 synthase was under the limit of detection and GD3 synthase activity was about sixfold lower than GM3 synthase, in NR the activities of GM3 and GD3 synthases were similar and both six-to ninefold lower than GM2 synthase. These results evidence a markedly different modulation of the ganglioside glycosylating system in cells of a common origin that through distinct differentiation pathways originate two closely related tissues of the optic system. In addition, they reinforce the relevance of the relative activities of key transferases in determining the pattern of gangliosides in different cell types.     

An Improved Method for the Injection of Crystalline Horseradish Peroxidase in Neuroanatomical Studies

A simple, inexpensive method for the injection of crystalline horseradish peroxidase is described that uses glass micropipettes. The horseradish peroxidase is expelled from the pipette by a fine wire inserted to the end of the pipette. A clay plug prevents diffusion of HRP during the needle's descent. The technique enables the investigator to reliably produce small, densely labeled injection sites with minimal diffusion. Both the retrograde and anterograde transport seen using this method compare favorably with that seen using iontophoretic or pressure injection methods.     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

Taste Disks are Induced in the Lingual Epithelium of Salamanders during Metamorphosis

Morphological changes of oral cavity during metamorphosis withspecial reference to the taste organ were examined in Ezo salamanders(Hynobius retardatus) and axolotls (Ambystoma mexicanum), andcompared with those in bullfrogs (Rana catesbeiana). The non-distensibletongue of salamanders changed the structure progressively duringmetamorphosis: a small area of the rostrum protruded and developedcaudally with recession of the flat area of the tongue. Theprotrusion that developed on the tongue had numerous papillae,as seen in the frog tongue. The apical region of the papillaeoccasionally had a cell mass similar to the taste disk of frogs(termed a taste disk-like cell mass). On the flat area of thetongue, the barrel-shaped taste buds of larval salamanders weretransformed into taste buds with a wider receptor area. Thebarrel-shaped taste buds decreased progressively during metamorphosis,while taste disk-like cell masses increased. Neuronal labelingwith an antibody to neuron-specific enolase and fluorescentcarbocyanine dye showed that the taste disk-like cell massesin metamorphosed salamanders were innervated by the glossopharyngealnerve (nerve IX). Nerve IX responded to taste stimulation aswell as mechanical stimulation applied to the rostral tongue.During metamorphosis the salamanders undergo transformationand rearrangement of taste organs on the tongue possibly asan adaptation to the terrestrial environment. Chem. Senses 22:535–545, 1997.     

The Search for Ways to Improve the Catalytic Activity of Encapsulated Horseradish Peroxidase

Biophysics - Abstract—We consider a previously proposed method for encapsulation of enzymes, which employs the layer-by-layer adsorption of oppositely charged polyelectrolytes onto composite...     

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development,axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.     

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.     

On the Fine Structure of Trypanosoma cruzi in Tissue Cultures of Pigment Epithelium from the Chick Embryo. Uptake of Melanin Granules by the Parasite*

Trypanosoma cruzi has been cultured in pigment epithelial cells of the iris from the chick embryo. Melanin granules, identical with those of the host cells were found in the intracellular, amastigote (leishmania) forms. In many of the intracellular forms cytostome-like structures were seen, often in intimate contact with the pigment granules of the host cells, which suggests the uptake of melanin granules through the cytostomes by the process of intracellular phagotrophy.     

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.