Glycosaminoglycans in early chick embryo

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.     

Induction of increase in intracellular calcium concentration of embryonic cells and acceleration of morphogenetic cell movements during amphibian gastrulation by a 50-Hz magnetic field

The influence of an alternating electromagnetic field (EMF) on early development of amphibian embryos was examined. When the embryos developed under the influence of a low-frequency EMF (50 Hz, 5-30 mT), the rate of early development was accelerated. The effect of EMF was exerted preferentially at the gastrula stage, and the period of gastrulation was shortened. Histological observations showed that EMF promoted morphogenetic cell movements during the gastrulation. The concentration of intracellular free Ca2+ ([Ca2+]i) in the embryonic cells under the influence of EMF was analyzed using Fura-2, an indicator of the intracellular concentration of calcium ions. The influence of EMF on [Ca2+]i was analyzed in embryonic cells isolated from blastula, gastrula, and neurula, EMF increased a [Ca2+]i particularly in the cells isolated from gastrula. Our results suggest that EMF specifically increased the [Ca2+]i of gastrula cells, thereby, accelerating the rate of morphogenetic cell movements during gastrulation.     

Immunohistological localisation of monoclonal antibody R 24-recognized ganglioside Gilac2* in early chick embryos

The spatio-temporal cellular expression and biosynthesis of ganglioside Glac2 was investigated in early chick embryogenesis. For demonstration of embryonic Glac2-biosynthesis, chick embryos of stage 0 and of stages 4-5 were incubated in vitro in the presence of radioactive sugar precursors. It was found that chick embryos synthesize Glac2 as early as at the blastula stage as well as at the gastrula stage, both within the area pellucida and the area opaca. In contrast to the biosynthetical findings immunohistochemical staining of the chick embryos at various stages by aid of the mouse monoclonal antibody (mAb) R 24, specific for the immunoepitope NeuAc alpha, 8NeuAc alpha, 3Gal beta less than, as present on the ganglioside Glac2, revealed a spatio-temporal cellular pattern of expression of this ganglioside in early chick embryos. Immunohistochemical staining of the chick embryo at stage 0 shows that all cells of the embryo, the extraembryonic epiblast and the yolk endoderm included, are mAb R 24-positive. At the intermediate streak stage (stage 3), the cranial part of the deep layer, the so-called endophyll, is strongly mAb R 24-positive, whereas at the end of gastrulation (stage 5), mAb R 24-recognized epitopes appear to be restricted to a narrow band of deep-layer cells in the endophyllic crescent and to the yolk endoderm of the area opaca. At this stage, no labelling by the antibody is observed in cell layers of the future embryo. The beginning of neurulation (stage 7) is characterized by the expression of the mAb R 24-recognized epitope in the notochord, whilst the deep layer in the cranial part of the neural fold still expresses this epitope. No ecto- or mesodermal structures are stained by the antibody at this developmental stage. During further development (stage 12 and 13), mAb R 24-reactivity is restricted to the cranial part of the embryo with a preferential staining of cells of endodermal origin. At these stages, the notochord expresses mAb R 24 binding sites only in its cranial region. The spatial and temporal correlation between the presence of mAb R 24-recognized epitopes and the morphogenetic positioning of tissues may be indicative for a possible role of the ganglioside Glac2 in corresponding cellular interactions.     

Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge

In the gastrula stage embryo, the epiblast migrates toward the primitive streak and ingresses through the primitive groove. Subsequently, the ingressing epiblast cells undergo epithelial-mesenchymal transition (EMT) and differentiate into the definitive endoderm and mesoderm during gastrulation. However, the developmental mechanisms at the end of gastrulation have not yet been elucidated. Histological and genetic analyses of the ventral ectodermal ridge (VER), a derivative of the primitive streak, were performed using chick and mouse embryos. The analyses showed a continued cell movement resembling gastrulation associated with EMT during the early tailbud stage of both embryos. Such gastrulation-like cell movement was gradually attenuated by the absence of EMT during tail development. The kinetics of the expression pattern of noggin (Nog) and basal membrane degradation adjacent to the chick and the mouse VER indicated a correlation between the temporal and/or spatial expression of Nog and the presence of EMT in the VER. Furthermore, Nog overexpression suppressed EMT and arrested ingressive cell movement in the chick VER. Mice mutant in noggin displayed dysregulation of EMT with continued ingressive cell movement. These indicate that the inhibition of Bmp signaling by temporal and/or spatial Nog expression suppresses EMT and leads to the cessation of the ingressive cell movement from the VER at the end of gastrulation.     

Serum protein synthesis in the early chick embryo

Isolated yolk-sacs of chick embryos secreted serum proteins when incubated in buffered chick Ringer's solution. The presence of serum transferrin, two embryo-specific alpha-globulins, and a prealbumin were demonstrated by acrylamide gel analysis. Yolk-sacs from embryos explanted at 11-13 somites (40 hr preincubation) and cultured for 48 hr secreted in addition a protein with the mobility of serum albumin. Incubation of yolk-sacs in the presence of radioactive valine indicated that serum proteins were synthesized as early as the primitive streak stage. By incubating isolated yolk-sacs and embryos from 48-hr explants in the presence of radioactive valine, the synthesis of serum proteins was found to be restricted to the yolk-sac at this stage of development. Culturing explants on various nutrient proteins as well as protein starvation medium altered the relative synthesis of several serum proteins. We have proposed that morphological and biochemical changes in embryos resulting from altered nutrition may be mediated by the proteins of the serum.     

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.     

Cardiac progenitor migration and specification

The heart is the first organ to function during vertebrate development and cardiac progenitors, are among the first cell lineages to be established from mesoderm cells emerging from the primitive streak during gastrulation. Cardiac progenitors have been mapped in the epiblast of pre-streak embryos. In the early chick gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. However, migration routes of cardiac progenitors have never been directly observed within the embryo and the factor(s) controlling their movement are not known. Furthermore, it is not understood how signals controlling cell movement are integrated with those that determine cell fate. Long-term video microscopy combined with GFP labelling and image processing enabled us to observe the movement patterns of prospective cardiac cells in whole embryos in real time. Embryo manipulations and the analysis of explants suggest that Wnt3a plays a crucial role in guiding these cells through a RhoA dependent mechanism involving negative chemotaxis. Wnt3a is expressed at high levels in the amniote primitive streak and ectopic signalling activity caused wider movement trajectories resulting in cardia bifida, which was rescued by dominant-negative Wnt3a. Our studies revealed Wnt3a-RhoA mediated chemo-repulsion as a novel mechanism guiding cardiac progenitors. This activity can act at long-range and does not interfere with cardiac cell fate specification.     

Nerve growth factor induces neural differentiation in undifferentiated cell of early chick embryos

Nerve growth factor (NGF) induced differentiation in postnodal pieces (PNPs) of stage 4 chick embryos. This induction was highly selective for neural tissue; no other structures developed in the NGF-treated PNPs. Furthermore, the number of PNPs showing neural differentiation was dependent on the concentration of NGF, but there was no correlation between the concentration of NGF (5-100 ng/ml) and extent of neuralization. The neural inducing capacity of NGF could be abolished by anti-NGF antibody. NGF-induced neural differentiation was accompanied by elevated intracellular levels of cyclic AMP. Exogenous cyclic AMP (175 micrograms/ml) was able to stimulate neural differentiation but, unlike NGF, induced other structures (e.g., notochord and pulsatile tissue). Overall results suggest that cells from chick embryos at developmental stages much earlier than previously thought are responsive to NGF and NGF or a a closely related substance may serve as a neural inducer in the chick embryo.     

Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos

BACKGROUND: Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. RESULTS: A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. CONCLUSIONS: Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.     

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.     

Activation of the serotonergic system in chick spinal cord during hatching.

The objectives of the present study were to determine the levels of serotonin (5-HT), its major catabolic metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) in chick spinal cord before, during, and after hatching and also to determine if changes in the levels of these chemicals are directly related to the hatching behavior. The levels of 5-HT, 5-HIAA, and NE were measured by high performance liquid chromatography with electrochemical detection in whole spinal cords of 20-day-old "pre-hatching" embryos, 21-day-old "normal hatching" embryos, 0-day-old "post-hatching" chicks, and 0-day-old "glass egg hatching" chicks. NE was measured but no significant differences were found in NE levels among experimental groups. The concentration of 5-HT was elevated in chick embryo spinal cords during normal hatching compared to pre-hatching embryos and post-hatching chicks. The concentration of 5-HIAA was elevated during and after normal hatching compared to pre-hatching embryos. However, neither 5-HT nor 5-HIAA levels were found to be elevated in chick spinal cords during glass egg hatching compared to pre-hatching embryos or post-hatching chicks. Therefore, there appears to be an activation of the serotonergic system in chick spinal cord related to the specific event of hatching but this activation is not directly related to the movements common to both hatching and glass egg hatching.     

The cholinergic system in the early development of chickens. 1. Choline acetyltransferase and acetylcholinesterase as the key regulators of acetylcholine metabolism

Acetylcholine esterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activities were studied in the early chick embryos. Gastrulation is accompanied by a sharp increase in the AChE activity which was most pronounced in anterior hypoblast. Three molecular of AChE (4.7, 6.8 and 10.9 S) were identified in the extract of chick embryos using a sucrose density gradient centrifugation. The CAT activity remained unchanged during gastrulation but increased twice at the end of gastrulation.     

Differential expression of D(1A) and D(1B) dopamine receptor mRNAs in the developing avian retina

In the chick retina, the D1 dopaminergic system differentiates very early, as shown by receptor-mediated increases in intracellular cyclic AMP concentration and the presence of [(3)H]SCH23390-specific binding sites. Here, we characterized, by RT-PCR, the expression of defined D1 receptor subtypes D(1A), D(1B), and D(1D) during the development of the chick retina. Total RNA was extracted from retinas of 6-day-old embryos (E6) to 1-day-old hatched chickens and reverse-transcribed. The resulting cDNA was amplified using D(1A)-, D(1B)-, or D(1D)-specific primers, and the PCR-amplified products were analyzed by electrophoresis. The fragment corresponding to D(1A) receptor was detected in developing retina as early as E7, whereas the fragment corresponding to D(1B) was observed starting around E10. No PCR product corresponding to D(1D) was observed in the retina, although it was detected in chick brain. As synaptogenesis in chick retina begins after E11 and [(3)H]SCH 23390 D1 binding sites increase after this stage, the present results show that expression of D(1B) receptor increases during synaptogenesis, whereas D(1A) is the receptor subtype associated with the D1-like actions of dopamine early in retina development.     

Whole-embryo culture of E5.5 mouse embryos: development to the gastrulation stage

This study reports establishment of an in vitro culture system for E5.5 mouse embryos that supports development to the gastrulation stage and allows the use of experimental approaches to study gastrulation during mouse embryogenesis. Recent experiments suggest that the extraembryonic tissues may play a critical role for gastrulation from as early as E5.5. To apply whole embryo culture to E5.5 embryos and analyze gastrulation, it is essential to optimize the conditions so that most of the embryos develop to the gastrulation stage in culture. For this purpose, we established a protocol in which embryos were isolated using micromanipulator and cultured with 50-75% rat serum. Although cultured embryos tended to grow a larger extraembryonic portion, more than 80% of them developed the primitive streak and induce mesoderm, which corresponds to the mid-streak stage.     

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos. © 1995 Wiley-Liss, Inc.     

The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

Expression of class I histone deacetylases during chick and mouse development

Histone deacetylases (HDACs) are a family of enzymes which regulate the acetylation state of nucleosomal histones, as well as non-histone proteins. By altering local chromatin architecture, HDACs play important roles in shaping cell differentiation and morphogenesis. Expression of class I HDACs during early chick development has so far not been analyzed. Here, we report the expression profile of chick class I HDACs from the onset of gastrulation (HH2) to day 4 of development and compare it to relevant stages during mouse development. Visualized by in situ hybridization to whole mount embryos and tissue sections, we found tissue-specific overlapping temporal and spatial expression domains for all four class I HDACs in chick and mouse, although species-specific differences could be identified. All class I HDACs in both species are highly expressed in the developing brain. In particular, HDAC1 is expressed at sites of anterior and posterior neural tube closure most obvious in the hot spot-like expression of HDAC1 in HH12 chicken embryos. A significant species-specific spatio-temporal expression pattern was observed for HDAC8. Whereas HDAC8 is exclusively found in fore- and midbrain regions during early mouse embryogenesis, the chick ortholog shows an expanded expression pattern, suggesting a more diversified role of HDAC8 in the chick system. Our results present a basis for further functional analysis of class I HDACs in chick development.     

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.