Fibroblast growth factor during mesoderm induction in the early chick embryo

A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.     

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.     

Stereological study of the early ultrastructural differentiation of chick embryo neuroepithelial cells during neurulation

The neuroectodermal cells of chick embryos have been analyzed during neurulation by stereological and morphometrical ultrastructural methods in an attempt to describe their cytometric evolution. A profound change of cellular form coefficient was observed which is related to the typical process of columnarization of these cells. At stages 7 and 8, the nucleus appeared round in shape, probably due to a loss of pressure of the vitelline inclusions. In this sense, the volume density of these inclusions falls during this period. There was also a significant increase of the nuclear surface density, the significance of which is discussed on the basis of the nucleo-cytoplasmic interchanges and the differentiation process. At the same time, an increase in the number of mitochondria was observed, which is related to the neural folding process. Simultaneously, the amount of rough endoplasmic reticulum increases, presumably related to the remarkable changes of the embryonic extracellular matrix.     

Acetylcholinesterase activity in neural crest cells of the early chick embryo

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.     

Hyaluronate-cell interactions during differentiation of chick embryo limb mesoderm

The mechanism of interaction of hyaluronate with the surface of cells from embryonic chick limbs was studied using cell cultures of mesoderm from various developmental stages. The mode of interaction of hyaluronate with the cell surface changed at the onset of mesodermal cell condensation prior to differentiation of cartilage and muscle. At this time hyaluronate binding sites appeared on the cells and continued to be present on differentiated chondrocytes but not on myotubes. Direct measurement of hyaluronate binding was made using stage 24 mesodermal cells and membranes isolated from cells derived from various limb stages. The stage 24 cells and membranes from stage 22, 24, and 26 cells exhibited hyaluronate binding, but not membranes from stage 19 mesoderm cultures. At stage 38, membranes from chondrocyte cultures exhibited the highest hyaluronate binding, and membranes from myoblasts and fibroblasts intermediate binding, whereas membranes from myotube-enriched cultures lacked binding activity. No significant competition of hyaluronate binding by chondroitin sulfate was observed. Occupied hyaluronate binding sites were measured by the displacement of radiolabeled cell surface hyaluronate with exogenous, unlabeled hyaluronate. Very little hyaluronate was displaced from mesodermal cells derived from the youngest embryos, namely, stage 19 or stage 20-21. However, greater than 50% of cell surface hyaluronate was displaced from stage 22 and 24 mesodermal cells. The addition of exogenous hyaluronate to stage 26 mesoderm, the stage of onset of cartilage differentiation, and to stage 38 chondrocytes resulted in displacement of large proportions of both hyaluronate and chondroitin sulfate. Addition of exogenous chondroitin sulfate did not cause displacement of significant amounts of cell surface hyaluronate or chondroitin sulfate. These results indicate the presence and developmental modulation of specific binding sites for hyaluronate on limb cells during their differentiation.     

Epiblast origin and early migration of neural crest cells in the chick embryo

Chick embryos carrying transplants labeled with tritiated thymidine demonstrate that the neural crest originates in the anterior epiblast, at the junction of areas destined for epidermis and neural tube. As the neural tube begins to fold and the axis lengthens, cells along this junction are drawn dorsomedially; at the seven-somite stage they begin to separate from the epithelium of the head, and migrate into the angle between the epidermis and the neural tube. The paraxial mesoderm already populating this angle originates in more posterior and medial portions of the epiblast than do the neural crest cells; after invagination at the primitive streak, it migrates anterolaterally, ventral to the ectoderm layer, until it too is folded dorsomedially into the angle between the epidermis and the neural tube.     

Morphological differentiation of mitochondria in the early chick embryo: a stereological analysis

The morphological evolution of mitochondria in three cell types of chick embryo in neurulation was analyzed by stereological methods. Mitochondria, showing a random distribution, were characterized by moderate electron-dense matrices and normal cristae. The numerical density of mitochondria significantly increased in the neuroectoderm and epiblastic cells while their volume density remained unchanged. The mitochondria in mesoderm cells were ellipsoidal (axial ratio 2:1) at stages 5 and 8 although they underwent an elongation in neuroectoderm and epiblastic cells (axial ratio from 2:1 to 1.6:1). The individual size of "average mitochondria" in the mesoderm cells was smaller than in other cell types. The total V/S (volume/surface) ratio of mitochondria decreased during neurulation. These morphological changes have been discussed emphasizing the possible metabolical role of mitochondria during morphogenesis.     

Regulation of programmed cell death during neural induction in the chick embryo

To study early responses to neural inducing signals from the organizer (Hensen's node), a differential screen was performed in primitive streak stage chick embryos, comparing cells that had or had not been exposed to a node graft for 5 hours. Three of the genes isolated have been implicated in Programmed Cell Death (PCD): Defender Against Cell Death (Dad1), Polyubiquitin II (UbII) and Ferritin Heavy chain (fth1). We therefore explored the potential involvement of PCD in neural induction. Dad1, UbII and fth1 are expressed in partly overlapping domains during early neural plate development, along with the pro-apoptotic gene Cas9 and the death effector Cas3. Dad1 and UbII are induced by a node graft within 3 hours. TUNEL staining revealed that PCD is initially random, but both during normal development and following neural induction by a grafted node, it becomes concentrated at the border of the forming neural plate and anterior non-neural ectoderm and downregulated from the neural plate itself. PCD was observed in regions of Caspase expression that are free from Dad1, consistent with the known anti-apoptotic role of Dad1. However, gain- and loss-of-function of any of these genes had no detectable effect on cell identity or on neural plate development. This study reveals that early development of the neural plate is accompanied by induction of putative pro- and anti-apoptotic genes in distinct domains. We suggest that the neural plate is protected against apoptosis, confining cell death to its border and adjacent non-neural ectoderm.     

Cardiac differentiation induced by dopamine in undifferentiated cells of early chick embryo

Evidence suggests that neurotransmitters can act as possible chemical signals involved in cell division and morphogenetic movements long before neurons appear in the embryo. However, whether they are playing a role in differentiation is now unknown. It was recently observed (M. Sarasa and S. Climent, 1987, J. Exp. Zool. 241, 181-190) that the neurotransmitter dopamine exerted a stimulating effect on cardiac differentiation in the chick in ovo. We show here that dopamine acts as a specific inducer of heart muscle differentiation in vitro. When cells of the gastrula of embryos treated with dopamine were dissociated and reaggregated, the aggregates obtained almost entirely underwent cardiac muscle differentiation. Also, when small postnodal pieces obtained from the most posterior region of the gastrula were cultivated in the presence of dopamine, they differentiated into myocardic tissue instead of following their fate map. Therefore, dopamine can trigger a process that both causes undifferentiated cells to differentiate into heart muscle and compels cells already determined to another way of differentiation to become myocardic tissue.     

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.     

Effect of the notochord on proliferation and differentiation in the neural tube of the chick embryo

After implantation of a notochord fragment lateral to the neural tube in a 2-day chick embryo, at 4 days the ipsilateral neural tube half was increased in size and axons left the neural tube in a broad dorsoventral area (van Straaten et al. 1985). This enlargement appears to coincide with an increased area of AChE-positive basal plate neuroblasts, as determined with scan-cytophotometry. The effect was ipsilateral and local: clear effects were seen only when the implant was localized less than 80 microns from the neural tube and over 120 microns from the ventral notochord. In order to investigate the expected enhancement of proliferation, the mitotic density and the number of cells at the site of the implant at 3 days was determined and the mitotic index calculated. All three parameters showed an increase. It was concluded that the cell cycle was shorter in the implant area relative to the control area, at least during the third day. At 4 days the number of cells was still increased, predominantly in the basal plate. It appeared that the numerical increase was for the larger part due to neuroblasts. The synergism of two notochords thus resulted in enhancement of proliferation and differentiation in the neural tube. It is suggested that the notochord merely regulates and arranges the surrounding sclerenchymal cells, which are the effective cells in the regulation of neural tube development.