Stage-specific gene expression in early chick embryo

Inhibition of DNA replication by aphidicolin in the chick morula interferes with its progression to a normal blastula and prevents induction of the first morphogenetic cell movements of primitive streak formation. Embryos in aphidicolin synthesize some polypeptides typical of blastula but do not display all the characteristic features of morula to blastula transition. Inhibition of DNA replication interferes with the sequential synthesis of maternally coded polypeptides and with the activation of the embryonic genome in the chick embryo.     

Molecular cloning and early expression of chick embryo SCO-spondin

SCO-spondin is a multidomain glycoprotein secreted by the subcommissural organ (SCO). It belongs to the thrombospondin type 1 repeat superfamily and has been identified in several vertebrate species. We report the cloning of the chick SCO-spondin ortholog and examine its temporal and spatial expression during early embryogenesis from Hamburger and Hamilton (HH) stage 12 to HH stage 21. Chick SCO-spondin cDNA contains a long open reading frame encoding a predicted protein of 5255 amino acids. Northern blot analysis has revealed SCO-spondin mRNA as a band of about 15 kb. Many conserved domains have been identified, including 27 thrombospondin type 1 repeats, 13 low-density lipoprotein receptor type A domains, one EMI domain (a cysteine-rich domain of extracellular proteins), three von Willebrand factor type D domains, and one cystine knot C-terminal domain. Whole-mount in situ hybridization enabled the first signal of mRNA expression to be detected at HH stage 17, exclusively in a thin area of the prosencephalon roof plate. During the following stages of development, SCO-spondin expression remained restricted to this region. The multidomain structure of SCO-spondin and its early expression suggest that it plays a role in developmental processes in the central nervous system.     

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.     

CEPU-1 expression in the early embryonic chick brain

We describe the expression pattern of CEPU-1, a cell adhesion molecule of the immunoglobulin superfamily, in the early chick embryo brain. An initially broad domain of expression, encompassing forebrain, midbrain and anterior hindbrain, is subsequently narrowed down to a ring-shaped domain at the midbrain-hindbrain boundary, co-localizing precisely with the expression of Wnt1 at the isthmus. In addition, CEPU-1 is expressed in the dorsal aspect of rhombomere 4 and its emigrating neural crest cells. Later in development, we also find CEPU-1 expression in other parts of the developing nervous system such as sensory ganglia and in the ventral aspect of forebrain, midbrain and hindbrain.     

Limbness in the early chick embryo lateral plate

In order for the limb to be useful in the evaluation of early determinants of morphogenesis, it is necessary to understand some of the characteristics associated with "limbness" and, more importantly at the beginning at least, it is necessary to know what regions of the early embryo exhibit limbness qualities. Previous investigators have assumed, without direct experimental evidence, that the flank does not have limbness qualities, even at early stages of development. However, there are a few studies suggesting that the early flank does possess limbness qualities. The purpose of the present study was to determine how extensively the qualities of limbness exist in the early chick embryo. Tissues from the future neck, wing, flank, and leg regions were grafted to host celoms and evaluated for their abilities to form limbs. Limbs developed from all four regions of stage 11-14 embryos, but after stage 14 only grafts from the wing and leg regions formed limbs.     

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.     

Immunohistological study of the expression of glucagon in dorsal pancreatic endoderm in the early chick embryo

An immunohistological study demonstrated that glucagon first appears in the dorsal pancreatic endoderm of the chick embryo at stage 16 of Hamburger and Hamilton during normal development. It was also shown that the self-differentiation potency of the isolated dorsal endoderm to express glucagon in vitro in the absence of adjoining tissues appears at stage 11.     

Lead-induced early lesions in the brain of the chick embryo

A single dose consisting of a solution of lead nitrate containing 1 mg lead in 0.1 ml distilled water was injected into the air chamber of chicken eggs on the tenth day of incubation. The injected embryos were killed 24, 48, or 72 hours after the injection. Control embryos were injected with a similar volume of distilled water or of an equimolar calcium nitrate solution on the tenth day and killed 72 hours after. Portions of the optic lobes were processed for their electron microscopic study. Multiple hemorrhagic foci appeared 24 hours after the administration of lead. Red blood cells were found to exit the blood vessels through the spaces between otherwise normal-looking endothelial cells; tight junctions were always found blocking the interendothelial spaces except during the migration of red blood cells through them. Vacuolization of the cytoplasm and mitochondrial swelling and disorganization were observed in the endothelial cells only during the third day after the injection. Similarly, histological and ultrastructural alterations of nerve cells were only found on the third day after the treatment. Hemorrhagic foci were never found in control embryos. Our observations are consistent with the idea that the hemorrhages constitute the primary lesion produced by lead. They also suggest that the hemorrhages are not the result of rupture of previously altered endothelial walls but occur by diapedesis through the interendothelial clefts.     

Regional specification of the endoderm in the early chick embryo

In the avian embryo, the endoderm, which forms a simple flat-sheet structure after gastrulation, is regionally specified in a gradual manner along the antero-posterior and dorso-ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA , a posterior endoderm marker, but not cSox2 , an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid-hindgut endoderm expressed cSox2 but not CdxA . At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2 , but anteriorly grafted presumptive mid-hindgut endoderm showed no changes in its posterior-specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid-hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid-hindgut.     

Acetylcholinesterase activity in the myotome of the early chick embryo

Summary The myotome of early chick embryos was investigated histochemically by means of the acetylcholinesterase (AChE) reaction.Light-microscopically, at the cervical level, the myotome was first recognized and AChE activity demonstrated at stage 13 (2 day-old embryo). Subsequently, the myotome elongated ventro-laterally along the inner surface of the dermomyotome and reached the ventro-lateral end of the dermomyotome at stage 17 to 18 (3 day-old embryo). AChE activity in the myotome showed subsequent increase in intensity during the course of development. The myotome consisted mainly of AChE-positive cells displaying enzymatic activity along the nuclear membrane and within the cytoplasm. In contrast, almost all cells of the dermomyotome and the interstitial cells were AChE-negative.Electron-microscopically, the myotome cells of the 2 day-old embryo and the cells in the dorso-medial portion of the myotome of the 3 day-old embryo were morphologically undifferentiated; AChE activity was detected in the nuclear envelope and in single short profiles of the endoplasmic reticulum (ER). On the other hand, in the 3 day-old embryo the cells in the ventro-lateral portion of the myotome showed AChE activity in the nuclear envelope, numerous profiles of the ER and some Golgi complexes. These AChE-positive cells were regarded as developing myogenic cells based on their morphological characteristics.The present findings indicate (i) that the appearance of AChE activity in the cytoplasm is the first sign of the differentiation of myogenic cells, and (ii) that in these myogenic cells the increase in AChE activity is based on the development of the ER.     

Tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.