Aristotle (384-322 BC): the beginnings of Embryology.

Aristotle made important contributions to many fields-biology, physics, metaphysics, logic, ethics, rhetoric, psychology, aesthetics, poetry- that are now cultivated by specialized experts, but he never lost sight of the aim of unifying knowledge, of understanding the world as an organized whole. Aristotle was the first to combine wet, field biology with daring cosmological thinking. He is the father of natural history and the first embryologist known to history. Aristotle's classic treatises History of Animals/Περί ζῴων á¼±στορίαι, and On the Generation of Animals/ Περί ζῴων γενέσεως "enjoyed for more than fifteen hundred years an authority altogether without parallel". Over the last four decades, the introduction of molecular techniques has gradually overturned the entire structure of the biological sciences. Biology, initially a science of inventory and classification in the hands of the 19th-century comparative naturalists, has become a science of codes and regulatory circuits. Aristotle was the first to codify laws of pure logic, and so he founded what is today known as ' proof theory' in mathematics. Aristotle was an inveterate collector and a classifier, the master scientist of his time. His main concern was to classify "the ultimate furniture of the world", under basic headings and categories, a powerful human strategy to organize knowledge for comprehension and action. This was part of Aristotle's attempt to create a theory of reality, one strongly opposed to Plato's otherworldly doctrine of the ideal 'forms'. To many generations of thinkers in the great era of Scholastic philosophy, Aristotle was known simply as "The Philosopher".

Hyaluronan receptor CD44: developmentally regulated expression and role in the early chick embryo.

CD44 is a membrane glycoprotein and is the main receptor for hyaluronan. We studied CD44 expression and spatio-temporal distribution by RT-PCR and immunofluorescence, and used an anti-CD44 blocking antibody to perturb CD44-depended signalling programs in the early chick embryo. The intense CD44 levels we detected in the morula embryo (XI) were of particular interest, suggestive of a maternally stored transcript. Intriguingly, the early presence of CD44 seemed to be essential for the rapid synthesis of hyaluronan. At stage XIII (blastula), CD44 expression was intense in the epiblast and hypoblast. During gastrulation (HH3-4), the cells ingressing into the primitive groove and migrating, and the blood islands, expressed CD44 intensely. At HH8, the folding neural plate showed polarity regulation of CD44 expression, and expression was also intense in neural crest, notochord, and blood islands. During early organogenesis, CD44 was expressed intensely in the developing cranial and caudal neural tube that showed polarity regulation, in optic stalks, otic vesicles, pre-and migratory neural crest cells, ganglia, notochord, pharynx, gut, liver, aortae, heart, somites, vascular area, amnion and chorion, and was distinct in extracellular matrix of cranial neural tube and otic vesicle lumens. Antibody-mediated perturbation of CD44 function resulted in unorganized extracellular matrix, loss of tissue spaces, grossly abnormal notochord, intermingling of clumped neuroectoderm and mesenchyme, absence of somites and blood vessels and inhibition of neural crest cell emigration. CD44 has various pivotal roles in matrix integrity and tissue patterning, consistent with its known biochemical features and interactions with hyaluronan, growth factors, receptors and other signaling molecules.

Decorin developmental expression and function in the early avian embryo.

Decorin, a proteoglycan, interacts with extracellular matrix proteins, growth factors and receptors. Decorin expression and spatio-temporal distribution were studied by RT-PCR and immunofluorescence, while decorin function was examined by blocking antibodies in the early chick embryo. Decorin was first detectable at stage XIII (late blastula). During gastrulation (stage HH3-4), decorin fluorescence was intense in epiblast cells immediately adjacent to the streak, and in migrating cells. Decorin fluorescence was intense in endoderm and strong at mesoderm-neural plate surfaces at stage HH5-6 (neurula). At stage HH10-11 (12 somites), decorin fluorescence was intense in myelencephalon and then showed distinct expression patterns along the myelencephalon axes by stage HH17. Decorin fluorescence was intense in neural crest cells, dorsal aorta, heart, somite and neuroepithelial cells apposing the somite, nephrotome, gut and in pancreatic and liver primordia. Antibody-mediated inhibition of decorin function affected the head-to-tail embryonic axis extension, indicating that decorin is essential for convergent extension cell movements during avian gastrulation. Decorin was also essential for retinal progenitor cell polarization, neural crest migration, somite boundary formation and cell polarization, mesenchymal cell polarization and primary endoderm displacement to the embryo periphery. The embryonic blood vessels were deformed, the dorsal mesocardium was thinned and the cardiac jelly was abnormally thickened in the heart. Decorin is known to modulate collagen fibrillogenesis, a key mechanism of matrix assembly, and cell proliferation. Decorin also appears to be essential for the coordination of cell and tissue polarization, which is an important feature in organ patterning of the embryo.

Spatial and temporal expression of perlecan in the early chick embryo.

Perlecan is a major heparan sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules. The expression and spatiotemporal distribution of perlecan was studied by RT-PCR, immunoprecipitation and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Combined RT-PCR and immunohistochemistry demonstrated the expression of perlecan as early as stage X and its presence may be fundamental to the first basement membrane assembly on the epiblast ventral surface at stage XIII (blastula). Perlecan fluorescence was intense in the cells ingressing through the primitive streak and was strong lining the epiblast ventral surface lateral to the streak at stage HH3-4 (gastrula). At stage HH5-6 (neurula), perlecan fluorescence was low in the neuroepithelium and stronger in the apical surface of the neural plate. At stage HH10-11 (12 somites), perlecan fluorescence was intense in the neuroepithelium and was then essentially nondetectable in the neuroepithelium, and the intensity had shifted to the basement membranes of encephalic vesicles by stage HH17. Perlecan immunofluorescence was intense in neural crest cells, strong in pharyngeal arches, intense in thymus and lung rudiments, intense in aortic arches and in dorsal aorta, strong in lens and retina and intense in intraretinal space and in optic stalk, strong in the dorsal mesocardium, myocardium and endocardium, strong in dermomyotome, low in sclerotome in somites, intense in mesonephric duct and tubule rudiments, intense in the lining of the gut luminal surface. Inhibition of the function of perlecan by blocking antibodies showed that perlecan is crucial for maintaining basement membrane integrity which mediates the epithelialization, adhesive separation and maintenance of neuroepithelium in brain, somite epithelialization, and tissue architecture during morphogenesis of the heart tube, dorsal aorta and gut. An intriguing possibility is that perlecan, as a signaling molecule that modulates the activity of growth factors and cytokines, participates in the signaling pathways that guide gastrulation movements and neural crest cell migration, proliferation and survival, cardiac cell proliferation and paraxial mesoderm (somitic) cell proliferation and segmentation.

Entactin and laminin gamma 1-chain gene expression in the early chick embryo.

The expression patterns of entactin and laminin gamma1 chain genes were examined by in situ hybridization of their mRNAs in the early chick embryo from stage X (morula) to stage HH10-11 (10-somites). The entactin and laminin gamma1 transcripts were found in abundance in the embryo at stage X. Entactin polypeptides were detected in embryos at stage X by immunoprecipitation. The expression of the laminin transcripts was intense and of entactin milder in the epiblast and in the hypoblast of embryos at stage XIII (blastula). During gastrulation (stage HH3-4), the laminin gamma1 and entactin cRNAs gave strong signals in the cells ingressing through the primitive streak, in the migrating mesenchymal cells and the cells of the lower layer. At the neurula stage (stage HH5-6), punctate groups of cells expressed laminin gamma1 strongly in the neural ectoderm, while the signal of expression was milder and more uniform in chordamesoderm. The entactin cRNAs gave a strong punctate pattern of mRNA expression in the neural ectoderm, in mesoderm and in endoderm in embryos at the late gastrula stage (HH4), but mRNA expression was mild in the neural plate and in mesoderm and gave no signal in endoderm and lateral ectoderm in embryos at stage HH6 (neurula). At the 10-somite stage, the laminin gamma1 cRNAs gave strong signals in the neural tube and in neural crest cells migrating along the neural tube ventrally and low signals in ectoderm, intense signals in the myotome and milder signals in the dermatome and sclerotome of somites and intense signals in the mesonephric tubules. The punctate pattern of entactin expression was notable in cells at all stages studied. Ubiquitous expression of laminin gamma1 and entactin genes during the morula and blastula stages becomes restricted to specific cell populations as the first cell commitments start.

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo.

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.

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.

Monensin inhibits the first cellular movements in early chick embryo.

In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.

Gene expression in chick morula.

The polypeptides synthesized during the morula stage in the chick embryo are insensitive to transcriptional inhibition by α-amanitin. Protein synthesis seems to depend predominantely, if not exclusively, on the recruitment of maternal mRNA, rather than on embryonic gene expression in chick morula. The morula embryo expresses the heatshock polypeptides when stressed at 43°C. The heat-induced polypeptides are isoforms of polypeptides that are synthesized normally. These polypeptides are α-amanitin sensitive and appear to mark the first major expression of the embryonic genome in the chick embryo.

Differential heat shock gene expression in chick blastula.

The component areas of chick blastula show differential expression of heat shock genes. The area opaca (ao), marginal zone (mz) and central area (ca) components of the epiblast display distinct quantitative and minor qualitative differences in the heat-induced and heat-repressible proteins, but are clearly different from the primary hypoblast (endoderm) in their expression of a given stress protein (hsp) as a response to heat shock. The major proteins synthesized in the component areas of epiblast in response to heat shock include hsp 18, 24, 70 and 89 Kd. Two-dimensional electrophoresis shows that each of these proteins consists of multiple charged species. The hypoblast expresses only hsp 70 Kd at non-significant levels and shows marked inhibition in the level of synthesis of heat-shock-repressible proteins. Heat shock during the blastula stage results in an increase in the size of the blastoderm and disrupts normal embryonic development. The heat shock genes provide an important molecular marker, which attests to regional specification in the chick blastula.

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis.

Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[35S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.

Determination of Erythroid Cells in Unincubated Chick Blastoderm: stem cells/erythrocytes/unincubated chick blastoderm/critical mass).

Portions the size of 1/6 to 1/32 part of unincubated blastoderm cultured in an albumen-salineagar solid medium form erythroid cells under conditions which suppress normal co-ordinated movements for mesoderm induction. Anterior and posterior regions of unincubated blastoderm have the same potentiality to form erythroid cells. The marginal zone seems to be the contributor of prospective erythroid cells. Portion 1/16 part of unincubated blastoderm forms morphologically distinct erythroid cells of the primitive and definitive lines as in normal development in ovo. It seems progenitor cell(s) is pre-programmed to a particular pattern of differentiation and/or includes differentiation to various erythroid cell types as an obligatory step. This system provides novel experimental possibilities in the study of erythroid cell determination and differentiation.