Conflicting data on intervillous circulation in early pregnancy.

According to classic embryological textbooks intervillous circulation is established early in the first trimester. This process starts with trophoblastic invasion of the decidua in which proteolytic enzymes facilitate the penetration and erosion of the adjacent maternal capillaries with formation of the lacunae. After the lacunar or previllous stage trophoblast invades deeper portions of endometrium with belonging spiral arteries. This gradual process finishes with direct opening of the spiral arteries in the intervillous space under the fully developed placenta. This classic concept of establishment of the intervillous circulation was challenged in 1987 and 1988 by the experiments of HUSTIN and SHAAPS. The authors believed that blood flow in the intervillous space is absent in incompletely development before 12 weeks of gestation. After the introduction of the new generation of far more sensitive color Doppler devices in the last few years, our group and several others reported a positive finding of intervillous circulation during the first trimester of pregnancy.

Ventral ectodermal ridge and ventral ectodermal groove: two distinct morphological features in the developing rat embryo tail.

The ventral ectodermal ridge (VER) is a thickening of the surface ectoderm on the ventral side of the embryonic tail which resembles the apical ectodermal ridge of the limb bud. The morphological characteristics of the ventral part of the embryo tail were investigated in 10.5- to 14-day rat embryos by light microscopy of serial semithin sections and by scanning and transmission electron microscopy. In 10.5- to 11.5-day embryos the thickening of the ventral surface ectoderm includes the complete ventral midline of the tail and can be divided into two parts. The posterior part is elevated and represents the ventral ectodermal ridge. The anterior part is, in contrast to the ridge, concave, and we have termed it the ventral ectodermal groove (VEG). The cloacal membrane is located at its anterior end. Contacts between the VER and the mesenchymal cells are visible until an intact basal lamina is formed at 11.5 days. Similarly, the VEG is connected by elongated cell processes with the ventral part of the tail gut. Gap junctions are present between the apical parts of ridge and groove cells. The VEG flattens and disappears in 12-day embryos. At this stage the ridge is at its maximum height, simultaneously undergoing extensive cell death. The VER is no longer visible in 14-day rat embryos.

Morphological evidence for secondary formation of the tail gut in the rat embryo.

The secondary body formation is a developmental mechanism occurring in the caudal part of the embryo in which embryonic structures arise from a mass of mesenchymal cells without previous formation of germ layers. The formation of the tail gut by this mechanism was investigated on transverse serial semithin and ultrathin sections of 12-, 13-, 14- and 15-day rat embryo tails. The tail gut, together with the tail portion of the notochord, originates from an axial mass of condensed mesenchymal cells named tail cord. Formation of the tail gut involves the appearance of large intercellular junctions among tail cord cells, and rearrangement of these cells around a newly formed lumen. Mesenchymal characteristics of these cells are gradually lost, and they simultaneously acquire the morphology of epithelial cells. Some cells of the tail cord, located ventral to the tail gut, do not participate in the tail gut formation and form a separate mass of cells without any definitive morphogenetic fate. This surplus group of cells is first evident in 12-day embryos, and it increases in mass during the following 3 days. In 15-day embryos, after the tail gut has completely disappeared, the surplus cells represent all that remains of the tail cord. The mesenchymal-epithelial transformation of the tail cord cells into the cells of the tail gut, and the appearance of the surplus cells, could be considered as the main morphological arguments for the secondary formation of the tail gut.

Morphogenetic features in the tail region of the rat embryo.

The secondary (direct) body formation is a mechanism of development in which morphogenesis of various organs occurs directly from a mass of undifferentiated mesenchymal cells (blastema) without previous formation of germ layers. It is characteristic of the posterior end of the embryonic body, i.e. of the tail bud of tailless and the tail of tailed mammals. Development of the neural tube occurring by this mechanism (secondary neurulation) has been previously explained. We investigated the morphogenetic mechanism by which two other axial structures in the rat tail develop: the tail gut and the notochord. Both structures develop from an axial condensation of undifferentiated mesenchymal cells (tail cord) of tail bud origin. The tail gut forms in a similar way to the secondary neural tube: cells in the ventral part of the tail cord elongate, acquire an apicobasal polarity and form a rosette-like structure around a lumen in the centre. The notochord forms by detachment of a group of cells of the tail cord dorsally to the developing tail gut. The peculiarities of this morphogenetic mechanism in comparison with those in other parts of the embryo are discussed. Causal (including evolutionary) explanations of this mechanism are ruled out.

Differentiation of the secondary elastic cartilage in the external ear of the rat.

The cartilage in the external ear of the rat belongs to the group of secondary cartilages and it has a unique structural organization. The chondrocytes are transformed into typical adipose cells, the proteoglycan cartilage matrix is reduced to thin capsules around the cells and the rest of the extracellular matrix is occupied by a network of coarse elastic fibers. It appears late in development (16-day fetus) and needs more than one month for final development. The differentiation proceeds in several steps which partly overlap: the appearance of collagen fibrils, elastin fibers, the proteoglycan matrix, and the adipose transformation of chondrocytes. The phenotype of this cartilage and the course of its differentiation are very stable, even in very atypical experimental environmental conditions. The only exceptions are explants in organ culture in vitro and perichondrial regenerates. In these conditions the development of elastic fibers is slow and poor while the production of the proteoglycan matrix is abundant. The resulting cartilage then displays structural characteristics of hyaline cartilage rather than those of the initial elastic one.

Zagreb research collection of human brains for developmental neurobiologists and clinical neuroscientists.

The aim of this paper was to offer for the first time a selective and systematic description of the "Zabreb Neuroembryological Collection" of human brains and to illustrate the major results of our research team. Throughout these 16 years of continuous and systematic research, we have applied different techniques for demonstrating the cytoarchitectonics (Nissl staining), neuronal morphology (Golgi impregnation), synaptogenesis (EM analysis), growing pathways (acetylcholinesterase histochemistry) and transmitter-related properties of developing neuronal populations (immunocytochemistry and acetylcholinesterase histochemistry) on several hundred human brains ranging in age from the 5th week post-conception to 90 years. The combination of classical and modern research techniques applied to the constantly growing developmental collection, as well as the continuous evaluation of our data in the light of experimental work in non-human primates, has led to the discovery of an early synaptogenesis within the human cortical anlage and hitherto undescribed transient subplate zone; our results also provided the first comprehensive evidence concerning the timing and pattern of development of afferent fiber systems in the human cortex. All this enabled us to offer a well-documented and coherent reconstruction of major histogenetic events in the human brain. We concluded that structural remodeling and reorganization of the brain, from the transient patterns of the fetal organization through the postnatal phase of transient overproduction of circuitry elements to the final maturation, is the crucial principle of development. Fetal neuronal elements (afferents, synapses and postsynaptic neurons) display transient patterns of laminar, vertical and modular organization and transient cellular interactions and competition in the subplate zone are crucial for the formation of cortical connections. The elucidation of the nature and timing of these histogenetic reorganizational events in the human brain represents the first step towards determining the neurobiological basis of the emergence of behavior, neural functions and cognition in human fetuses, infants and children, which takes place during perinatal and early postnatal life.

Origin of the notochord in the rat embryo tail.

The origin of the notochord in the rat tail was investigated on transverse serial semi-thin and ultra-thin sections of 12- and 13-day embryo tails. It was found that the notochord develops from a mass of condensed mesenchymal cells which is located ventrally to the secondary neural tube, and which subsequently splits into a) a thin cord which becomes notochord and b) a thick portion which gives rise to the tail gut. By analogy with the secondary neurulation and the secondary gut formation, one might therefore speak of a secondary notochord formation in the tail. It occurs in close relationship with the formation of the tail gut.

Development of Cajal-Retzius cells in the human auditory cortex.

We analysed the differentiation and areal distribution of Cajal-Retzius (C-R) cells in the human auditory cortex using acetylcholinesterase (AChE) technique on specimens ranging between 10 weeks of gestation (w.) and the 3rd postnatal month. AChE-reactive cells appear in the marginal zone of the prospective auditory cortex as early as 10 1/2 weeks of gestation. Analysis of primary and associative auditory cortex in subsequent stages of gestation and during early postnatal life reveals an age-dependent decrease in cell-packing density of C-R cells and an increase in thickness of the marginal zone. Large AChE-reactive cells were readily found in the early postnatal cortex. These and our previous data on the human frontal associative cortex demonstrate the presence of AChE-reactive C-R cells in both primary and associative cortical areas during late fetal and early postnatal life. The postnatal changes in the morphology and distribution of AChE-reactive C-R cells may serve as cellular parameters of the postnatal cortical maturation in man.

On the ultrastructure of the developing elastic cartilage in the rat external ear.

Selected ultrastructural features of chondrocytes and the extracellular matrix in the developing elastic cartilage of the external ear were studied in rat fetuses and young animals. The cytoplasmic lipid droplets were first observed in the 19-day fetus. They increase in number and size during the first post-natal week. The elastogenesis proceeds in the sequence: oxytalan fibers (17-day fetus), elaunin fibers (1-day rat), elastic fibers (5-day rat). Intermediary stages between the randomly oriented individual microfibrils and bundles of microfibrils (oxytalan fibers) were also observed.

Morphogenetic behaviour of the rat embryonic ectoderm as a renal homograft.

Halves of transversely or longitudinally cut primary ectoderm of the pre-primitive streak and the early primitive streak rat embryonic shield developed after 15-30 days in renal homografts into benign teratomas composed of various adult tissues, often in perfect organ-specific associations. No clear difference exists in histological composition of grafted halves of the same embryonic ectoderm. The primary ectoderm of the pre-primitive streak rat embryonic shield grafted under the kidney capsule for 2 days displayed an atypical morphogenetic behaviour, characterized by diffuse breaking up of the original epithelial layer into mesenchyme. Some of these cells associated into cystic or tubular epithelial structures. The definitive ectoderm of the head-fold-stage rat embryo grown as renal homograft for 1-3 days gave rise to groups of mesenchymal cells. These migrated from the basal side of the ectoderm in a manner which mimicked either the formation of the embryonic mesoderm or the initial migration of neural crest cells. This latter morphogenetic activity was retained in the entire neural epithelium of the early somite embryo but was only seen in the caudal open portion of the neural groove at the 10- to 12-somite stage. The efficient histogenesis in grafts of dissected primary ectoderm and the atypical morphogenetic behaviour of grafted primary and definitive rat embryonic ectoderm were discussed in the light of current concepts on mosaic and regulative development, interactive events during embryogenesis and positioning and patterning of cells by controlled morphogenetic cell displacement.

Ultrastructure of elastic cartilage in the rat external ear.

The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy. The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40--100 nm) vesicles. Scarce glycogen granules and bundles of 6--7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant. Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments. The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a "stellate reticulum". The complex of these two structures appears in the scanning electron micrographs as a a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.