The proximal border of the human respiratory unit, as shown by scanning and transmission electron microscopy and light microscopical cytochemistry.

The epithelial cell types present in respiratory (= distal alveolarized) and terminal (= distal nonalveolarized) bronchioles in adult human lung were characterized with scanning and transmission electron microscopy (SEM, TEM) and light microscopic cytochemistry, using specific antibodies against surfactant protein SP-A and mucins, and Alcian blue/periodic acid-Schiff (AB/PAS) staining. In the respiratory bronchiole, two epithelial cell populations share the same basal lamina: one pseudostratified columnar with ciliated, secretory, and basal cells and the other predominantly simple cuboid with some interspersed flat (type I) cells. The columnar secretory cells show the ultrastructure of mucous cells. Light microscopically, they react with mucin antibodies and contain primarily periodate-reactive acid mucins. The mucous cells are the distal secretory cells described by Clara (1937). The cuboid cells are identified as type II (precursor) cells based on ultrastructural criteria for embryonic type II cells (Ten Have-Opbroek et al., 1988a, 1990a), including a cuboid cell shape, a large and roundish nucleus, rough and smooth endoplasmic reticulum (ER), osmiophilic multivesicular bodies, and dense bodies. These dense bodies in turn frequently exhibit--like those in embryonic type II cells--internal vesicles or lamellae, variability in size and shape, a specific relationship to ER and a widespread cytoplasmic distribution. Finally, the cuboid cells show a cytoplasmic staining pattern for SP-A. The terminal bronchiole is lined by the columnar cell population. In the respiratory bronchiole, the columnar (bronchial) and cuboid (alveolar) cell populations occupy distinctly different zones (pulmonary artery zone versus remaining wall). The alveolar part of the respiratory bronchiole (called alveolar tubule) defines the proximal border of a true respiratory unit.

Ultrastructural characteristics of inclusion bodies of type II cells in late embryonic mouse lung.

As we reported earlier, type II alveolar epithelial cells make their appearance in the early embryonic mouse lung around day 14.2, and show distinctive ultrastructural features. The present study focuses on the ultrastructural characteristics of the inclusion bodies by investigating embryos aged 17-19 days (birth on day 19), using transmission electron microscopy. Late embryonic type II cells appear also as low-columnar or cuboid cells having large, approximately round nuclei and cytoplasm displaying typical features of a differentiated cell. The inclusion bodies show a widespread distribution and are extremely variable in appearance. Schematically we discern five main types, namely cytoplasmic, granular/flocculent, multivesicular, dense, and (multi)lamellar, which occur with intermediate and composite forms. All these inclusion bodies frequently contain glycogen particles, and show a structural relation to profiles of endoplasmic reticulum which are wrapped around them. Other distinctive properties are the osmiophily of multivesicular inclusion bodies, and the presence of vesicles in many dense inclusion bodies. The possible interrelationship, and the differences in various aspects of electron density, suggest that the five main types of inclusion bodies may represent different stages in the formation of mature multilamellar bodies.

Ultrastructural features of type II alveolar epithelial cells in early embryonic mouse lung.

Immunofluorescence studies of type II alveolar epithelial cells indicate that they first appear in the pseudoglandular period of mouse lung development (around day 14.2). They are the only cell type to line the prospective pulmonary acinus at this time. The ultrastructural characteristics of this cell are defined by investigating embryos aged 13-16 days with transmission and scanning electron microscopy. Early embryonic type II cells appear as low-columnar or cuboid cells having large, approximately round nuclei and distinct ultrastructural features, including a well-developed Golgi apparatus with many associated vesicles, multivesicular bodies, dense bodies, and large apical and basal glycogen fields. These fields represent a distinctive property of the cell. Frequently, they show compartmentalization due to the presence of membrane systems, and association with dense bodies of various sizes.

The inadequacy of existing theories on development of the proximal coronary arteries and their connexions with the arterial trunks.

Coronary arterial development was studied in complete microseries of 20 human embryos and microseries of the hearts from 18 rat embryos. We never observed more than two coronary arterial orifices; these always originated from the facing aortic sinuses. In the human embryos these coronary orifices were variably identified between 16-19 mm crown-rump length, but were invariably present above 19 mm crown-rump length. In rat embryos, the orifices were variably identified at 13-17 mm and invariably present above 17 mm crown-rump length. In both human and rat embryos the left coronary orifice was observed significantly earlier. In all the embryos septation at arterial orifice level was complete. At the stages in which identification of the coronary orifices was variable, the proximal epicardial segments of the left and right coronary arteries could usually already be identified, in human as well as in rat embryos. On the other hand, a coronary orifice was never seen in the absence of a proximal coronary artery. At all stages studied (in human embryos from 10 mm crown-rump length and in rat embryos from 11 mm crown-rump length) vascular structures could be identified in the epicardial covering of the heart. The present theories on proximal coronary artery development are inadequate to explain either these data or the known possible congenital abnormalities of the coronary arteries. Our study offers a detailed chronology of development of these proximal coronary arteries and mostly supports dual coronary arterial development. The process by which the coronary orifices are brought into contact with the main coronary arteries still remains to be explained.

Scanning electron microscopy substantiates histology in showing the inadequacy of the existing theories on the development of the proximal coronary arteries and their connections with the arterial trunks.

Development of proximal coronary arterial segments and coronary arterial orifices was studied by scanning electron microscopy in 20 rat embryos and by light microscopy in serial sections of 20 human and another 18 rat embryos. Neither by scanning electron microscopy nor by light microscopy did we observe more than two coronary arterial orifices. These coronary orifices were always situated in the sinuses of the aorta that faced the pulmonary artery. In the human embryos the coronary orifices emerged between 37-39 days of gestation (16-19 mm crown-rump length, Streeter horizon XVIII-XIX) and were invariably present beyond 39 days (19 mm crown-rump length, Streeter horizon XIX). In rat embryos, the coronary orifices emerged in both scanning electron microscopy and light microscopy at 15-17 days of gestation (13-17 mm crown-rump length) and were invariably present beyond 17 days (17 mm crown-rump length). In both human and rat embryos, either by scanning electron microscopy and light microscopy, the left coronary orifice was observed significantly earlier. In all the investigated embryos, human as well as rat, septation at arterial orifice level was complete, including the earliest stages studied. Light microscopy showed that at the emerging stages of the coronary orifices, the proximal epicardial segments of the left and right coronary arteries could already be identified in a peritruncal ring of epicardial vasculature, before the coronary orifice was observed. This was the case in human as well as in rat embryos. Thus, a coronary orifice was never seen in the absence of a proximal coronary artery. The present theories on development of the proximal coronary arteries and coronary orifices do not offer an adequate explanation for either these data or the known possible congenital abnormalities of the coronary arteries. Our study supports dual proximal coronary arterial development. These two proximal coronary arteries develop out of a peritruncal ring of vascular structures on to the aorta. The process by which the coronary orifices actually develop remains to be explained.

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker.

The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectin-coated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.

The formation of mesoderm and mesectoderm in presomite rat embryos cultured in vitro, using WGA-Au as a marker.

Presomite rat embryos cultured in vitro were injected with the cell marker wheat germ agglutinin-gold in order to find out whether the ectoderm already formed mesodermal cells. These labelled so-called mesectodermal cells were found in all embryos studied, ranging in age from 8.7 to 9.3 days post coitum. In embryos younger than 9.0 days, the entire head fold ectoderm produced mesectodermal cells. From 9.0 days onwards, the neural crest and surface ectoderm placodes were recognizable as separate entities, both producing mesectodermal cells. The early onset of mesectodermal cell formation and the numerous and continuous manufacture led us to the conclusion that mesectodermal cells are deposited at their definitive location and that subsequent long-distance migration is unnecessary.

Wheat germ agglutinin-gold as a novel marker for mesectoderm formation in mouse embryos cultured in vitro.

The routes of movement of mesectoderm cells in mammalian embryos have not yet been investigated experimentally due to technical problems. However, the recent development of in vitro culture methods have made an experimental approach to this problem in mouse and rat embryos possible. We have used combined lectin and colloidal-gold (WGA-Au) probe as a nontraumatic, easily detectable mesectoderm marker. The probe is introduced into the amniotic cavity by microinjection. All of the cells lining the cavity, including the mesectoderm precursors, phagocytose the colloidal gold, which is then stored in membrane-bound vesicles. The probe remains inside the target mesectoderm cells after their migration into the mesoderm compartment. Vesicles containing gold are detectable in both ultrathin and semithin sections. The applicability of WGA-HRP as a probe was also assessed because of the many properties it shares with WGA-Au, but it proved to be unsatisfactory for this purpose because it is transferred between cells and also to the extracellular spaces.

The neural crest in presomite to 40-somite murine embryos.

The production of cells by the neural crest is studied light-microscopically in 10 microns and 1 micron serially sectioned mouse and rat embryos, ranging in age from presomite to 40-somite stages. In the head region, mesectoderm formation starts in a pre-neural plate stage. It continues to the 20-somite stage. This implies that the contribution of the neural crest to the head mesoderm must be considerable. In the trunk, the neural crest only produces cells after adhesion of the neural walls. Mesectoderm formation continues for a long time, slowly retreating in a caudal direction. At the 40-somite stage, mesectoderm formation still occurs in the most caudal part of the trunk. Compared to the head, the contribution of the neural crest in the trunk seems to be less important than that of the primitive streak.