Spatial and temporal relationships between cell death and tissue overgrowth in imaginal wing disks of the Drosophila mutant l(3)c43hs1.

The temperature-sensitive mutant l(3)c43hs1 is lethal at the restrictive temperature late in the last larval instar and has wing disks that show excessive growth when larvae are reared at 25 degrees C. Such mutant disks give rise to defective wings showing duplications and deficiencies. Abnormal folding patterns are localized to the region between the wing pouch and the area where adepithelial cells are found; the disks retain an epithelial morphology. Apoptotic cell death is distributed throughout the wing disks without any obvious concentration of dead cells in a specific area. Cell death is seen as early as 12 hr after a shift to the restrictive temperature. Temperature shift experiments also show that cell death precedes the onset of overgrowth, but since the spatial distribution of death is not localized to the regions of abnormal folds, it is unlikely that cell death and overgrowth are causally related.

Localized cell death in Drosophila imaginal wing disc epithelium caused by the mutation apterous-blot.

Drosophila melanogaster carrying the mutation apterous-blot have blistered wings. Trypan blue stains a patch of dead cells localized to the wing pouch of imaginal discs and the same area shows acid phosphatase (AcPase) activity suggesting that the cell death is lysosomal. Autophagic vacuoles and other secondary lysosomes show AcPase activity within the disc epithelium and enzyme activity is found in fragments of dead cells which have been extruded basally. The cell death, although extensive and confined to the presumptive wing region, does not result in loss of adult structures.

Correlations between endocrine gland ultrastructure and hormone titers in the fifth larval instar of Manduca sexta.

Correlations were made between endocrine gland ultrastructure and circulating hormone titers of Manduca sexta to investigate the mechanisms of hormone biosynthesis and secretion. Both the prothoracic glands (PTG), which secrete ecdysone, and the corpora allata (CA), which secrete juvenile hormone (JH), were studied. In the prothoracic glands, the intracellular spaces increase in area and reach their maximum size following the major ecdysteroid peak in the fourth and fifth larval instars. Within the intercellular spaces are multivesicular sacs (MVS), structures which are clusters of vesicles bounded by another membrane. Since these sacs are largely depleted of their internal vesicles after the second cycle of tropic hormone stimulates the PTG to secrete ecdysone, the MVS probably release a gland cell product at this time. In the CA, concentric whorls of smooth endoplasmic reticulum are present in larval glands, when the JH titer is high, but are absent from pupal CA when the JH titer is low. The peak of JH at Days 6-8 of the fifth larval instar occurs after an increase is seen in the neurosecretory cell axon diameters suggesting that the CA are stimulated by a brain hormone to release JH. The number of Golgi complexes increases in pupal CA and dense bodies are present in pupal but not larval glands. These Golgi complexes may be involved in the manufacture of lysosomal enzymes which degrade JH within the gland itself.

Correlations between epidermal cell structure and endogenous hormone titers during the fifth larval instar of the tobacco hornworm, Manduca sexta.

Epidermal cell morphology and cuticle production in Manduca sexta are directly influenced by both ecdysterone and juvenile hormone. Up to day 6 of the last larval instar, post-molt endocuticle is continuously deposited even though cells undergo a partial and temporary separation from the overlying cuticle at the time when a small ecdysteroid peak is detected (approximately day 3.5). At about days 6--7 when another, larger ecdysteroid peak is present, apolysis occurs accompanied by the appearance of edcysial droplets. Following apolysis, layers of pupal cuticle are deposited. Increased quantities of rough endoplasmic reticulum characterize the epidermis at times of peak endocuticle deposition (day 3, larval cuticle; day 9, pupal cuticle). Dense pigment inclusions are found in epidermis from the day of ecdysis to the last larval instar until they are eliminated 5 days later. These dense bodies migrate from cell apex to base in the absence of juvenile hormone (or in the presence of a negligible amount of juvenile hormone) and probably contain insecticyanin.

Comparison of two types of endothelial cells in long term culture.

Bovine aortic endothelial cells have been serially subcultured and maintained for ten months (24 passages) without any apparent loss of endothelial-like properties. There appear to be two endothelial cell types; one type is mononucleated, 40-60 mu in diameter, and the other (giant) cell type, multinucleate and 300-600 mu in diameter. Both cell types were identified as endothelial cells by established criteria; 1. ultrastructurally, by the presence of Weibel-Palade bodies, microfilaments and pinocytotic vesicles and 2. immunologically, by the presence of factor VIII antigen and thrombosthenin.

Epidermal cell development during the pupal-adult metamorphosis of Hyalophora cecropia.

To establish a base for studying the hormonal control of insect epidermal cell activity, the ultrastructure of abdominal epidermis was analyzed during the normal pupal-adult development of Hyalophora cecropia. Adjacent epidermal cells could be distinguished on the basis of organelle content and staining intensity, suggesting that this monolayer is not composed of a homogenous cell population. At the onset of adult development the form of the epidermal cell is transformed from that typical of a quiescent cell with free ribosomes and few mitochondria to one which is metabolically active and possesses numerous apical membrane microvilli, rough endoplasmic reticulum and numerous mitochondria. On about day 5 of pharate adult development the apical plasma membrane is no longer folded but becomes folded again several days later when cuticulin and endocuticle are deposited. On about day 7, giant autophagic vacuoles are discerned that may be important in cellular reprogramming. After adult ecdysis, the epidermal cells continue to deposit endocuticle.

Culture of arterial endothelial cells: characterization and growth of bovine aortic cells.

Arterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially all (90-95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3-5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12-14 months (30-35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32-34 hours and 29-31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluenct cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.