Induction of supernumerary molting by starvation in Manduca sexta larvae

Studies were carried out on the effects of prolonged starvation on the development of fifth- (last-) instar larvae of the tobacco hornworm Manduca sexta. Following ecdysis, larvae were starved for varying lengths of time and subsequently fed normal diet. The percent of starved larvae molting to sixth instars increased, while the percent survival decreased with increasing length of the starvation period. When larvae were provided with agar as a source of water during the starvation period, the percent survival increased, but the percent undergoing supernumerary molting decreased. The optimal condition for maximum survival and supernumerary molting appeared to be 3 days of starvation with 0.5–1.0 g of agar provided on day 0. The endocrine basis for the supernumerary larval molt induced by starvation is briefly discussed.

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Zusammenfassung Es wurden Studien durchgeführt über die Effekte längeren Hungerns auf die Entwicklung des fünften (letzten) Larvenstadiums bei Manduca sexta. Nach der Häutung wurden die Larven während unterschiedlicher Zeit ohne Nahrung gelassen und dann mit normalem Futter versehen. Der Prozentsatz Häutungen für ein sechstes Larvenstadium nahm mit der Dauer des Hungerns zu, während die Ueberlebensrate abnahm. Wenn Agar als Quelle für Wasser gereicht wurde, wurden beide Effekte vermindert. Die optimalen Verhältnisse für maximales Überleben und zusätzliches Häuten scheinen bei 3 Tagen Hunger mit 0.5–1.0 Gramm Agar am Tage 0 zu sein. Die endokrinologische Basis für die zusätzliche Larvenhäutung wird kurz diskutiert.

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Supported by grants from NSF (PCM 77-25417) and from Organized Research, Texas A & M University.     

Chondrogenesis of mandibular mesenchyme from the embryonic chick is inhibited by mandibular epithelium and by epidermal growth factor

This study documents the role of mandibular epithelium and epidermal growth factor (EGF) in the initiation, maturation and maintenance of Meckel's cartilage using percent 3H-thymidine-labelled cells as an index of proliferative activity and distribution of labelled cells, chondrocyte size and relative amount of extracellular matrix as indices of chondrogenesis. Mandibular mesenchyme from embryos of H.H. stages 18, 22, 25 was cultured for 2 to 10 days (a) unseparated from mandibular epithelium, (b) in isolation, or (c) after recombination with mandibular epithelium in the presence or absence of 5-40 ng/ml EGF. Epithelium delayed both initiation of chondrogenesis and maturation of already formed cartilage. The 3H-thymidine-labelling index was reduced in cartilage that differentiated in the presence of mandibular epithelium. Epithelium influenced the timing of mesenchymal differentiation (a) by delaying cytodifferentiation through prolonging high levels of proliferation, and (b) by directly affecting differentiation itself. EGF, especially at 10-20 ng/ml, affected both proliferation of mesenchyme and chondrogenesis in mesenchyme cultured with or without epithelium. All observed effects of epithelium on intact tissues could be duplicated by exposing isolated mesenchyme to EGF at 10 ng/ml, i.e. a role for EGF in chondrogenesis is suggested.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.     

Sonic hedgehog在鸡胚法氏囊发育过程中的表达研究

Sonic hedgehog(Shh)是Hedgehog(Hh)家族中的一员,在胚胎发育和器官形成过程中发挥重要作用.法氏囊是鸟类所特有的中枢免疫器官,在机体的免疫防御方面发挥重要作用.利用切片原位杂交的方法探究Shh基因在鸡胚法氏囊发育过程中的表达模式,检测发现Shh基因主要在鸡胚法氏囊的囊下上皮细胞、血管周围上皮细胞以及网状细胞中表达.     

Sonic hedgehog expression in developing chicken digestive organs is regulated by epithelial–mesenchymal interactions

Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell–cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh . These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial–mesenchymal interactions in developing digestive organs.     

Retention of epithelial basal lamina allows isolated mandibular mesenchyme to form bone

The initiation of bone formation in the avian mandible requires that neural crest-derived cells undergo an inductive interaction with mandibular epithelium. To examine the role of the epithelial basal lamina in that interaction, mandibles were separated into their epithelial and mesenchymal components following exposure to the chelating agent, EDTA. Transmission and scanning electron microscopy was used to show that the basal lamina was retained as a continuous layer over the mesenchyme. Osteogenesis was initiated when such EDTA-isolated mesenchyme was grafted to the chorioallantoic membranes of host embryos. In contrast, mesenchyme isolated using trypsin and pancreatin failed to form bone. It is concluded that the property of mandibular epithelium which permits osteogenesis resides within the basal lamina.     

Induction of male germ cell-like lineage from chicken fetal bone marrow stem cells with chicken testis extract

Bone marrow-derived stem cells (BMCs) are able to differentiate into multilineage cells such as muscle, bone, cartilage, fat, and nerve cells. In the present study, we investigated the differentiation capability of chicken BMCs into germ cells by using retinoic acid (RA) and chicken testis extract (chTE). The chicken BMCs were isolated from fetal chicken femurs on post-fertilization day 20, cultured in vitro, and treated with RA and chTE, respectively. The cultured chicken BMCs displayed fibroblast-like morphology and were positive for mesenchymal-specific markers such as CD44, CD90, and CD105 at the mRNA level. RT-PCR and immunocytostaining revealed that both RA and chTE treatments induced the expression of early-germ-cell markers such as Stra8, Dazl and DDX4. The increase of germ cell-specific gene expression after chTE treatment indicates that testicular environment-derived proteins may induce in vitro germ-cells. In addition, we performed a microarray analysis to identify differentially expressed genes (DEGs) in RA and chTE, respectively. A total of 1,629 DEGs were obtained and the chTE treatment showed very lower numbers of DEGs than the RA treatment. Collectively, our results indicate that chicken BMCs have the potential to differentiate to male germ cells in vitro with testis derived proteins.     

Metabolism of prostaglandin E2 by mouse embryo palate mesenchyme cells in vitro

Mouse embryo palate mesenchyme cells synthesize a number of prostaglandins, particularly prostaglandin E2 (PGE2). However, the ability of such cells to metabolize prostaglandins was unknown. By use of radiolabeled PGE2 we determined that palate mesenchyme cells have little ability to degrade that prostaglandin in vitro but are able to metabolize products formed from its spontaneous degradation.     

Cell polarity in precartilage mouse limb mesenchyme cells

The patterns of orientation of individual mesenchyme cells have been evaluated in the hindlimb of the mouse embryo during the period of transition from early aggregation (Day 12) to cartilage formation (Day 13). Orientation was measured by determining the angular relationship between the Golgi-nucleus axis of each cell relative to either the longitudinal limb axis or the center of the cartilaginous aggregate. Patterns were assessed qualitatively and quantitatively in horizontal, vertical, and transverse sections of the proximal, middle, and distal precartilage mesenchyme. These analyses showed that the mesenchyme cells are oriented predominantly toward the longitudinal axes of both the early (Day 12) and late (Day 13) aggregates.     

Prostaglandin synthesis by primary cultures of mouse embryo palate mesenchyme cells

Evidence is presented for a concomitant storage of α-Neo-endorphin and dynorphin immunoreactivities in neurons of the rat brain. Antisera were raised against the structurally related opioid peptides dynorphin(1–17) and α-Neo-endorphin. Both antisera were highly specific for their respective antigen. Thus, the α-Neo-endorphin antisera did not crossreact with dynorphin and the dynorphin antisera did not crossreact with α-Neo-endorphin. Both antisera were also not cross-reactive with leu-enkephalin which is contained within the sequence of both dynorphin and α-Neo-endorphin. The antisera were used for immunofluorescent staining of frozen sections through brains from rats which had been treated with colchicine 48 hours prior to death. Both antisera revealed strong and specific immunoreactivities of magnocellular neurons in the supraoptic, retrochiasmatic supraoptic and paraventricular nuclei. Neuronal fiber systems in various areas of the brain were also labeled by the two antisera. Consecutive immunostaining of the same sections, first with dynorphin antisera and — after electrophoretic elution of the antibodies — with α-Neo-endorphin antisera or vice versa, showed that immunoreactivities for the two peptides are contained within the same hypothalamic magnocellular neurons. The neuronal fiber systems for α-Neo-endorphin and dynorphin also showed a close overlap. These studies demonstrating colocalization raise the question as to whether the two peptides have a common origin from a single precursor molecule.     

Induction of the high-affinity nerve growth factor receptor on embryonic chicken sensory nerve cells by elevated potassium

Culture medium with elevated K⁺ has been shown to enhance the survival of neurons isolated from several different regions of the nervous system. Nerve growth factor binds to binding sites on sensory and sympathetic neurons through two sites, one of high-affinity (K _d13×10^–11 M) and the other of low-affinity (K _d22×10^–9 M). Equilibrium binding data generated on dissociated cells derived from E9 chicken embryo dorsal root ganglia, has shown that there is a two-fold increase in the number of high affinity (type I) receptors, with no effect on the affinity, when cells are incubated for 2 hours in buffer containing 59 mM K⁺. There does not appear to be a significant change in the affinity or the number of low-affinity binding sites. This two-fold increase in type I receptors is dependent on temperature, Ca²⁺, and active protein synthesis. There does not appear to be an intracellular pool of the type I receptor sufficient to account for this increase. The induction is not observed on sensory nerve cells cultured in 59 mM K⁺ for 24 hours, either in the presence or absence of nerve growth factor. Additionally, the induction in the number of type I receptors requires that both nerve growth factor and K⁺ be present simultaneously. Taken in total, this data suggests that there may be a critical period in which the sensory neurons require nerve growth factor exposure to respond. Evidence is presented which indicates that nerve growth factor responsive cells are able to elicit neurites after an acute exposure to nerve growth factor of as little as 4 hours. Finally, there is an approximate two-fold decrease in the concentration of nerve growth factor needed to elicit maximal fiber outgrowth, consistent with the two-fold increase in the number of type I receptors.Abbreviations NGF nerve growth factor - 7S NGF the high molecular weight form of NGF - NGF the -subunit of 7S NGF - ¹²⁵I-NGF ¹²⁵I-labeled NGF - mNGF–_rAb polyclonal rabbit IgG raised against mouse NGF - DRG dorsal root ganglia - K_d the equilibrium dissociation constant - N the maximal number of binding sites for the ligand NGF - NGFR the biologically relevant receptor through which the neurite outgrowth and neuron survival are mediated - GBS Gey's balanced salts - HKGBS high K⁺ GBS - PBG phosphate buffered GBS - HKPBG high K⁺ PBG - CFHKPBG Ca⁺² free high K⁺ PBG - PBG-cyt c PBG containing 2 mg/ml cytochrome c - HKPBG-cyt c HKPBG containing 2 mg/ml cytochrome c - AbU antibody unit - BU biological unit PBS, phosphate buffered saline - HKPBS high K⁺ PBS Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Induction of tubules in rat metanephrogenic mesenchyme in the absence of an inductive tissue

Differentiation of metanephrogenic mesenchyme to renal tubular epithelium requires induction by the ureteric bud in vivo or any of several embryonic tissues in vitro. In an effort to eliminate the tissue requirement in embryonic induction, extracellular matrices and soluble factors were analyzed individually or in combination for their ability to stimulate tubulogenesis in uninduced metanephrogenic mesenchyme from 13-gestation-day rat embryos. These evaluations have established that pituitary extract and epidermal growth factor (EGF) in concert with a matrix can promote morphogenesis of mesenchymal rudiments in culture. While type I collagen, laminin, or fibronectin matrices all promoted tubulogenesis in the presence of pituitary extract and EGF, type IV collagen proved the most effective. Under these conditions, tubules were induced in 23/24 mesenchymal rudiments by 9 days in culture. Mesenchyme was not induced prior to explanation since it formed no tubules when cultured in a medium that allowed tubulogenesis in intact embryonic kidneys. Preliminary characterization of the undefined factor in pituitary extract was consistent with a protein of molecular weight greater than 100,000 but less than 300,000. When uninduced metanephrogenic mesenchyme from mouse was used instead of rat tissue, a similar pattern of morphogenesis was not observed, suggesting that the described medium is inappropriate for promoting differentiation in mouse or, less likely, that different mechanisms mediate differentiation in rat and mouse. These studies show that embryonic induction can occur in explanted rat renal mesenchyme in an appropriate environment and does not require the presence of an inductive tissue.     

Fishing for jaws in early vertebrate evolution: a new hypothesis of mandibular confinement

The evolutionary origin of the vertebrate jaw persists as a deeply puzzling mystery. More than 99% of living vertebrates have jaws, but the evolutionary sequence that ultimately gave rise to this highly successful innovation remains controversial. A synthesis of recent fossil and embryological findings offers a novel solution to this enduring puzzle. The Mandibular Confinement Hypothesis proposes that the jaw evolved via spatial confinement of the mandibular arch (the most anterior pharyngeal arch within which the jaw arose). Fossil and anatomical evidence reveals: (i) the mandibular region was initially extensive and distinct among the pharyngeal arches; and (ii) with spatial confinement, the mandibular arch acquired a common pharyngeal pattern only at the origin of the jaw. The confinement occurred via a shift of a domain boundary that restricted the space the mesenchymal cells of the mandibular arch could occupy. As the surrounding domains replaced mandibular structures at the periphery, this shift allowed neural crest cells and mesodermal mesenchyme of the mandibular arch to acquire patterning programs that operate in the more posterior arches. The mesenchymal population within the mandibular arch was therefore no longer required to differentiate into specialized feeding and ventilation structures, and was remodelled into a jaw. Embryological evidence corroborates that the mandibular arch must be spatially confined for a jaw to develop. This new interpretation suggests neural crest as a key facilitator in correlating elements of the classically recognized vertebrate head ‘segmentation’.     

Glycoconjugates in the secretory epithelium of the chicken mandibular gland

Summary In the secretory epithelium of the chicken mandibular gland, glycoconjugates have been studied by means of histochemical methods of light and electron microscopy. In light microscopy, a series of histochemical procedures have been employed which included lectin—peroxidase—diaminobenzidine methods and a digestion technique with neuraminidase or-amylase. In electron microscopy, a battery of methods were used that corresponded to those employed in light microscopy. In the secretory cells of the chicken mandibular gland, vicinal diol- and sulphate-containing glycoconjugates with sialic acid,-d-mannose,-d-glucose and-d-galactose residues were visualized and the possible histophysiological significances of such glycoconjugates were discussed with special reference to the functions of the salivary gland.