pdx-1 function is specifically required in embryonic beta cells to generate appropriate numbers of endocrine cell types and maintain glucose homeostasis

The pdx1 gene is essential for pancreatic organogenesis in humans and mice; pdx1 mutations have been identified in human diabetic patients. Specific inactivation of pdx1 in adult beta cells revealed that this gene is required for maintenance of mature beta cell function. In the following study, a Cre-lox strategy was used to remove pdx1 function specifically from embryonic beta cells beginning at late-gestation, prior to islet formation. Animals in which pdx1 is lost in insulin-producing cells during embryogenesis had elevated blood glucose levels at birth and were overtly diabetic by weaning. Neonatal and adult mutant islets showed a dramatic reduction in the number of insulin(+) cells and an increase in both glucagon(+) and somatostatin(+) cells. Lineage tracing revealed that excess glucagon(+) and somatostatin(+) cells did not arise by interconversion of endocrine cell types. Examination of mutant islets revealed a decrease in proliferation of insulin-producing cells just before birth and a concomitant increase in proliferation of glucagon-producing cells. We propose that pdx1 is required for proliferation and function of the beta cells generated at late gestation, and that one function of normal beta cells is to inhibit the proliferation of other islet cell types, resulting in the appropriate numbers of the different endocrine cell types.     

Molecular characterization of cell types in the developing,mature, and regenerating fish retina

The fish retina has become a powerful model system for the study of different aspects of development and regeneration. An important aspect in understanding retinal anatomy and function is to trace the development of various cell types during embryonic stages. Several markers that detect the cessation of proliferative activity have been used in studies of cellular birth days, in order to follow the temporal progression of retinogenesis. Moreover, by using cell type-specific markers, the onset of differentiation can be determined by identifying the earliest time points for which immunolabeling is observed. Additionally, fish retinal regeneration research holds the potential of providing new avenues for the treatment of degenerative diseases of the retina. Retinal markers constitute powerful tools in studies of retinal regeneration, because they allow characterization of the cell types involved in nerve tissue regeneration, providing insights into different aspects of this process. In this review, after presenting several structural and histological aspects of the mature and developing fish visual system, data on the use of various neurochemical markers specifically indicating cell types of the fish neural retina are summarized. This will be done through a review of the pertinent literature, as well as by drawing on our own experience gathered through recent studies on fish retinogenesis.     

Interactions between cations in modifying the binding of hexokinases I and II to mitochondria

Summary Interactions between cations in modifying the binding of hexokinases I and II to mitochondria was examined with reference to the intracellular condition. Mitochondria-binding of either of hexokinases I and II, both prepared from mouse ascites ELD cells, was markedly increased by Mg2+ as has been known well. However, even in the absence of Mg^s+, marked binding was attained by 100 mM K⁺ alone especially for hexokinase I, which seemed generally more ready to bind to mitochondria. On the other hand, the effect of Mg²⁺ to increase the binding was reduced by the addition of K⁺, and the decreasing effect of K⁺ was much more marked for hexokinase II than I. These results indicate that, in addition to Mg²⁺, monovalent cations as represented by K⁺, also have marked effect on the binding, and the effect is different for each of hexokinases I and II, which may be responsible for the difference in the intracellular distribution between these hexokinases.     

Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture: II. Synaptic mechanisms

Dissociated rat sympathetic neurons and skeletal myotubes were grown in mass cultures and microcultures as described in the accompanying paper (C. A. Nurse, 1981, Develop. Biol.88, 55–70). Excitatory synaptic interactions developed between neuron and neuron and between neuron and myotube. Electrical coupling occurred rarely. More often, the interaction was chemical and as shown in the accompanying paper, cholinergic. At the chemical neuronmyotube junctions, spontaneous miniature potentials (mejp's), sensitive to d-tubocurarine, occurred infrequently (1–20/min) and their discharge appeared random; their amplitude distribution was skew at all ages (up to ca. 4 weeks) even when the myotube was innervated by a single neuron in microculture. The evoked postsynaptic potentials (ejp's) in the myotubes were sensitive in conventional ways to the extracellular calcium (Ca_o) and magnesium (Mg_o) concentrations, and several tests suggested that transmission was quantal. In a few cases where a single neuron innervated a myotube in microculture, the estimated mean quantal unit size (assuming “Poisson” release) was similar to the mode of the spontaneous mejp amplitude histogram, suggesting that many of the spontaneous units were similar to the evoked units. At several junctions the quantal content m_o, estimated by the “failure” method, varied nonlinearly with Ca_o over the range 0.2–1.2 mM; data could be fitted by a power relation where the power ranged from 2.6 to 5.2.     

Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture. I. Cholinergic transmission

Sympathetic neurons, dissociated from superior cervical ganglia of newborn rats, and skeletal muscle cells were grown together in mass cultures containing many neurons (ca. 1000–3000) and myotubes, and in microcultures containing only one to three neurons and one or a few myotubes. When these neurons grow under the influence of certain nonneuronal cells many of them acquire cholinergic functions; in the absence of this influence they remain adrenergic. In the present study, the influence of the skeletal muscle cells was so effective that under certain conditions more than 75% of the neurons expressed cholinergic function as judged by their ability to form excitatory cholinergic synapses with myotubes (from rat and chick) and with each other. Stimulation of single neurons often gave rise in the myotubes to simple (direct) postsynaptic potentials (ejp's) and/or complex responses comprising a burst of ejp's that evoked one or more spikes; it appeared that these complex responses involved the activation of interneuronal pathways. In microcultures, a single neuron often made cholinergic synapses with itself (“autapse”) and/or with another neuron as well as with one or more myotubes. The nicotinic blocking agents, tubocurare (dTC), α-bungarotoxin (α-BuTx), and hexamethonium (C₆), attenuated or abolished the ejp's at moderate concentrations; the muscarinic blocker, atropine, was effective only at high concentrations. At several neuron-myotube junctions, the acetylcholine (ACh) receptors had dTC sensitivity similar to adult extrajunctional receptors; however, when different junctions were pooled the average dTC sensitivity was intermediate between that of adult end plate and extrajunctional receptors. The junctional C₆ sensitivity was much higher than expected from the action of the drug at the adult mammalian end plate. As in other studies, chemical transmission from neuron to neuron was also nicotinic cholinergic, but the nicotinic receptors on the myotubes were pharmacologically distinct from those on the neurons.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.     

Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet cell types

Cells within rat islets of Langerhans are typically organized as a core of B-cells, surrounded by the other cell types. When mixed in culture, primary islet cells and insulinoma (RIN2A) cells form aggregates where B-cells are centrally located, surrounded by non-B-cells, while RIN-cells segregate as the outermost layer. To gain insight into the molecular basis underlying this nonrandom cellular organization, the aggregation properties of the three cell populations were studied. Isolated islet cells were separated into B-cells and non-B-cells by autofluorescence-activated cell sorting (FACS). In a short-term aggregation assay, primary B-cell aggregation in the absence of calcium was only 19 +/- 3.7%, compared to the 67 +/- 2.9% seen in the presence of calcium (mean +/- SEM; P less than 0.001; n = 7). By contrast, non-B-cell aggregation and RIN cell aggregation in the absence of calcium (62 +/- 2 and 66 +/- 2%, respectively) were only slightly less than with calcium (70 +/- 3 and 76 +/- 3%). The surface density of the Ca2(+)-independent neural CAM (NCAM) was therefore measured by flow cytometry and found to be 2.64 +/- 0.82-fold higher in non-B-cells, compared to that in B-cells (P less than 0.01; n = 3). Even higher levels were found on RIN cells. In the three cell types, NCAM-140 was the only molecular form detected by immunoblotting. In conclusion, differences in the calcium dependency of aggregation and in the levels of NCAM are demonstrated among islet B-cells, non-B-cells, and RIN cells. Because cell-cell adhesion is crucial for the maintenance of adult tissue, these aggregation specificities might contribute to the concentric segregation of islet cell types in culture and to the nonrandom distribution of cells within rat islets.     

Interactions of collagen types I and II with chondroitin sulfates A-C and their effect on osteoblast adhesion

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants. The main aim of this study was to compare the ability of the collagen types I and II to bind preparations of the chondroitin sulfate types A-C (CS A, CS B, CS C). In addition, the effect of the three CS preparations on the extent of collagen incorporated into fibrils and the morphology of collagen fibrils was investigated, as was the influence of collagen fibril coatings containing CS A-C on titanium surfaces on the adhesion of primary rat osteoblasts. Fibrils of both collagen types bound a higher mass of CS C than CS B and a greater mass of CS B than CS A per milligram of fibrils formed. Fibrils of collagen type II bound a higher mass of CS B and C than collagen I fibrils. The proportion of collagen incorporated into fibrils decreased with increasing CS A and CS C concentration but not with increasing CS B concentration. All three CS preparations caused collagen I and II fibrils to become thinner. CS A and CS B but not CS C appeared to stimulate the formation of focal adhesions by osteoblasts after incubation for 2 hours. These results could be of importance when selecting collagen type or CS type as materials for implant coatings or tissue engineering scaffolds.     

Interactions between nutrition and reproduction in the management of the mature male ruminant

In mature male sheep and goats, changes in feed intake seem to have little effect on gonadal endocrine function but induce profound changes on sperm production. These outcomes are due to changes in size of the seminiferous tubules and in spermatogenic efficiency. Except with severe underfeeding, there are only minor changes in the endocrine function of the testis (testosterone production) unless season-long treatments are imposed. For cattle, nutrition clearly affects testicular development and the production of spermatozoa in young bulls, as it does in other species but, after the period of rapid growth has ended, there appears to be little or no response to nutrition. We are developing a clear picture of the metabolic signals, neuroendocrine processes and hormonal control systems that are involved, particularly for the mature male sheep. The energetic components of the diet, rather than protein, seem to be responsible, so we have envisaged a model of the relationship between energy balance and reproduction that has 4 'dimensions': genotype, structure (organs), communication (chemical and neural signals, nutrient sensing) and time (dynamics, metabolic memory, programming). We have linked these perspectives to 'resource allocation theory' and incorporated them into strategies for 'clean, green and ethical animal production'. In contrast to the clear outcomes with respect to spermatogenesis, the effects of nutrition on sexual behaviour are more difficult to define, perhaps because the behaviour is affected by a complex mix of physiological factors and because of flawed methods for quantifying male behaviour. For example, sexual behaviour is compromised by severe feed restriction, but male sexual behaviour requires intensive motor activity so a decline in libido could be caused by general weakness rather than specific nutritional limitations. The interaction between sexual activity and feeding behaviour also complicates the issue under field conditions. At the other end of the scale, overweight males can show reduced sexual success because they have difficulty courting and mounting. For this reason, exercise can enhance the fertilising capacity of rams. This will be important in extensive mating systems where males need to assemble and guard a harem and then mate many times for several weeks. For artificial insemination centres, there seems to be very few data on the nutritional management of males, but problems with overfed animals appear to be a risk. Future research should concentrate on the intra-testicular systems mediating the effects of nutrition on the production of spermatozoa.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Conditional expression of fibroblast growth factor-7 in the developing and mature lung

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)(7)-cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta x (Teto)(7)-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.