ALBERT

Notes: Summary Enhancement of photovoltage has been found in bone and its two major constituents when placed in a magnetic field in standard Hall geometry. Infrared light has been used for carrier excitation. From data obtained in this manner, values for the light absorption constant, extinction constant, mass absorption constant, and efficiency have been estimated. All these parameters increase with the increase of magnetic field. A possible explanation for the observed phenomena is presented.

Notes: Summary To test the importance of 24-hydroxylation of vitamin D3 on bone mineralization, rat pups born to vitamin D-deficient females were given either 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3 for 16 days beginning at the time of weaning. Following such treatment analysis of blood samples revealed no detectable 24R,25-(OH)2D3 and 1,25-(OH)2D3 in the rats given the difluoro compound while revealing the expected 24,24-difluoro-25-hydroxyvitamin D3 and 24,24-difluoro-1,25-dihydroxyvitamin D3. The rats given 25-hydroxyvitamin D3 had the expected levels of 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3. Following sacrifice at day 17, postweaning bone mineralization and modeling were studied in long bones using histological methods. Bones taken from vitamin D-deficient rats at the beginning and end of the experimental period had lesions typical of rickets. These included wide growth plates, excessive amounts of osteoid, and metaphyseal fibrosis. Following treatment with either 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, bone mineralization returned to normal. Growth plate widths and the amount of osteoid on bone surfaces were both substantially reduced and to a similar degree in both treatment groups. Normal cartilage core formation and trabecularization of the metaphyseal primary spongiosa were also restored to a similar degree in both groups. In effect, no difference was observed in any bone parameter studied between the 25-hydroxyvitamin D3- and the 24,24-difluoro-25-hydroxyvitamin D3-treated animals. These results provide strong evidence that 24-hydroxylation of the vitamin D molecule plays little or no role in the modeling and mineralization of bone.

Notes: Summary The hormonal metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], exerts its biological effects by binding to a cytosolic receptor protein. Such a protein has been demonstrated in vitamin D3 target organs including fetal rat calvariae and more recently in rat osteogenic sarcoma cells. In this study we have compared the binding of 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] to that of 1,25-(OH)2D3 in fetal rat calvariae and osteogenic sarcoma (OS) cells. Sucrose density sedimentation, DNA-cellulose chromatography, and intracellular uptake studies have been employed to evaluate these interactions. In cytosol preparations from calvariae, [3H]-1,25(OH)2D3 bound to a 3.3S macromolecule and to a much greater extent to a 5.8S macromolecule while both [3H]25(OH)D3 and [3H]24,25(OH)2D3 bound to the 5.8S macromolecule. By incubating intact calvariae and OS cells with labeled metabolites and thus establishing binding intracellularly prior to cell disruption, we have found that the 3.3S protein which has high specificity for 1,25(OH)2D3 occurs inside the cells; the 5.8S protein, however, does not occur inside the cells but is generated after cell disruption. The [3H]-1,25(OH)2D3-receptor complex adsorbed to DNA-cellulose and was eluted from this affinity resin at 0.28M KCl. In contrast, [3H]25(OH)D3 and [3H]-24,25(OH)2D3 binding activity did not adsorb to DNA-cellulose. We conclude that, in contrast to the 3.3S protein, the 5.8S macromolecule does not fulfill receptor criteria but is rather generated by the experimental manipulation of the bone cells. Our data suggest that the vitamin D3 actions on bone are mediated only via the 3.3S receptor, and hence quantitative but not qualitative differences of the effects of the various metabolites are feasible.

Notes: Abstract A method is presented for separating osteoclasts from the heterogeneous population of bone and marrow cells. Cell suspensions were prepared from femora of young rabbits by mechanical dispersion. The starting cell suspension typically contained only 1.0%±0.5 osteoclasts. Following an initial 45 min of unit gravity sedimentation in a lucite chamber osteoclasts were primarily distributed in fractions 2–5. A second 45-min sedimentation of these pooled fractions yielded cell suspensions containing greater than 30% osteoclasts (as much as a 50-fold increase over starting percentages). Linear scan analysis, however, revealed that osteoclasts accounted for 73.14%±0.58 of the cell colume. Subsequent in vitro experiments demonstrated linear incorporation of3H-leucine into TCA precipitable protein for cells comprising the osteoclast fraction. Concomitant radiautographs revealed radioactive label in isolated osteoclasts.

Notes: Summary Male rats weighing 100 g were assigned to groups and injected daily for 10 days with vehicle (control), 0.4, 2.0, 4.0, 10.0, or 20.0 mg/kg/day of ethane-1-hydroxy-1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl2MDP). The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation parameters. From the data, information about hard tissue resorption was deduced. All doses of Cl2MDP and doses of 2.0 mg EHDP/kg/day and greater caused significant increases in percentage of hard tissues with Cl2MDP being more effective than similar doses of EHDP in decreasing bone resorption. Osteoclast population parameters were increased with all doses of both Cl2MDP and EHDP with Cl2MDP having a greater effect than similar doses of EHDP. Decreases in the proliferation of the osteoprogenitor pool parallel the decreases in osteoblasts and bone formation parameters. These decreases in osteoprogenitor pool proliferation do not account for the increases with diphosphonates in osteoclast population parameters.

Notes: Summary The stimulation of bone resorption, assessed by the release of45Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.

Notes: Summary The extractability of125I-labelled plasma albumin from bone pieces and from powdered bone has been compared after both in vivo and in vitro incorporation. The results show that albumin is more readily extracted from bone pieces than from bone powder which implies that tissue disruption exposes additional protein adsorption sites. It is suggested that incorporation of plasma albumin into calcified matrix during bone formation occurs mainly as a result of its strong interaction with bone mineral.

Notes: Summary Cortical bone remodeling along the femur diaphysis was determined in normal female rats (Sprague-Dawley) with the tetracycline technique. Three segments on the cortical bone circumference (the anterolateral, the medial, and the posterior) were found to be most suitable for the study of the remodeling process. Oxytetracycline was administered at age 60 and 75 days, and groups of animals were killed at age 75, 85, 95, and 105 days. The accumulated endosteal growth during age 60 to 75 days in the anterolateral segment was found to increase uniformly in the distal direction along the femur diaphysis. A method is described where this accumulated endosteal growth is used. This method eliminates the use of calipers to determine the section level and makes it possible to study comparable sections even after varying periods of time. The proximal part of the diaphysis showed periosteal apposition in all three segments. The periosteal apposition turned into resorption in the distal part of the diaphysis in the anterolateral and medial segments, whereas the periosteal appsition increased in the posterior segment. The endosteal growth increased in the distal direction in the anterolateral and medial segments. Irregular OTC bands made measurements of endosteal remodeling in the posterior segment impossible. The cortical width decreased in the distal direction along the femoral shaft. Comparison between the different age groups is described and also the relation between the accumulated endosteal growth and the diameter of the medullary cavity.

Notes: Summary The elaboration of bone matrix glycoprotein by osteoblasts of alveolar bone was investigated by radioautography after the intravenous injection of3H-fucose into young rats. At selected times after injection, animals were sacrificed by intracardiac perfusion and demineralized specimens were prepared for light and electron microscope radioautography. At 5 and 10 min after injection, when the blood fucose level was high, silver grains were restricted to the spheroidal and cylindrical saccules of the Golgi apparatus. At 20 min membrane-limited secretory granules were also labeled. By 35 min, the blood fucose level had dropped and silver grains were detected over the apical cortical cytoplasm, in association with secretory granules located therein. Some grains were present over osteoblast processes and the adjacent prebone matrix. By 4 h most of the silver grains had left the cell. At that time they were observed over prebone, adjacent to osteoblast processes, as well as over the prebone-bone junction where a distinct band of label was noted. In demineralized preparations an electron-dense granular material was present at the prebone-bone junction in association with collagen fibrils. These findings provide evidence that osteoblasts in alveolar bone synthesize fucose-containing glycoprotein and indicate that the addition of3H-fucose occurs in the Golgi apparatus. The glycoprotein passes to the apical cortical cytoplasm by way of membrane-limited secretory granules, is exteriorized, and accumulates at the site where prebone transforms into bone (the prebone-bone junction). Since this is also the site of the calcification front, the deposition of labeled glycoprotein may be related to the deposition of bone mineral.

Notes: Summary A short review is given of the water in bone. Various analyses of bone water content are discussed, and its possible location is considered in relation to the behaviour of water in isolated components of bone. Some of the difficulties encountered in examining such microscopic phenomena as water structure in a heterogeneous system such as bone are also discussed.

Notes: Summary The present study is an investigation of the mechanism of hypercalcemia and hyperphosphatemia induced by the intravenous injection of lead acetate (Pb-Ac). A total of 118 male rats were injected with 30 mg/kg of Pb-Ac, or with 16.5 mg/kg of sodium acetate as the control. The levels of serum calcium, phosphorus and lead were then determined at various time periods after the injections. Serum calcium and phosphorus levels increased with time after Pb-Ac injection and the maximum values of calcium (17 mg%) were found after 1 h and of phosphorus (13.5 mg%) after 30 min. Both calcium and phosphorus levels reverted to the normal range after 12 h. The maximum net rates of increase of calcium and phosphorus were found immediately after Pb-Ac injection. At that time, deposition of lead at the calcifying sites of bone and incisor dentin was demonstrated by a histochemical examination. In other experiments the changes in the calcium and phosphorus contents in the medium after shaking bone powder in serum with Pb-Ac in an in vitro system were studied. It was confirmed that the calcium and phosphorus were displaced from the bone mineral, the extent of the displacement being correlated with the concentration of the Pb-Ac added to the medium, and that these displacements were very rapid reactions. These results suggest that hypercalcemia and hyperphosphatemia following Pb-Ac injection results from a direct action of lead on the bone mineral.

Notes: Summary Recently published bone fluoride values from Iowa are very high compared to earlier reports, suggesting an increase in fluoride intake. Reanalysis of the Iowa specimens shows levels one-fourth those reported by the Iowa laboratory indicating an error in the original report. Seventeen bone specimens, collected from long-term residents of Rochester, New York, drinking 1 ppm F− water, had a mean value of 2085±270 ppm F− on an ashed-weight basis. This value is not significantly different from that predicted by the data of Zipkin et al. in 1958. These data, therefore, do not support the contention that there has been an increase in fluoride intake.

Notes: Summary The net rate of loss of45Ca from plasma was monitored in normal, thyroparathyroidectomized (TPTX), and nephrectomized rats. Also studied was the effect of a 24 h intravenous infusion of PTH in TPTX rats. The45Ca was injected after an overnight fast and 1 to 4 h prior to the time the first blood sample was obtained. In normal rats, the net rate of loss of45Ca from plasma was slower than in TPTX rats for at least 12 h after radionuclide injection. Nephrectomy did not affect this difference between control and TPTX rats until 8 h after45Ca injection or about 18 h after nephrectomy. At this time a reversal occurred and the net rate of plasma45Ca loss in nephrectomized rats with parathyroids became faster than in TPTX nephrectomized rats. When PTH was infused at a constant rate (3 mU/g body weight/h) plasma calcium concentrations rose gradually for approximately 12 h after which a constant plasma calcium level was maintained with continued PTH infusion. During the period when plasma calcium concentrations were rising, the net rate of loss of45Ca from plasma was slower than in TPTX controls infused only with saline. However, when plasma levels stabilized, plasma45Ca disappearance rates changed such that the rate loss of45Ca from plasma became faster in PTH-infused than in TPTX controls. It is concluded that PTH increases calcium efflux from the same compartment in bone which receives calcium from plasma, thereby returning some recently deposited45Ca to plasma. In the non-equilibrated state, this returns additional45Ca to plasma causing anet decrease in the rate of loss of45Ca from plasma. However, in the equilibrated condition, when both calcium efflux from bone and influx into bone are in balance but at higher flux rates, PTH stimulates the diffusion of45Ca into other bone compartments.

Notes: Summary A conventional spectrophotometer was used to do radiographic photodensitometry of bone mass. An experiment to produce osteoporosis in guinea pigs required quantification of changes in bone mass. Lack of a densitometer necessitated finding another method for quantification. The method developed involved using industrial film in a closed X-ray system. Optical density of X-rays was then read in a spectrophotometer at nine femur locations. Highly significant correlations between optical density and femur calcium content and femur dry weight per millimeter were obtained. Histological sections from an animal with low optical density measurements confirmed bone loss by showing decreased cortical width and loss of trabecular bone. In addition to using readily available equipment, this method offers the advantage of being able to detect bone loss in specific areas of the bones. In addition, bones are left intact and can be used for other purposes.

Notes: Summary Carbonate-containing hydroxyapatite, enamel, and bone were irradiated by an X-ray and investigated between 77° and 350°K by means of electron spin resonance (ESR) spectroscopy. The ESR spectrum of enamel irradiated at 77°K in vacuum and observed at the same temperature was almost the same as that of the carbonate-containing hydroxyapatite. The temperature dependence of signal intensities confirms a spin-energy exchange between the mineral and organic constituents in bone, but in enamel no or very little spinenergy exchange between the mineral and organic constituents. Considerable similarity among the ESR spectra of enamel, bone, and carbonate-containing apatite was obtained after X-ray irradiation in air at 300°K with both an X-band and a Q-band ESR spectrometer. The Q-band spectrum can be interpreted in terms of two paramagnetic species. One is identified as a CO 3 3− anion radical which has an axial symmetry withg factors of 2.0029 and 1.9972. The other species is found to be centered atg=2.0019.

Notes: Abstract Photovoltaic effect (infrared and visible light) is observed in bone and its two major components, collagen and apatite, at room temperature. A dimunition in the magnitude of photovoltage is observed after exposure to ultraviolet light in all the cases. The drift mobility of the charge carriers is obtained by measuring I versus V relationships in sandwich samples and relating them to the permitivity of the medium. Lifetime of the injected carriers is measured in the usual way. The data are consistent with the hypothesis that the effects are due to protonic conduction phenomena.

Notes: Summary Mineral particle cluster, corresponding to calcospherites or matrix vesicle-initiated clusters in calcifying cartilage and dentin and to collagen bundle-related mineralization in lamellar bone, have been isolated from NaOCl solutions used to dissolve the organic matrix in preparation of anorganic mineralizing fronts for scanning electron microscopy.

Notes: Summary Osteoclastic bone resorption involves the solubilization of the mineral salts and the degradation of noncollagen bone matrix and collagen fibrils. As no recognizable collagen fibrils have ever been reported within cytoplasmic vacuoles in osteoclasts, it is generally assumed that the collagen fibrils are digested extracellularly in the resorption zone. The extent to which lysis occurs extracellularly and whether or not the osteoclasts phagocytose the degradation products remain to be established. In the present communication, a hypothesis is presented suggesting the possibility that osteoclastic resorption of bone involves the participation of two different cell types. According to this hypothesis, osteoclastic bone resorption is initiated by osteoclasts that demineralize areas of bone and degrade noncollagen bone matrix. After the osteoclasts have moved away or become partially detached from the demineralized site, the exposed collagen fibrils are phagocytosed by mononuclear, fibroblast-like or monocyte-derived cells.

Notes: Summary Plasmaα 2HS-glycoprotein is specifically accumulated in calcified tissues. In the present studies this glycoprotein was isolated from plasma and after iodination with iodine-125 was injected intravenously into young rabbits. The tissue distribution and plasma disappearance rate of this radioactively labeled material were determined. Of the various tissues studied, bone showed the greatest retention of labeled glycoprotein expressed as percentage of the injected dose per gram tissue relative to the plasma content. The rate of loss of iodinatedα 2HS-glycoprotein from plasma was similar to that ofα 2HS-glycoprotein labeled endogenously by using14C-glucosamine or3H-glucosamine. The uptake of exogenously labeled3I-α 2HS-glycoprotein into bone tissue expressed as a percentage of the injected dose was similar to that of endogenously labeled14C-α 2HS-glycoprotein. These results suggest that the125I-labeled material can be used to study further the metabolism ofα 2HS-glycoprotein by bone tissue.

Notes: Summary Mithramycin suppresses bone resorption. Its effect on the synthesis and release of β-glucuronidase (a referent for lysosomal enzymes) in mouse calvarial explants was studied in an in vitro culture system. A newly described medium (designated as KT medium) was introduced in this specific study. Mithramycin initially inhibited the release of β-glucuronidase into the medium and resulted in an ultimate accumulation of this enzyme in the bone. These results suggest that inhibition of bone resorption by mithramycin may be attributed to interference in release of lysosomal enzymes from bone cells.

Notes: Summary Thaw-mount autoradiographic studies after injection of3H-1,25-D3 were conducted on 18-and 20-day-old rat fetuses. In maxillary bones, ribs, and tibia, nuclear concentration of radioactivity was found in osteoprogenitor cells and osteoblasts. Osteocytes and chondrocytes in epiphyseal plates were either unlabeled or weakly labeled. In competition experiments, nuclear concentration of radioactivity was blocked by the injection of a high dose of nonradioactive 1,25-D3 prior to the administration of the labeled hormone, but not by a similar dose of nonradioactive 25-D3. The results are interpreted as indicating that osteoprogenitor cells and osteoblasts are target cells for the direct action of 1,25-D3 on fetal bone.

Notes: Summary The role of vitamin D in early skeletal development was studied by measuring serum calcium and phosphorus, osseous tissue quantity and mineralization, and endochondral bone elongation in rat fetuses and pups from vitamin D-replete and vitamin D-deficient mothers. At the 20th day of pregnancy there was a slight, yet significant, increase in the amount of osteoid on trabecular bone surfaces in fetuses from vitamin D-deficient mothers. The fetal bones otherwise appeared normal in spite of severe skeletal changes in the vitamin D-deficient mothers. After parturition, the importance of vitamin D in skeletal development becomes progressively more obvious. Serum calcium levels were slightly, yet significantly, lower in vitamin D-deficient than in vitamin D-replete pups and these levels continued to fall in the vitamin D-deficient pups through lactation and after weaning. At 3 days postpartum, there was a small, yet significant, increase in the amount of osteoid on bone surfaces of the vitamin D-deficient pups. The relative amounts of osteoid in the vitamin D-deficient pups continued to increase through lactation and after weaning when compared with vitamin D-replete pups. By the 14th day of lactation and at later periods, there were significant reductions in metaphyseal mineralized tissues in the vitamin D-deficient pups when compared with the vitamin D-replete pups. At weaning and after weaning, there were substantial increases in growth plate thickness and decreases in longitudinal bone growth in the vitamin D-deficient pups when compared with the vitamin D-replete pups. The results from this study indicate that vitamin D does not appear to play a major role in fetal skeletal development. However, after birth, vitamin D becomes progressively more important with age for normal bone development, mineralization, and endochondral growth.

Notes: Summary When demineralized bone matrix powder is implanted subcutaneously in the rat, the early responses involve the appearance and proliferation of mesenchymal cells at the site of implantation, followed by cartilage and bone formation. The ability of cells to migrate to the implant suggests that chemotaxis may be a critical event in this process. Therefore, using the modified Boyden chamber assay, we tested extracts of demineralized bone matrix for chemotactic activity. We have identified and partially purified, on molecular sieve chromatography, a heat labile and trypsin-sensitive protein (Mr=60,000–70,000) that is a potent chemoattractant for mouse calvaria, osteoblast-like cells (MMB-1), but not for monocytes (putative osteoclast precursors). These findings suggest that chemotactic protein(s) have a significant role in the recruitment of osteoprogenitor cells to a site of bone repair.

Notes: Summary Uptake of tetracycline by enamel in the short-term was studied at an advanced stage of crown formation and after completion of crown formation in deciduous molars in the cat. Both secretory phase enamel and bands of postsecretory, maturation phase enamel labeled rapidly. The pattern of labeling mimicked that seen in the continuously growing, rootless incisor teeth of the rat, with narrow doublets fusing to form narrow bands with wide unlabeled intervals in the short term. This is a physiological demonstration which indicates that cyclical activity and changes may occurin vivo during the maturation phase of amelogenesis in rooted teeth. It is also noted that dentine did not, and that some circumscribed patches of bone did label in the same animals in the same time interval. Short-term tetracycline labels are lost following conventional histological processing, but are retained after freeze-drying or air-drying.

Notes: Summary It has long been known that the stress history of bone tissue influences its structure; however, the nature of this relationship remains largely uncharacterized. The objective of this work was to induce a quantifiable change in the stress history ofin vivo bone tissue and examine subsequent changes in structural and material properties that might occur. Continuous compressive loads were applied to the diaphysis of adult mongrel dogs for 2 months. The loads, ranging from 12–130 N, were superposed on the normal activity of the animals by implanting spring loading devices on the diaphysis of the femur. After the animals were sacrificed, mid-diaphysial specimens were subjected to compression testing to determine a structural bulk stiffness. The cross-sectional areas of original bone tissue and new bone deposition were then determined. The ash weights of selected specimens were also determined. The results indicate that a positive correlation between the increase in cross-sectional area and the superposed stress does exist. The new bone apposition was found almost entirely on the periosteal surface. Very little evidence of internal remodeling or endosteal movement was observed. The new tissue was found to have a lower ash weight and appeared to have a disorganized microstructure. Mechanical testing also suggests that the newly deposited tissue is far less stiff than the mature original bone.

Notes: Summary A new semiautomatic technique combining advantages of the manual and fully automatic methods is described for obtaining quantitative static and dynamic histologic data of bone. The hardware consists of a photomicroscope, digitizing platen, digitizer, plotter/printer, floppy disc drive, and computer. The microscope is equipped with a drawing tube through which the image of the digitizing platen is projected over the optical field. The investigator selects and traces all histologic structures to be measured by moving a cursor on the digitizing platen which is visible by its projection over the histologic field. The results on accuracy and static and dynamic precision of this method show that static and dynamic parameters of bone are obtained with a degree of error (〈20%) well within the acceptable range for biologic measurements. Comparison of this method with the grid technique according to Merz and Schenck showed that for almost all micromorphometric parameters comparable absolute data are obtained. Due to the higher precision of our method, however, the number of optical fields evaluated in obtaining these comparable data could be reduced to 25% of the number of fields evaluated by the Merz and Schenck technique. The time requirements for quantitative evaluation of a histologic slide of bone by our technique are 40–50 min; 20–25 min is needed for quantitative evaluation of osteocytes.

Notes: Summary Quantitative bone histology was done in undecalcified sections of iliac crest bone specimens obtained from 84 normal American individuals. Samples were obtained within 12 h after death in a vertical and horizontal manner from both the right and left iliac crests. In addition to the determination of normal values of micromorphometric parameters of bone in these healthy American subjects, the following studies were carried out: (a) comparison of variance of micromorphometric parameters of bone obtained from the right versus left iliac bone (40 pairs), (b) comparison of micromorphometric parameters of bone obtained in a vertical versus horizontal manner (12 pairs), (c) evaluation of variance with increasing distance from the compact zone in bone samples obtained in a vertical manner (44 pairs), (d) analysis of variation between bone samples obtained more anteriorly versus posteriorly along the iliac crest (N=40), (e) comparison of differences in micromorphometric parameters obtained from age-matched men versus premenopausal women (N=12), and (f) plotting of histograms for assessment of distribution of micromorphometric parameters. The results show that histomorphometric data of bone cannot be easily compared when different techniques are employed for obtaining bone samples. Sampling variations are kept smaller when bone specimens are obtained in a vertical manner. Anterior/posterior variation does not cause major sampling error. If ranges of variation are taken into account, quantitative bone histology is a valuable tool for assessment of bone structure and bone cells.

Notes: Summary The linear attenuation coefficient (μ in cm1) of trabecular bone was modeled for different conditions of bone and marrow composition in order to assess their influence on computed tomography (CT) quantitation. A large relative change (10% of TBV at 15% TBV) of bone concentration resulted in small changes of μ: 2.3% at 60 keV, 3.4% at 44 keV, 5.2% at 29 keV. Relative changes of trabecular bone volume (TBV) on the order of 3% could be detectedin vivo by CT were it not for errors of relocation and for compositional influences on accuracy. The μ (and density) depended critically not only on amounts of bone substance and marrow but on their compositions. Normal variation in the composition of bone substance produced an uncertainty in μ equivalent to 0.5 to 1% TBV. Increases of yellow marrow produced a decrease of μ which could be mistaken for a decrease of bone concentration. The biological variation (90% confidence limit) of marrow composition gives an uncertainty at 15% TBV of about 2.4% TBV at 60 keV, 1.7% at 44 keV, and 1.3% at 29 keV. These correspond to relative uncertainities of 16, 11, and 9% respectively. These factors help explain the large accuracy errors (30%) observed in all studies of trabecular bone where single-energy CT was used. Marrow composition also can affect precision of bone measurement. Systematic shifts of red and yellow marrow could mask biological changes such as those occurring with aging or treatment.

Notes: Summary We have analyzed the sequences of soybean leghemoglobin genes as an initial step toward understanding their mode of evolution. Alignment of the sequences of plant globin genes with those of animals reveals that (i) based on the proportion of nucleotide substitutions that have occurred at the first, second, and third codon positions, the time of divergence of plant and animal globin gene families appears to be extremely remote (between 900 million and 1.4 billion years ago, if one assumes constancy of evolutionary rate in both the plant and animal lineages) and (ii) in addition to the normal regulatory sequences on the 5′ end, an approximately 30-base-pair sequence, specific to globin genes, that surrounds the cap site is conserved between the plant and animal globin genes. Comparison of the leghemoglobin sequences with one another shows that (i) the relative amount of sequence divergence in various coding and noncoding regions is roughly similar to that found for animal globin genes and (ii) as in animal globin genes, the positions of insertions and deletions in the intervening sequences often coincide with the locations of direct repeats. Thus, the mode of evolution of the plant globin genes appears to resemble, in many ways, that of their animal counterparts. We contrast the overall intergenic organization of the plant globin genes with that of animal genes, and discuss the possibility of the concerted evolution of the leghemoglobin genes.

Notes: Summary The nucleotide sequence ofDrosophila melanogaster 5S RNA has been determined and appears to be homogeneous both in the KC cell line and in the insect at different developmental stages. Experimental evidence on the conformation of this molecule is in agreement with a general class of 5S RNA models.

Notes: Summary If a phenotypic character is under stabilizing selection, the selective disadvantage of a nonoptimal genotype will decrease exponentially to zero as the proportion of phenotypic variation that is environmental in origin -V e /V p - increases. Under the modified mutation-drift hypothesis of genetic polymorphism, the proportion of mutations that are effectively neutral and average heterozygosity should increase with this ratio. Invertebrates, because of their small size, fast development, and low degree of homeostasis (relative to vertebrates), are expected to show a larger environmental component of phenotypic variation than vertebrates. This may help explain why invertebrates are in general more genetically variable than vertebrates and why, when laboratory populations ofDrosophila are maintained in heterogeneous environments, genetic variability is lost less rapidly than when they are kept in constant conditions.

Notes: Summary The evolutionary behavior of two mitochondrial enzymes (L-glycerol 3-phosphate:cytochrome c oxidoreductase E.C.1.1.1.95,αGPO, and L-malate: NAD+ oxidoreductase, E.C.1.1.1.37, m-MDH) obtained from several temperate and tropicalDrosophila species was examined by comparing their catalytic properties, which related to temperature (Km-Ea-Q10-Thermostability). MitochondrialαGPO or m-MDH obtained either from temperate or from tropical species was found to exhibit similar catalytic properties while for both cytosolic enzymes, theαGPDH and s-MDH, Km patterns were similar among species from the same thermal habitat and different between thermal habitats. In combination with other observations reported in the literature these facts support the view that the function, and probably the structure, of mitochondrial enzymes are better conserved in evolution than those of the corresponding enzymes found in the cytosol. It is proposed that the relative invariance of the mitochondrial enzymes structure is probably linked to a necessary relative invariance of molecular interactions inside the mitochondrion.

Notes: Summary The 68C puff is a highly transcribed region of theDrosophila melanogaster salivary gland polytene chromosomes. Three different classes of messenger RNA originate in a 5000-bp region in the puff; each class is translated to one of the salivary gland glue proteins sgs-3, sgs-7, or sgs-8. These messenger RNA classes are coordinately controlled, with each RNA appearing in the third larval instar and disappearing at the time of puparium formation. Their disappearance is initiated by the action of the steroid hormone ecdysterone. In the work reported here, we studied evolution of this hormone-regulated gene cluster in themelanogaster species subgroup ofDrosophila. Genome blot hybridization experiments showed that five other species of this subgroup have DNA sequences that hybridize toD. melanogaster 68C sequences, and that these sequences are divided into a highly conserved region, which does not contain the glue genes, and an extraordinarily diverged region, which does. Molecular cloning of this DNA fromD. simulans, D. erecta, D. yakuba, andD. teissieri confirmed the division of the region into a slowly and a rapidly evolving protion, and also showed that the rapidly evolving region of each species codes for third instar larval salivary gland RNAs homologous to theD. melanogaster glue mRNAs. The highly conserved region is at least 13,000 bp long, and is not known to code for any RNAs.

Notes: Summary Injection of α-ecdysone into the larval haemolymph of late third instar larvae ofD. virilis induces both the extrusion of secretory proteins and the inactivation of the enzyme glutamine-fructose-6-phosphate-aminotransferase (E.C. 2.6.16) in the salivary glands. In the presence of actinomycin D or cycloheximide the hormone is ineffective. If before adding these inhibitors RNA synthesis is allowed to proceed for 1.5h, or protein synthesis for 2h after ecdysone injection, however, the protein extrusion and the enzyme inactivation do occur. It is proposed that ecdysone controls these two cytoplasmic events at the transcriptional level by the activation of specific Correlations with puff activities are discussed.

Notes: Summary 1. The developmental potentials of dissociated cells of the different regions of the male foreleg disc ofDrosophila melanogaster were analysed. To this end, various amounts of foreleg disc material were dissociated together with an excess of heavily irradiated wing discs (“feeding layer”), and the reaggregates were cultured for 10 days in the abdomens of adult hosts prior to metamorphosis. 2. The foreleg disc cells were in most cases unable to regenerate missing structures in a circular direction within the leg segments. Instead they strongly tended to adopt the specifications of more distal leg segments (distal transformation), irrespective of the region of origin of the ancestor cells within the disc. 3. The distal transformation occurred mainly, if not exclusively, during an early phase (“initial phase”) in the reaggregates. 4. The extent of distal transformation was most pronounced in those series in which the foreleg cells were initially least diluted by the “feeding layer” cells. 5. Cells of the lower lateral quadrant were very poor both in proliferative activity and in the extent of distal transformation, compared to cells of the three remaining quadrants. In the experiments with a low initial dilution of the foreleg cells, cells of the lower medial quadrant underwent distal transformation much more distinctly than cells of the upper medial and the upper lateral quadrants. 6. Allotypic structures occurred exclusively in reaggregates of the upper medial and upper lateral quadrants. In these implants, however, the frequency of transdetermination was extremely high. 7. Two alternative mechanisms are discussed which could have led to the general occurrence of distal transformation. They differ in the basic assumption of whether or not the “feeding layer” cells were able to interact with the leg cells to influence their regulative behaviour. In addition, interactions among the leg cells themselves seemed to stimulate proliferation to varying degrees and may account for the observed differences in the degree of distal transformation.

Notes: Summary Three-hundred and twenty fertile,pal-induced Y-chromosome mosaic males and females were obtained. Fractional analysis of the sons of 55 somatically mosaic flies that were also germinally mosaic tentatively suggests that the number of functional primordial germ cells inDrosophila melanogaster is variable and that it is seldom greater than 24. From the observed 0.17 frequency of germinal mosaicism it was estimated that the average number of pole cells at the end of blastoderm formation is 45. At present, the germ cells afford the only opportunity to compare genetic estimates of the number of blastoderm or primordial cells with available histological counts. The good agreement between them suggests that both the fractional and the mosaic frequency methods for estimating primordial or blastoderm cell numbers of various larval and imaginal anatomical structures provide reasonably close approximations of the actual values.

Notes: Summary 1. Histological analyses were made of imaginal discs and histoblasts during the larval development ofDrosophila melanogaster to determine the number of cells, the patterns of cell division and the growth dynamics in these adult primordia. Histological studies were also made of the imaginal rings which are the primordia of the adult salivary gland, fore-and hindgut, the anlage cells of the midgut and several larval and embryonic tissues. 2. In the newly-hatched larva, the immature eye-antenna, wing, haltere, leg and genital discs contain about 70, 38, 20, 36–45 and 64 cells respectively. These numbers include cells destined to form cuticular elements as well as peripodial, tracheal and nerve cells and probably the progenitors of adepithelial cells. The number of cells counted in the various imaginal disc anlagen is 1.5 to 4 times higher than the numbers deduced from genetic mosaic analyses by other investigators and reasons for these differences are given. 3. About 12 h after fertilization, mitosis ceases in all tissues of the embryo except the nervous system. After the larva hatches, mitosis resumes in most of the imaginal anlagen and in some larval tissues. The time of resumption of mitosis in the imaginal anlagen was determined after treating the larvae with colchicine for 2 h. 4. Among the imaginal discs, the eye disc is the first to begin cell division, at about 13–15 h after the hatching of the larva (first instar) followed by the wing (15–17 h), the haltere (18–20 h), the antenna, leg, and genitalia (24–26 h, early second instar), and finally the labial and dorsal prothoracic discs (52–54 h, early third instar). The cell doubling time for various discs was calculated from cell counts and the times agree closely with the doubling times deduced from clonal analyses by other workers: e.g., 7.5 h for the cells of the wing disc. 5. The imaginal ring of the hindgut first shows cell division early in the second instar. The imaginal rings of the foregut and salivary glands, the anlage cells of the midgut and the cells of the segmental lateral tracheal branches begin to divide early in the third instar. 6. The histoblasts which are the anlagen of the integument of the adult abdomen do not increase in number from the time of larval hatching until about 5 h after pupation when they begin to divide. Their behaviour contrasts with that of the histoblasts of the other dipterans such asCalliphora, Musca andDacus, which begin to divide during the second instar. 7. The histoblasts are an integral part of the larval abdominal epidermis and, unlike imaginal disc cells, secrete cuticle during larval life. Each hemisegment consists of an anterior dorsal, a posterior dorsal, and a ventral histoblast nest containing about 13, 6 and 12 cells respectively. The 62 histoblasts in each larval segment represent about 7–8% of the total number of cells that form the integument of that segment. 8. The number of cells in a particular type of histoblast nest was constant for both male and female larvae and among the different abdominal segments, except that the anterior dorsal group of the first and the seventh segments contains fewer cells than those of the other segments. Although the male and female adultDrosophila lack the first abdominal sternite and the male lacks the seventh abdominal tergite and sternite, the ventral histoblast nests of the first and the dorsal and ventral nests of the seventh abdominal segments are present in the larval stages as well as in the prepupa and have the same morphology and cell number as similar nests in the rest of the abdominal segments. 9. The cells of the imaginal discs increase in volume about six-fold and their nuclei increase in volume three-fold between the time of hatching and the initiation of mitosis. The histoblasts increase in volume about 60-fold and their nuclei increase in volume about 25-fold between larval hatching and pupariation. 10. Prior to each cell division, the nuclei of the columnar cells of the disc epithelium and of the histoblasts appear to migrate toward the apical surface of the epithelium. The cells round up and shift toward the apical region where mitosis occurs. After cytokinesis, the daughter cells move back to deeper positions in the epithelium. Because the nuclei of the non-dividing cells continue to lie deep in the epithelium, this intermitotic migration of nuclei gives these epithelia a pseudostratified appearance. 11. Analyses of the growth of larval cells and of organs confirmed the observations of earlier investigators that cell division occurs only in a few larval tissues, whereas growth in the rest of the larval tissues is by cell enlargement and polyteny. During larval life, cell division was detected only in the central nervous system, gonads, prothoracic glands, lymph glands and haemocytes. Each tissue began mitosis at a characteristic stage in larval life. The larval cells that did not divide, grew enormously, e.g., epidermal cells increased in volume 150-fold and their nuclei increased in volume 80-fold. 12. The adepithelial cells, which give rise to some of the imaginal muscles, were first identified between the thick side of the imaginal dise epithelium and the basement membrane at the beginning of the third larval instar (50–52 h). The origin of these precursors of mesodermal structures was analysed and evidence is presented that the adepithelial cells come from the disc epithelium. The question of the origin of the mesoderm of cyclorrhaphan Diptera is reviewed and it is suggested that the imaginal disc ectoderm may become segregated from the rest of the embryo before gastrulation has occurred, that is before the mesoderm has been established.

Notes: Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis. The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos. Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development. All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae. Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.

Notes: Summary The lineages of cells on the second-leg basitarsus ofDrosophila melanogaster were analyzed by examining gynandromorphs andMinute mosaics. Bracts lie proximal to bristles on the adult basitarsus, yet bract precursor cells were found to originate lateral to bristle precursor cells. In 6 of the 8 longitudinal rows of bristles on this segment, the bract cells arise ventral to the bristle cells; in the others they arise dorsally. The lateral cell origins are interpreted as reflecting a pattern of lateral cell movements associated with evagination of the leg disc. An unusual discrepancy was observed in the relative frequencies of male vs. female bracts and bristles in gynandromorphs. The discrepancy suggests that there is a cell-autonomous sexual difference in either the time at which cells begin moving during evagination or the speed with which they move. On the basis of the results, it is reasoned that the bristle pattern of the basitarsus does not originate in its final form. Prior to evagination, the bristle cells of each row are apparently closer together than in the final pattern, and the rows are farther apart. Evidence is presented which suggests that the bristle cells of each row may originally be arranged in a jagged line which is later straightened by cell movements. The two locations where the anterior/posterior compartment boundary of the second leg passes through the basitarsus were found to vary relative to the bristle pattern. If this boundary is assumed to be a fixed line of positional values, then the extent of the observed variability — which is estimated to be ± 1 or 2 cell diameters — provides a measure of the precision of patterning around the circumference.

Notes: Summary We have analysed the effect of temperature on both developing and adult eye cell clones homozygous forshi ST139, a temperature-sensitive mutant ofDrosophila melanogaster. The mutant gene, autonomous in its cellular expression, causes structural modifications of ommatidial cells when adult clones of cells are exposed to the restrictive temperature (29°C) for several days. However, the mutant phenotype reverses to normal within 4 days at the permissive temperature (20°C). The results of pulse, shift-up and shift-down experiments show that the temperaturesensitive period for developing compound eye cells is from the late second instar up to the early pupa. Cytodifferentiation of compound eye cells is blocked by restrictive temperature treatment during this period, whereas cell proliferation does not seem to be directly affected. These results are discussed with regard to the other known aspects of the phenotype observed in mutant individuals.

Notes: Summary The differentiation of muscles in primary cultures of cells fromDrosophila melanogaster embryos was investigated. In early cultures, and in the absence of exogenous ecdysone, two main classes of muscle were found. Comparison, by light and electron microscopy, of one of these classes (the “myotube” class) with muscles from third instar larvae shows that this class corresponds to the muscles of the body wall of the larva. When α- or β-ecdysone is added to the cultures, these undergo a number of metamorphic changes. Most of the larval muscles disappear, and two new types of muscle form. Ultrastructural and light microscopic examination of these two types indicates that they correspond to the two classes of skeletal muscle (fibrillar and tubular) found in adult flies.

Notes: Summary The pathway of adult sensory nerves has been analysed in three experimental situations: (i) in flies with grossly abnormal thoracic morphology resulting from X-irradiation early during development, (ii) in flies which had been subjected to surgical operations late in the larval period, (iii) in homoeotic mutants. The results provide experimental support for a simple mechanism in which developing adult axons join the nearest larval nerve and are guided by it up to the central nervous system. In particular, experimental interference with normal development can result in nerves from different segments, or from dorsal and ventral appendages, joining each other and entering the central nervous system together.

Notes: Summary The early morphogenesis of the eye-antennal disc ofDrosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial membrane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.

Notes: Summary The mature labial disc, when implanted into a larva of the same age, undergoes metamorphosis along with the host and produces one lateral half of the medi- and distiproboscis. On the basis of results obtained from transplanted disc halves (including the separate peripodial membrane) a tentative fate map of the labial disc was constructed, which shows most of the presumptive mediproboscis to be located in the dorsal, and most of the presumptive distiproboscis in the ventral part of the disc. The distal protion of the peripodial membrane also contains imaginal anlagen, viz. part of the mediproboscis, prementum, and labellar cap anlagen. The involvement of this part of the peripodial membrane was checked by a careful histological analysis of labial disc development during the first ten hours after prepupation. The results were compared with the situation described forCalliphora imaginal discs. In addition, a detailed morphological analysis was made of the proboscis of the homoeotic mutantproboscipedia (pb). At 27°C,pb changes the distiproboscis into a “telopodite” (leg segments distal to the coxa); the (unchanged) prementum may therefore correspond to the coxa. At 15° C, the tarsus of this homoeotic “telopodite” is replaced to a greater or lesser extent by an arista. The present analysis thus confirms (a) the fundamental morphological correspondence of the medi- and distiproboscis with the labium of other insects, and (b) the fundamental developmental correspondence of the labial, antennal, and leg discs.

Notes: Summary A temperature-sensitive period during early embryogenesis for three stocks carrying thetuh-3 gene suggests that it is a homoeotic mutation involved in the initial determination of the eye-antennal disc rather in maintenance of the determination.

Notes: Summary The morphology of the evaginating female genital disc ofDrosophila melanogaster was examined at different stages of metamorphosis. The observations show that the internal genital organs are derived from the anterior half of the disc and that their morphogenesis is mainly a protrusion of the different primordial areas of the disc epithelium. The external genital and anal derivatives originate from the posterior half of the disc, which undergoes complex rearrangements during metamorphosis. The disc opens along the posterior margin and the dorsal and ventral epithelia evert and thereby completely reverse their anteroposterior orientation. Dramatic elongation has been observed during the formation of the seminal receptacle. The cells of the repressed male genital primordium do not form any recognizable structures and are assumed to be eliminated during metamorphosis.

Notes: Summary The effects of cytidine and cytidine analogs were studied inDrosophila embryonic cell cultures and two wild-type established cell lines, Oregon-R and Schneider line 2. Primary embryonic cultures have been shown to be an excellent system for the study of embryonic development; a number of cell types undergo normal differentiation in vitro. Treatment of these cultures with putative teratogens resulted in an inhibition of muscle and/or neuron differentiation in our study. Treatment of these cells with cytidine and seven other analogs had no effect on neuron and muscle differentiation. The compound 5-azacytidine, when added to primary cell cultures, inhibited normal differentiation at subtoxic doses while inducing the production of three proteins that comigrate with the heat-shock proteins, hsp 23, 22a and 22b. 5-Azacytidine did not stimulate differentiation in Oregon-R or SchneiderDrosophila cell lines. The in vitro blockage of differentiation by 5-azacytidine suggests that it may act as a teratogen.

Notes: Summary In a search for embryonic lethal mutants on the second chromosome ofDrosophila melanogaster, 5764 balanced lines isogenic for an ethyl methane sulfonate (EMS)-treatedcn bw sp chromosome were established. Of these lines, 4217 carried one or more newly induced lethal mutations corresponding to a total of 7600 lethal hits. Eggs were collected from lethal-bearing lines and unhatched embryos from the lines in which 25% or more of the embryos did not hatch (2843 lines) were dechorionated, fixed, cleared and scored under the compound microscope for abnormalities of the larval cuticle. A total of 272 mutants were isolated with phenotypes unequivocally distinguishable from wild-type embryos on the basis of the cuticular pattern. In complementation tests performed between mutants with similar phenotype, 48 loci were identified by more than one allele, the average being 5.4 alleles per locus. Complementation of all other mutants was shown by 13 mutants. Members of the complementation groups were mapped by recombination analysis. No clustering of loci with similar phenotypes was apparent. From the distribution of the allele frequencies and the rate of discovery of new loci, it was estimated that the 61 loci represent the majority of embryonic lethal loci on the second chromosome yielding phenotypes recognizable in the larval cuticle.

Notes: Summary EMS induced temperature-sensitivelethal (2) giant larva, 1(2)gl, alleles were isolated by screening against a knownl(2)gl allele. Analysis of the lethal phase of thel(2)gl ts-deficiency heterozygotes demonstrated: (1) the majority of thel(2)gl tslarvae survive to late third instar, (2) at 29°C the majority of thel(2)gl tslarvae failed to pupate and only rarely did they differentiate adult cuticular structures, (3) at 15°C the majority of the larvae pupated and frequently differentiated adult cuticular structures. Examination of the imaginal discs ofl(2)gl tslarvae reared at 29°C revealed the presence of morphologically abnormal wing, haltere and leg imaginal discs. No morphologically abnormal discs were found in thel(2)gl tslarvae reared at 15°C. Studies on both the histology and the developmental capacity of the morphologically normal and abnormall(2)gl tsdiscs were performed. The morphologically normal discs are histologically normal and produce a full complement of adult cuticular structures. However, the morphologically abnormal discs contained both regions that maintained the normal monolayer epithelium and regions that had lost the normal tissue architecture. The implants obtained when the morphologically abnormal discs are injected into metamorphosing larvae contained only a limited number of the normal complement of adult structures and usually only structures found in the ventral wing hinge region were recovered. In addition, the “metamorphosed” morphologically abnormal discs contained undifferentiated tissue that gave rise to transplantable neoplasms when cultured in adults. The results of the studies on the pathology of thel(2)gl tslarvae are discussed with respect to the role of thel(2)gl tsfunction during normal development, the autonomy of the neoplastic development of thel(2)gl tstissues, and similarities between neoplastic development inDrosophila and mammals.

Notes: Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.

Notes: Summary We transplanted pole cells betweenDrosophila melanogaster, D. mauritiana andD. ananassae to investigate the ability of germ cells to develop in the gonad of a heterospecific host, and to study the interaction between somatic follicle cells and the cells of the germ line in producing the species-specific chorion. FemaleD. mauritiana germ cells in aD. melanogaster ovary produced functional eggs with normal development potential. The same is true for the reciprocal combination. FemaleD. ananassae pole cells in aD. melanogaster host only developed to a very early stage and degenerated afterwards. None of the interspecific combinations of male pole cells led to functional sperm. We could not determine at what stage the transplanted male pole cells were arrested. The cooperation of follicle cells and the oocyte-nurse cell complex in producing the chorion was studied using the germ-line-dependent mutationfs(1) K10 ofD. melanogaster, which causes fused respiratory appendages and an abnormal chorion morphology. Wild-type femaleD. mauritiana germ cells in a mutantfs(1) K10 D. melanogaster ovary led to the production of wild-type eggs withD. melanogaster-specific, short respiratory appendages. On the other hand,D. melanogaster fs(1) K10 germ cells in aD. mauritiana ovary induced the formation of eggs with mutant fused appendages which were, however, typicallyD. mauritiana in length. When.D. mauritiana pole cells developed in aD. melanogaster ovary, the chorion exhibited a new imprint pattern that differs from both species-specific patterns.

Notes: Summary In this paper we describe the aggregation of cells from embryo-derived cell lines ofDrosophila, measured by examining the ability of single cells to adhere to one another when suspended in culture medium and swirled on a rotary shaker. Using this method we demonstrated the presence of receptors for Concanavalin A, soybean agglutinin, and possibly wheat germ agglutinin on the surface of Schneider's line-2 cells. Our work provides basic descriptive and background information for further studies onDrosophila cells, including those isolated from imaginal discs.

Notes: Summary Electrophoresis of myosin extracts from larvae and adult tissues ofDrosophila melanogaster under non-dissociating conditions indicate that two of the bands seen are myosins. They stain for Ca2+ ATPase activity and when cut and re-run under dissociating conditions are found to contain a myosin heavy chain that co-migrates with rabbit skeletal muscle myosin heavy chain. One of the forms of myosin seen is found primarily in extracts from the leg. The other is common to the adult fibrillar flight muscles and the larval body wall muscles. The electrophoretic evidence for two myosin types is strengthened by the histochemical demonstration of two myofibrillar ATPases on the basis of their lability to acid or alkali preincubation. The myofibrillar ATPase in the leg and the Tergal Depressor of the Trochanter (TDT) are shown to be relatively acid labile and alkali stable. The larval body wall muscles and the adult fibrillar flight muscles have an ATPase which is acid stable and alkali labile. This distribution of the two myofibrillar ATPase coincides with that predicted by electrophoresis of extracts from whole tissue and also locates the two myosins to specific muscle types.

Notes: Summary The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of “in vitro”3H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.

Notes: Summary The derivatives of 110 mosaic genital discs of gynandromorphs have been analysed microscopically. It has been found that theanalia of both sexes are homologous and derive from a single primordium (see Fig. 1a). Whether male or female anal plates are formed depends on the genetic constitution of the cells. This is analogous to the development of male sex combs versus female transversal rows on the forelegs of gynandromorphs. In contrast, the data for thegenitalia (see Fig. 1 b) are best explained if it is assumed that there are two genital primordia in everyDrosophila embryo: a male primordium that will only develop into genitalia if populated by XY (or XO) nuclei, and a female primordium that will only do so if populated by XX nuclei. This model, as depicted in Figure 2, is compatible with all our gynandromorph data and also with observations onMusca andCalliphora where in fact two separate genital primordia are found.

Notes: Summary Five regions of the compound eye have been found to be preferential boundaries for clones of labelledMinute + cells, and to act restrictively on the growth of cell clones after a given developmental stage. One of these regions is topographically related to the line of pattern inversion existing at the level of the equator. The results of experiments showing independency of origin of restriction lines and line of pattern inversion are reported.

Notes: Summary When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In “06” fragments (Bryant 1975a) wound healing has occurred after 1–2 days of culture and the regeneration of missing structures after 2–4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days. When disc fragments are cultured in female abdomens and then exposed to3H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.

Notes: Summary The transdetermination capacities of leg discs ofDrosophila melanogaster were examined by mechanically disrupting and kneading whole discs from late third instar larvae and by culturing the resulting tissue mass for 10–14 days in adult female abdomens where the cells continued to divide. The grown implants were then dissected from the abdomens and injected into third instar larvae to undergo metamorphosis. After this treatment, prothoracic leg discs ofDrosophila melanogaster transdetermined with a high frequency (59% of all implants) to wing. Mesothoracic leg discs also transdetermined to wing, but at a very low frequency (4%). Metathoracic leg discs exhibited the same low frequency of transdetermination (4%), but in this case the direction of transdetermination was to haltere (Table 1,D. melanogaster). Very similar results were obtained with leg discs ofDrosophila nigromelanica (Table 1,D. nigromelanica), showing that the peculiar behavior of the three leg discs is not unique forDrosophila melanogaster. The homeotic mutation Polycomb (Pc 3) which partially transforms meso- and metathoracic legs into prothoracic legs did not significantly increase the frequencies of transdetermination in these leg dises and had clearly no effect on the direction of transdetermination (Table 1).

Notes: Summary The aldehyde oxidase staining pattern in wing discs ofDrosophila melanogaster bearing the genotypesap blt /ap blt andap blt andap blt /ap 73n showns changes from the wild-type pattern. Extensive areas of the presumptive dorsal posterior wing blade, which are normally unstained, have enzyme activity in these mutants. In wings of these genotypes, dorsal posterior structures are replaced by dorsal anterior wing structures. A strong correlation has been found between the frequencies of various staining patterns in the discs and the extent of transformation in the cuticular structures of the wing, which is consistent with the idea that aldehyde oxidase activity can be used as an indicator in the wing disc of this transformation. Unlike the homoeotic mutationengrailed, apterous has not been interpreted as a selector gene yet the work reported here shows thatapterous alleles can cause changes resembling those of theengrailed phenotype both in aldehyde oxidase staining behaviour and in the cuticular transformation.

Notes: Summary The chromosome which carries the mutationsexcombless (In(1)sx) affects males and females ofD. melanogaster. In the male foreleg basitarsi the number of sexcomb teeth is dramatically reduced from 10 to 0.7 and the number of transverse rows of bristles is increased from 6 to 8. Females homozygous forIn(1)sx show a normal bristle pattern in the foreleg basitarsus. The genital disc derivatives of both male and femaleIn(1)sx flies are strongly affected. While the external genitalia show a duplicated or a reduced bristle pattern, the internal genitalia are mostly absent. However, the sexually dimorphic tergites and sternites of the abdomen remain unaffected. The male-specific effect on the basitarsus and the general effects on the genital disc derivatives are proposed to represent two different phenotypic effects ofIn(1)sx which may derive from mutations at different gene loci in the inverted chromosome.

Notes: Summary Imaginal wing discs from late third-instar larvae were gammairradiated to induce clones of rapidly growingMinute − cells in a background of slowly growingMinute cells and culturedin vivo for periods up to 18 days. Clones in discs cultured for 16 to 18 days did not grow significantly larger than clones in uncultured controls, indicating that competition between populations of cells having potentially different mitotic rates does not occur in imaginal discs after their growth is completed.

Notes: Summary sev LY3,the only existing allele at thesev locus (1–33,2±0,2), behaves as strongly hypomorph or even as amorph. Ommatidia in asev compound eye have only seven receptor cells, the position of the R7 pattern element being vacant. Various criteria showing that the missing cell is R7 have been verified. These include (i) anatomical characteristics ofsev ommatidia; (ii) behaviour of central R cells insev rdgB double mutants; (iii) medullary projection of central R cell axons; and (iv) mitotic pattern ofsev imaginal discs. The analysis of morphogeneticsev-sev + mosaics has shown thatsev is expressed autonomously by R7 cells, indicating that thesev phenotype is not due to asev genotype of ommatidial pattern elements other than R7. The study of third instarsev imaginal discs has not brought any direct evidence for death of clustered presumptive R7 cells; however, clonal analysis of the developingsev compound eye has given evidence of developmental parameters comparable to those ofsev +, therefore favouring the hypothesis that R7 cells die insev mutants. On the other hand,sev + seems to be required for the determination of the R7 cells, since thesev phenotype cannot be uncovered during the last mitoses of heterozygous mutant cells.

Notes: Summary Morphogenetic movements of the intersexual genital disc of thedoublesex-dominant mutant ofDrosophila melanogaster were followed during metamorphosis. Intersexual genital discs contain well developed genital primordia of both sexes as well as an anal primordium, and all of these primordia evaginate simultaneously. The female genital primordium is deflected to the ventral side by the male genital primordium which is located anterior to it. Subsequently the anterior parts of the two genital primordia project their internal appendages in parallel in the anterior direction. The morphogenetic movements closely resemble those of the corresponding parts of normal males and females. The disc opens at the stalk along the posterior edge and the two genital primordia completely evert their posterior parts. These areas undergo complex rearrangements whereby the anlage for the male genital arch as well as that for the 8th tergite evert and move around the lateral side of the disc. They both fuse dorsally after enclosing the anal tube. The formation of the characteristic abnormalities of the intersexual genitalia seems not to result simply from spatial problems of the simultaneous evagination of the genital anlagen but rather to be a direct result of the ambiguous genetic signalling in the intersexual cells of these primordia.

Notes: Summary The genital imaginal disc ofDrosophila differentiates the terminalia, i.e. the genitalia and analia, of both sexes. It represents a composite anlage, containing a female genital primordium, a male genital primordium and an anal primordium. In normal males and females, only one of the two genital primordia differentiates; the other is developmentally repressed. Therefore, cell-lineage relationships between the male and female genital primordia can only be studied in sexual mosaics which differentiate female and male cells. We producedMinute (M)‖non-Minute(M+) gynandromorphs and selected those with sexually mosaic terminalia for a cell-lineage analysis. In these mosaics, either the male (XO) or female (XX) cells wereM + and thus had a growth advantage. The differential growth rates served as a tool to detect clonal restrictions. In control gynandromorphs (M +‖M +), the amount of female genitalia differentiated was largely independent of the amount of male genitalia present. In contrast, male and female anal structures, as a rule, added up to one full set. The same was true for the experimentalM‖M + gynandromorphs, but the contribution ofXX andXO cells to mosaic terminalia changed drastically due toM + cells competing successfully against the more slowly growingM cells. Specific subsamples ofM‖M + gynandromorphs showed thatM cells in a non-mosaic primordium are shielded from cell competition taking place in the neighbouring mosaic primordium. We conclude that the three primordia of the genital disc represent developmental compartments. In the genital primordia, even developmentally repressedM + cells compete successfully against developmentally activeM cells.

Notes: Summary In this paper we present an analysis of the behavior ofl(2)gl tsimaginal wing discs during culture in adult hosts. Thel(2)gl tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl +function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.

Notes: Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.

Notes: Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and α-glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.

Notes: Summary Changes in polytene chromosome 3 L puffing patterns in the fat body ofDrosophila melanogaster larvae and prepupae are compared to those in the salivary gland. While some general features are common to the two tissues, there are differences which reflect their different developmental roles. In vitro experiments with fat body chromosomes show that they have a distinct response to ecdysteroids which is different from that of salivary gland chromosomes, and which does not,in this culture system, reproduce the changes observed in normal development. In short term culture experiments, the fat body chromosomes appear more sensitive to ecdysteroids than the salivary gland chromosomes and, although 20-OH ecdysone is more active than ecdysone in these assays, the possibility is not excluded that ecdysone has a role in normal development as it appears to alter gene activity at physiological levels in these cells.

Notes: Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.

Notes: Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 μm−1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 μm−1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.

Notes: Summary The females produced in the crossesD. melanogaster×D. simulans andD. melanogaster×D. mauritiana are sterile and have reduced ovaries. Normal and fertile ovaries were produced when genetically marked pole cells ofD. melanogaster were transplanted into eggs which gave rise to the hybrid females. These results eliminate the possibility that the sterility of these hybrids is due to the somatic component, i.e. the follicular cells of the ovaries, or to other physiological causes. The results also suggest that the control of gonadal morphogenesis is dependent mainly on the germ line.

Notes: Summary Gynandromorphs with female XX-and male XO-areas result from the loss of an unstable ring-X-chromosome in the early cleavage mitoses of ring/rod-X-chromosome heterozygotes. The phenotypes of the recessive alleles on the rod-X-chromosome are expressed in the XO-areas. 377 larval gynandromorphs of the genotypeR(1)2, In(1)w vC /y w sn3Iz50e mal were examined and scored for the phenotypes of 13 paired and 10 unpaired structures (Table 2, Fig. 2). This was possible mainly by the cell-autonomous expression of aldehyde oxidase activity in soft tissues and by the comparison of the distribution of enzyme activity in wildtype and gynander larvae. The distances between pairs of structures were calculated in sturt-units (Tables 3 and 4). A morphogenetic fate map with the presumptive areas of larval structures was constructed (Fig. 3). The relative positions of the structures agree well with Poulson's fate map (Fig. 4). In addition, the distribution of phenotypes was scored in 380 adult gynandromorphs Table (5). The fate map (Fig. 5) which was constructed from these data is very similar to the fate map of larval structures. This similarity becomes even more pronounced if fate maps are constructed which contain only structures analogous in larva and imago (Table 6, Fig. 6). Therefore an attempt was made to set up an integrated morphogenetic fate map containing the presumptive areas of both larval and imaginal structures (Fig. 7). The possibilities of further blastoderm mapping are discussed.

Notes: Summary Females homozygous for a newly isolated mutation induced by ethyl methane sulphonate,fs(1)K10, lay abnormally shaped eggs in which the dorsal appendages of the chorion are enlarged and fused ventrally. The eggs are usually not fertilized and development is never normal beyond the blastoderm stage. The mutant was mapped to the tip of the X-chromosome with a meiotic position of 1–0.5 and a cytological location between 2B17 and 3A3. Using germ line mosaics constructed by transplantation of pole cells, it was shown that the abnormal morphology and the sterility are obtained only when the germ line is homozygous for the mutant.

Notes: Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.

Notes: Summary The bristle pattern of the second-leg basitarsus inDrosophila melanogaster was studied as a function of the number and size of the cells on this segment in well-fed and starved wild-type flies, in triploid flies, and in two mutants (dachs andfour-jointed) that have abnormally short basitarsi. The second-leg basitarsi of well-fed, wild-type flies from 22 otherDrosophila species were studied in a similar manner. There are typically 8 longitudinal rows of evenly-spaced bristles on the second-leg basitarsus, and in each row the number of bristles was consistently found to vary in proportion to the estimated number of cells along the segment, and the interval between bristles was found to vary in proportion to the average cell diameter on the segment. These correlations are interpreted to mean that the spacing of the bristles within each row is controlled developmentally, whereas the number of bristles is not. The interval between bristles is evidently measured either as a fixed number of cells or as a distance which indirectly depends upon cell diameter.

Notes: Summary Changes at the ultrastructural level during germ band extension in the embryo ofDrosophila melanogaster are described. Cytoplasmic connections between cells and the yolk sac are present during initial cellular movements. At this time, a continuous system of microfilaments is present adjacent to the membranes in the connections and at the periphery of the yolk sac. As germ band extension progresses, this system becomes discontinuous, and microfilaments are apparent only in the immediate vicinity of the connections. Cytoplasmic connections are disassembled at approximately the midpoint of extension; at the same time, extensive membrane associations develop between germ band cells and between these cells and adjacent yolk sac membranes. Positioning and orientation of cytoplasmic connections suggest that the yolk sac, via these connections, is actively involved in the cellular movements of early germ band extension.

Notes: Summary This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked mad-ts (mad-ts: Jürgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1–3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1 ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings. In the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.

Notes: Summary Peripheral tissue of the imaginal wing disc gives rise to the proximal mesothoracic structures of the adult. Pieces of peripheral tissue, which have no regenerative capacity when cultured as intact fragments, are capable of distal outgrowth (regeneration) after dissociation and reaggregation. This ability depends on the region of the disc periphery from which the fragment is taken. Extensive distal outgrowth occurs in reaggreages of a fragment containing equal proportions of tissue from anterior and posterior developmental compartments. The extent of outgrowth decreases as the proportion of posterior tissue is reduced, so that a fragment containing only anterior tissue shows no regeneration after dissociation. Limited distal outgrowth occurs in reaggregates of a wholly posterior fragment, but the regenerative capacity is increased greatly when a small amount of anterior tissue is included. It is concluded that distal outgrowth in the wing disc requires an interaction between cells of the anterior and posterior compartments.

Notes: Summary It has been known for many years that when a wing disc ofDrosophila is bisected, and the fragments cultured in adult females, regulation occurs and either a complete disc is regenerated or the fragment is duplicated. We have investigated how this regeneration process occurs. To establish which cells contribute to the regenerate, and thus determine if regeneration is the result of epimorphic regulation, fragments of discs, after culture in an adult for one to five days, were exposed to3H-thymidine to label replicating cells. Imaginal discs, both whole and as regenerating fragments, undergo some DNA replication which is distributed throughout the disc, but cut discs frequently show clusters of labelled cells around the wound, indicating that regeneration is probably epimorphic.

Notes: Summary Imaginal disc development in the non-pupariating lethall(1)npr-1, a mutant that maps to an ecdysone early puff site, is studied in situ, in vitro and in transplanted discs. Disc development is slightly abnormal from the middle of the third instar with severe abnormalities appearing after the rise in 20-hydroxyecdysone that triggers metamorphosis. The mutant discs only partly evaginate and do not undergo any of the detailed morphological changes characteristic of metamorphosis. Treatment of the mutant dises in vitro with colcemid and trypsin facilitates evagination but the appendages remain morphologically abnormal. A number of differentiative processes occur in mutant discs in situ and in discs transplanted into wild type hosts in spite of the absence of normal morphogenesis. Implications of the observations for normal disc development are discussed. Possible modes of action of thel(1)npr-1 gene are also discussed in light of the observation that the mutant gene maps to a locus which is thought to have a regulatory function in development.

Notes: Summary The temperature sensitive mutationfs(l)h is characterized at the restrictive temperature of 29°C by both a maternal effect responsible for the early embryonic lethality and pupal zygotic lethality. The two phenotypes are inseparable and map at a short deletion in the X chromosome (7Dl, 7D5-6). At semipermissive temperatures, hemizygous mutant females produce adults with morphological defects, such as organ deficiencies and homeotic transformations of haltere to wing and third leg to second leg. These defects depend on the maternal genotype and are governed by an early temperature sensitive period, which covers the end of oogenesis and the first hours of embryogenesis. Furthermore, this maternal effect mutation interacts with some dominant mutations of the bithorax system. These properties suggest thatfs(l)h is somehow involved in segmental determination.

Notes: Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1−33.8±0.7 forgs(1)N26 and 1−39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work. Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect. Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed. In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.

Notes: Summary The l(1)su(f)ts67g mutation has been shown to suppress the developmentally regulated expression of glue protein genes at 30°C. Transferring mutant larvae to the restrictive temperature before the end of the second larval instar results in the absence or extreme reduction of glue protein synthesis while general protein synthesis is unaffected. At the same time, the three glue protein correlated chromosomal regions 3C, 25B, and 68C continue to show prominent puffs. The results suggest that the mutation may be affecting the processing or translatability of specific mRNAs rather than the translational machinery itself.

Notes: Summary The mutationdicephalic (dic) affects follicle development and thereby alters the antero-posterior polarity of embryonic patterning. It maps at a single locus (3–46.0±1.0) and can be characterized as a semi-dominant maternal effect mutation with low penetrance. Indic follicles, the 15 nurse cells form two clusters located at opposite poles of the oocyte; the numerical distribution of the nurse cells among the clusters varies from 7:8 to 1:14. Thedic egg shell carries a micropyle (anterior marker) at either pole, but the misshapen respiratory appendages are restricted to one of the two poles in most eggs. The malformed eggs rarely yield larvae and these are always abnormal anteriorly and/or posteriorly. The segment pattern expressed in their cuticle may represent two anterior parts of opposite polarities (double head type), two posterior parts of opposite polarities (double abdomen type, rare) or show uniform polarity. Lability of organization at the cystocyte stage appears as the primary developmental defect of the mutant.

Notes: Summary Females ofDrosophila melanogaster, crossed with males ofDrosophila mauritiana, produce only female offspring. The male hybrid larvae grow very slowly, fail to pupate and die after prolonged larval life. Imaginal discs from these male hybrids transplanted into Drosophila melanogaster larvae can give rise to adult structures with normal patterns. Differentiation of hybrid imaginal disc tissue is improved by short term culture in non-hybrid larvae prior to metamorphosis, suggesting that the hybrid larval haemolymph is inadequate to sustain normal imaginal disc growth. This may represent the physiological basis of the reproductive isolating mechanism separating the twoDrosophila species

Notes: Summary The female genital disc ofDrosophila melanogaster was cut into distinct fragments, and the prospective fates of the fragments were determined by putting them through metamorphosis in host larvae. The dorsal epithelium contains the anlagen for the anal plates and parovaria, as well as the repressed male genital primordium. The ventral epithelium gives rise to all of the female genital structures except for the parovaria. The results were compared with published fate maps and observations made in experiments with sex-transforming mutations. This allowed us to establish a detailed three-dimensional fate map of the female genital disc, which shows a well-developed female genital primordium in the ventral epithelium, a repressed male genital primordium in the anterior part of the dorsal epithelium and an anal primordium in the posterior region of the dorsal disc epithelium.

Notes: Summary The mutationsvestigial (vg; recessive) andUltravestigial (vg U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense (“apoptosis”). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.

Notes: Summary Developmental changes in the distribution of gap junctions in early, mid and late third larval stage wing discs and in pupariation+6 h and pupariation+24 h stage wing discs fromDrosophila melanogaster were analyzed by quantitative electron microscopy. Gap junctions occur in all 12 intradisc regions examined in each of the five developmental stages. Their distribution is non-random and changes during development which suggests that they are developmentally regulated. The gap junctions are not static structures, rather they grow and regress during development. The changes tend to be gradual ones without sudden increases or decreases. Gap junctions continuously form and grow in size throughout the third larval stage and during the first 6 h following pupariation. Their surface density, number, percent of the lateral plasma membrane area, and absolute area as well as the lateral plasma membrane surface density all increase during this time. Between pupariation+ 6 h and pupariation+24 h all but one of these parameters decrease indicative of gap junctional breakdown. Gap junctions are most numerous and change least during development in the apical cell regions where intercellular contacts are close and stable. They change most in the basal cell regions where intercellular contacts tend to be looser and change during development. The most dramatic change is in the absolute area which increases by a factor of 23 between the early third larval stage and pupariation+24 h. At pupariation the rate of gap junction growth undergoes a transient increase before the phase of disassembly begins. Developmental changes in gap junction surface density are closely coupled with changes in the lateral plasma membrane surface density which suggests that these may be coregulated. Evidence from mutants suggests that when the number and density of gap junctions fail to increase in proportion to lateral plasma membrane growth, wing disc development will be abnormal. Our results support the idea that some minimum gap junction density is required for normal development and that this must increase as development proceeds. The results are consistent with the notion that gap junctions are involved in pattern formation and growth control and are discussed with respect to the acquisition of competence for metamorphosis, disc growth, disc morphogenesis and changes in the hormonal environment.

Notes: Summary Neurogenic mutations have been found to cause the neuralization of certain regions of the ectoderm and yet to permit normal development of the remaining embryonic cells. Thus, it seems that the activity of the wild-type alleles of these genes is dispensable in a considerable fraction of the embryo during wild-type development. This effect might be a consequence of the cells' position within the embryo; alternatively, it might be independent of the position but be due rather to the genetic activity experienced by the cells previous to their commitment. The results described in this paper indicate that genes controlling patterning along the embryonic dorso-ventral perimeter (dorsal and Toll) are epistatic to genes controlling neurogenesis, their activity deciding which ectodermal cells are susceptible to neurogenesis. Using alleles with low expressivity, evidence was obtained showing that the tracheal placodes define the boundary of the territory which has neurogenic abilities at thoracic and abdominal levels.

Notes: Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as ‘glue’ at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.

Notes: Summary The generalogical relationships of photoreceptor cells within the compound eye ofDrosophila have been studied using cell labelling, with either3H-thymidine or recessive mutations, during the third larval stage. It has been found that photoreceptor and secondary pigment cells arise from different precursor cells. Under the present experimental conditions, precursors of receptor cells give rise to about 8 elements which differentiate as R cells of two different groups. One of the cells differentiates as R7 and the remaining as any one of the R1 to R6. The last cells behave initially as equivalent, and can differentiate within the same or within different, but neighbouring, ommatidia. The class of R1 to R6 cell in which each one of these elements differentiates, seems to depend on the time of its origin. The implications of these findings for the formation of the ommatidial pattern are discussed.

Notes: Summary We report on the size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h oldDrosophila larvae. The results indicate that the radiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. We estimate that 1000 r reduces by 40–60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation; this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100 r. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.

Notes: Summary Immature ovaries ofDrosophila mercatorum were injected into young larvae and into adult males ofD. mercatorum, D. melanogaster, D. hydei, D. virilis, andZaprionius vittiger. These homo- and heteroplastic transplantations allow normal vitellogenesis to occur in the donor ovary. By SDS gel electrophoresis, we identified the major species-specific yolk proteins of mature eggs (stage 14) which were exclusively of donor-specific origin. Other experiments withD. hydei andZ. vittiger showed that, when females were used as hosts, the host-specific yolk proteins became incorporated into the donor eggs. When two immature ovaries, one ofD. mercatorum and one ofD. hydei, were co-cultured in males, again only the donor-specific yolk proteins were found in the mature eggs implying that these yolk proteins were not released into the host hemolymph. A parthenogenetic strain ofD. mercatorum was used to demonstrate the ability of transplanted immature ovaries to produce viable eggs which can give rise to fertile adults. The role of the species-specific yolk proteins is discussed with respect to the dual origin of these proteins during normal vitellogenesis, i.e., an autonomous synthesis within the ovary itself in addition to the well-known production by the fat body. Further experiments with pupae as hosts indicate that even in the absence of juvenile hormone and in the presence of high doses of ecdysone, vitellogenesis can proceed within the donor ovary. Based on these experiments, a new hyopthesis on the hormonal control of vitellogenesis inDrosophila is presented. We propose that yolk proteins derived from the fat body are controlled by juvenile hormone, whereas the independent and autonomous vitellogenesis within the ovary itself is controlled by endogenously synthesized ecdysone.

Notes: Summary We estimate the number of blastoderm cells which generate the thoracic imaginal discs ofDrosophila. At hatching the wing disc is twice the size of the haltere disc, but the results suggest that both discs develop from a similar number of blastoderm cells. Two homeotic mutations, which transform the haltere into wing, affect embryonic growth but not the primordial number. All the segmental primordia may be of similar size and each may be similarly subdivided into a larger anterior, and a smaller posterior polyclone.

Notes: Summary A hypothesis is presented which explains the segregation of germ cells from somatic cells, and the subsequent determination of both cell types with a single mechanism. This hypothesis is in part based on that of Meinhardt (1977) and can be summarized as follows: In the newly fertilized egg, the action of a sink in the pole plasm leads to the formation of an anterior-posterior gradient of an inhibitor. The concentration of this inhibitor in the posterior 20% of the egg is below that needed to repress synthesis of an activator. When, during the nuclear division stage, nuclei enter this posterior region, synthesis of the activator begins. As the activator is autocatalytic, this leads to the formation of a peak of activator in this region; and since the activator also catalyses the synthesis of the inhibitor, a peak of inhibitor is formed in the same place. The inhibitor then diffuses anteriorly through the periplasm, forming a posterior-anterior gradient. The presence of this inhibitor in the periplasm causes the nuclei that enter the periplasm to form blastoderm cells and to take up particular segmental states appropriate to their position, while those that remain in the yolk-containing plasm develop into vitellophages. The action of the sink in the pole plasm is postulated to result in the formation of the pole cells, and subsequently to direct some of these into forming cells of the germ-line.

Notes: Summary Cells of the line Kc, derived fromDrosophila melanogaster embryos, extend long processes when exposed to ecdysteroid hormones. We have devised a quantitative assay for this morphological response, using the subline Kc-H. The assay was used to characterize the conditions required for the response. A halfmaximal response is elicited by approximately 10−8M 20-hydroxyecdysone; the response is saturated by 10−7M 20-hydroxyecdysone, which causes detectable elongation within a few hours, and a maximal response after 2–3 days. The response occurs substantially normally in the absence of serum, during growth in suspension, and in over-crowded cultures. It is not elicited by cyclic nucleotides, vertebrate growth factors, or a variety of other non-ecdysteroid reagents. Of 60 ecdysteroid compounds tested, only those which were active in other insect test systems elicited the response, and the concentrations required were approximately proportional to the concentrations active in other in vitro systems. We conclude that the response of Kc cells to 20-hydroxyecdysone retains basic features of the ecdysteroid response of intact tissues and therefore that Kc cells are a useful model system for studying ecdysteroid action.

Notes: Summary TheDrosophila chorion contains an endogenous peroxidase activity which remains inactive until late stage 14 when it catalyzes the crosslinking of the chorionic proteins. Using explanted follicles developing in vitro, premature, but otherwise normal crosslinking can be induced with hydrogen peroxide and normal crosslinking can be prevented with peroxidase inhibitors. Inhibition or premature activation of the shell peroxidase allows characterization of chorionic filament specific proteins and establishes new criteria for the identification of eggshell components.

Notes: Summary Further IF screening ofDrosophila melanogaster geographic strains has revealed a variant of the s19 major chorion protein. Developmental analysis of F1 hybrids indicates that the source of the variation is found in the structural gene for this protein. The linkage group of the variant gene was determined to be the third, and the gene was localized by several methods of recombination analysis. The s19 gene was found to be tightly linked to thesepia locus, as had been previously found for the s18 gene (Yannoni and Petri 1980). Lack of recombination between the s19 and s18 genes in double heterozygotes suggested that these two genes are within 0.3 map units of each other. Although more precise localization of the s19 gene failed, the s18 gene could be more specifically located to the right ofsepia, betweensepia andhairy. Contrary to our prediction (ibid.), the s19 and s18 genes have been found to be tightly linked in spite of the fact that they display somewhat different developmental stage specificity.

Notes: Summary We describe a set of cells in the central nervous system of theDrosophila embryo which are restricted to the thoracic ganglia in the wildtype. Taking these cells as indication of thoracic identity, we find that the ventral cord of embryos homozygous mutant for different bithorax functions and for Polycomb undergoes homoeotic transformations equivalent to those observed in the larval cuticle.

Notes: Summary InDrosophila, the terminalia (i.e. internal and external analia and genitalia, except the gonads) are formed by the genital disc. Comparative studies suggested that this disc may have evolved through fusion of the imaginal primordia of the last 3 or 4 abdominal segments. The present report describes the clonal relationships within the complex genital disc. Genetically marked cell clones were induced in male and female embryos and larvae heterozygous for cell marker mutations. 1) Frequencies and sizes of clones suggest that the embryonic disc anlage consists of 14–17 precursor cells: 4–6 for the analia, some 7 for the male genitalia, and 3–4 for the female genitalia. These cells grow exponentially during larval development. 2) In both sexes, the clones were confined to either analia or genitalia, suggesting two separate cell lineages already established at blastoderm. 3) Internal and external genitalia remain in the same compartment at least up to 60 h (end of first instar). 4) A clonal restriction appeared around 84 h (mid second instar), separating a dorsal from a ventral part in the male genitalia. The ventral compartment comprises the ventral part of the lateral plate and clasper, hypandrium, and all internal genitalia. No such boundary was detected in the female. 5) In the female, analia and parovaria originate from the same precursors; another cell lineage forms eighth tergites, vaginal plates, oviduct, receptacle, and spermathecae. 6) In female analia, dorsal and ventral plate share common precursors at least up to 84 h. A medio-lateral boundary may appear at 84 h in the ventral anal plate. No clonal restriction was found in the male analia. 7) At all times, clones could cross between left and right sides of the symmetrical terminalia; they consistently did so via ventral structures. 8) The results are discussed in a phylogenetic context, and we propose that the clonal relations reflect the evolution of the complex genital disc.