The effects of transplantation and regeneration of sensory neurons on a somatotopic map in the cricket central nervous system

The restoration of the cercal afferent projection of crickets was examined after severing the cercal nerve or amputating the cercus and reimplanting it. After either maneuver the sensory neurons regenerated arborizations in the central nervous system (CNS) within about 1 month. In order to assess the role of the pathway taken to the CNS in controlling the growth of the terminal arborization, we transplantated left cerci to the right side of the host. The operation mismatched the mediolateral axes of host and graft tissues. In one-third of the neurons examined, the axon trajectories of the regenerated neurons were altered. The terminal arborizations in these cases were unusual; for example, one neuron arborized in an abnormal area as well as in its normal area. In rare instances this neuron arborized only in incorrect areas of the CNS. Thus, it appears that axon pathway can have an effect on the central structure of sensory neurons. However, in most cases after the surgery, the neurons were able to reach their proper target areas even by circuitous routes. The proximodistal coordinate of the map is isomorphic with sensory neuron age, because the most distal receptors are produced early in postembryonic development and new ones are added proximally at each molt. We tested the possibility that the order of differentiation was critical for generating the afferent projection with two experiments. First, the distal cercus including the distal members of the clavate array was amputated. The specimen regenerated an entire distal cercus including distal clavate receptors. When newly generated, distal neurons were stained, the terminal arbors were identical to the amputated neurons they replaced. In this case, both age and order of arrival were reversed from normal yet the topographic projection pattern was not altered. Second, we transplanted young cerci onto older specimens and then examined the regenerated arbors of the transplanted sensory neuron. The immature neuron arborized in the adult nervous system exactly as the mature homolog. Thus the age of a sensory neuron did not appear to be a controlling variable in the elaboration of a terminal arborization. The significance of these results is discussed in the context of two models for development of orderly neuronal connections.     

Development and regulation of substance P in sensory neurons in vitro

Substance P (SP), the putative neuropeptide mediator of pain sensation, is contained in small dorsomedial sensory neurons of the dorsal root ganglion. Using different culture techniques and a sensitive radioimmunoassay for SP, we studied the ontogeny and regulation of this functionally important neurotransmitter in these neurons, obtained from neonatal rats. In ganglion explants grown by two different techniques, SP increased two- to threefold during the first week in culture. This rise was predominantly due to mechanisms intrinsic to the ganglion since it occurred in a fully defined medium, in the absence of added nerve growth factor (NGF). Blockade of protein synthesis with cycloheximide prevented the increase in SP suggesting that ongoing protein synthesis was necessary. Furthermore, depolarization with veratridine blocked the increase in SP, an effect which was reversed by tetrodotoxin, suggesting that transmitter characteristics in sensory neurons may be regulated by depolarization and/or transmembrane sodium flux. After a week in culture on a collagen substratum, supplementary NGF was necessary for the continued rise in SP. However, raising the dose of the trophic factor had no incremental effect on SP content, suggesting that NGF was acting primarily on neuronal survival. To approach such questions at the cellular level, ganglia were dissociated and grown in cell culture. In all cultures, SP increased 1.5-fold during the first day. In the absence of NGF, however, SP and cell numbers fell progressively after the second day. NGF elicited parallel increases in cell survival and SP content, supporting the suggestion that NGF acts primarily through neuronal survival to increase SP. Veratridine blocked the increase in SP in a tetrodotoxin-reversible manner, without affecting neuronal survival, indicating that the effects of these agents do not depend on normal ganglionic cellular architecture. Consequently, depolarization probably affects ganglionic sensory neurons directly. Our studies suggest that the development of transmitter characteristics in primary sensory neurons may be regulated by multiple factors, including neuronal activity as well as trophic agents such as NGF.     

26S and 18S rRNA synthesis in bobbed mutants of Drosophila melanogaster

For the most part, bobbed mutations of Drosophila melanogaster consist of deletions of 26S and 18S rDNA located on the X and Y chromosomes. Studies on the synthesis of rRNA of third instar larvae and one day old adult females of three severe bobbed genotypes, indicate that no decrease can be detected, compared ot wild type strains. One of the bobbed mutants studied was a rather unusual type: these flies possess a quantity of rDNA that should confer upon them a near wild type phenotype whereas they actually show an extreme bobbed phenotype. The two other bobbed mutants are of a classical type: their severe bobbed phenotype corresponds to large deletions of rDNA. Two hypotheses can be proposed to explain the extreme bobbed phenotype of the flies, in spite of the fact that rRNA synthesis occurs normally. A regulatory phenomenon may interfere at the stages studied, but in earlier stages a net decrease in rRNA synthesis may have occurred producing an irreversible effect in the tissues affected by bobbed mutations (abdominal cuticle, bristles). The second hypothesis is that the rRNA produced may not be functional, perhaps because it is specific of earlier stages.     

Distribution of mutants in aggregates of cultured cells

The analysis of the distribution of mutants in an exponentially growing culture of cells that are aggregated into clumps of homogeneous size is described, given the mutation rate and a random process by which clumps divide to produce progeny. The mean and standard deviation of the proportion of clumps with a given number of mutant cells at a particular time are calculated. Since the standard deviation tends to be much smaller than the mean, the following conclusions can be drawn. Aggregation lowers the number of mutant-containing clumps in cultures grown to a standard number of cells, but raises the number of mutant-containing clumps in cultures grown to a standard number of clumps. In the absence of mutation, or at low mutation rates, clumps tend to become pure types (normal or mutant). The probability of finding pure, nonmutant-containing clumps, however, is approximately the initial fraction of nonmutant cells (given realistic forward and back mutation rates). Also, in terms of the given process, it is possible to compute the probability that all the cells in an aggregate descend from a single, common parent cell within a given number of generations, and thus to calculate the probability that all the cells in a clone grown from an aggregate descend from a single cell within a known number of generations.     

The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras

The quail-chick marker system has been used to study the early developmental stages of the ganglia located along cranial nerves VII, IX, and X. The streams of neural crest cells arising from the rhombencephalic-vagal neural crest were followed from the onset of their migration up to the localization of crest cells in the trunk and root ganglia of these nerves. It was shown that two different populations of crest cells are segregated early as a result of morphogenetic movements in the hypobranchial region. The dorsal population gives rise to the root ganglia of nerves IX and X located close to the encephalic vesicles, where the crest cells differentiate both into neurons and into glia. In contrast, the ventral stream of neural crest cells contributes together with cells from epibranchial placodes to the trunk ganglia (geniculate, petrous, and nodose ganglia) of cranial nerves VII, IX, and X. The successive steps of the invasion of the placodal anlage by crest cells can be followed owing to the selective labeling of the neural crest cells. It appears that the latter give rise to the satellite cells of the geniculate, petrous, and nodose ganglia while the large sensory neurons originate from the placodes. The nodose ganglion has been the subject of further studies aimed to investigate whether neuronal potentialities can be elicited in the neural crest-derived cells that it contains. The ability to label selectively either the neurons or the glia by the quail nuclear marker made this investigation possible in the particular case of the nodose ganglion whose neurons and satellite cells have a different embryonic origin. By the technique already described (N. M. Le Douarin, M. A. Teillet, C. Ziller, and J. Smith, 1978, Proc. Nat. Acad. Sci. USA75, 2030–2034) of back-transplantation into the neural crest migration pathway of a younger host, it was shown that the presumptive glial cells of the nodose ganglion are able to remigrate when transplanted into a 2-day chick host and to differentiate into autonomic structures (sympathetic ganglion cells, adrenomedullary cells, and enteric ganglia). It is proposed as a working hypothesis that neuronal potentialities contained in the neural crest cells which invade the placodal primordium of the nodose ganglion are repressed through cell-cell interactions occurring between placodal and crest cells.     

The acquisition of resistance to ecdysteroids in cultured Drosophila cells

Drosophila melanogaster K_c cells become refractory toward ecdysteroids after 4 days of exposure to the molting hormone, 20-OH-ecdysone. Associated with the appearance of hormonal insensitivity is a loss of ecdysteroid receptors. Hormone-resistant cells maintain a low level of receptor that is indistinguishable from that of responsive, hormonally naive cells. After extended periods in culture, ecdysteroid receptor content in previously exposed cells returns to that of naive control cells. The reappearance of receptor is coincident with the resumption of hormonally induced growth inhibition.     

The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster

Pulse labeling of proteins, in vivo, followed by indirect immunoprecipitation of the vitellogenin polypeptides, has shown that not only the thoracic and abdominal fat bodies but also the ovary devote a significant percentage of their synthetic capacity to vitellogenin (VG) production. These methods have also shown that ovarian stages 9 and 10 contribute the majority of VG synthesized by the ovary and that the follicular epithelium of these stages is the specific site of VG synthesis. In situ hybridization (of a probe containing the coding regions of the two larger polypeptides) to sections of ovaries confirmed that the VG mRNAs are abundant species in the cytoplasm of stage 9 and 10 follicle cells. In addition, two of the three polypeptides (VGP1 and VGP2) are produced at roughly equal levels by the follicle cells, but the smallest polypeptide (VGP3) is produced at one-fourth this level by these cells. Hybridization of cloned genomic probes (T. Barnett, C. Pachl, J. P. Gergen, and P. C. Wensink, 1980, Cell21, 729–738) to RNA bound on nitrocellulose filters has shown that the ovary contributes aproximately 35% of the total amount of the mRNAs coding for VGP1 and VGP2 but only about 10% of the mRNA for VGP3. The same procedure demonstrated that the levels of all three VG mRNAs during follicular development closely parallel VG polypeptide synthesis. Finally, culture of ovaries in males has shown that the mRNA levels accurately reflect the follicle cell contribution to VG synthesis.     

The mitochondrial genome of wild-type yeast cells. VII. Recombination in crosses.

Two Saccharomyces cerevisiae wild-type strains were crossed, and 26 diploid clones were obtained from (1) mass mating; (2) individual buds in zygote lineages; (3) individual zygotes. The mitochondrial DNAs from these diploids were investigated in their recombination and segregation by analyzing their restriction fragment patterns.Recombinant mitochondrial genomes were present in 75% of the diploid clones. Such recombinant genomes had unit sizes different from, yet within ± 5% of, the parental ones and showed EcoRI and HindII + III fragment patterns of parental types, two strong indications that both the gene complement and the gene order were very largely preserved in the progeny.Fragment patterns produced by HpaII and HaeIII were characterized by (1) fragments originating from the DNAs of both parents; and (2) new fragments, namely fragments absent in either parent. The new fragments appear to arise from unequal crossing-over events occurring in the spacers of allelic parental genetic units and usually have preferential localizations in the genome.These results provide the first evidence for physical recombinations of mitochondrial DNA in crosses of wild-type yeast cells, indicate that recombination is very frequent in crosses, and shed some light on mitochondrial segregation. They also have interesting implications for recombination phenomena in interspersed systems of unique and repetitive nucleotide sequences.     

Dissociate destruction of noradrenaline and dopamine descending projections in the thoracic spinal cord of the rat

Intracisternal administration of 6-hydroxydopamine to male Wistar rats produced a near complete depletion of noradrenaline levels, as measured by a radioenzymatic assay in micropunches sampled from the dorsal, lateral and ventral horns of the thoracic spinal cord. This drastic effect was reversed by pretreatment with desipramine, a pharmacological inhibitor of noradrenergic neuron uptake. Surprisingly, dopamine content was not significantly reduced. The question as to whether such a lack of concomitant dopamine decrease might be inherent to the dopamine assay itself could be answered by the results obtained with both pharmacological (reserpine) treatments and interference determinations in the dopamine assay. The relative potency of 6-hydroxydopamine to destroy noradrenergic and dopaminergic neurons might account for their differential behavior. Conversely, a large midbrain section performed by knife cut could decrease both dopamine and DOPAC (one of its major acid metabolites) in the thoracic lateral horn and partly in the ventral one. The noradrenaline content was not reduced. Results are discussed in light of recently reported data on dopaminergic descending projections to the spinal cord. The lesion procedures presented here seem to provide valuable tools to dissociate noradrenergic from dopaminergic spinal projections, which is necessary for further anatomical and functional studies on these systems.     

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T₁ and T₂, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T₂ directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306–314) while the T₁ translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T₁ and T₂ RNAs, radiolabeled T₁ and T₂ RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

The polarity of axon growth in the wings of Drosophila melanogaster

We have analyzed the growth of axons in the wings of the mutants Hairy wing and hairy of Drosophila melanogaster. These mutants produce many supernumerary bristle organs and sensilla campaniformia, whose axons grow between the two wing epithelia and can be visualized in both pupal and adult stages. The sensory axons of wild-type animals follow two paths in the wing, within longitudinal veins L1 and L3, and always grow with a distal to proximal polarity. In the mutants, all axons following these two paths likewise grow with correct polarity. Axons elsewhere in the wing, however, are found to grow in many different directions, including from proximal to distal and hence directly away from the central nervous system. A variety of patterns of axon growth and fasciculation are seen in different individuals. Only if the supernumerary axons encounter the two normal paths do they reliably grow toward the base of the wing. We conclude that these two paths provide polarity information for axon growth, information which is either not used or not available elsewhere in the wing in spite of the obvious morphological polarization of every epithelial cell. The time course of neural differentiation suggests that the normal sensory cells of mutant wings, which grow axons relatively early, may be the source of polarity information for the later-differentiating supernumerary cells.     

Biosynthesis of medium chain fatty acids in Drosophila melanogaster

Adult Drosophila melanogaster synthesizes dodecanoic and tetradecanoic acids in vivo, along with the more common 16- and 18-carbon fatty acids. The radiolabeled C12 and C14 fatty acids synthesized from sodium [1-14C]acetate are found primarily in the diacylglycerol and triacylglycerol fractions. Partially purified fatty acid synthetase (FAS) synthesizes C14, C16, and C18 fatty acids (as the free acids) at 0.2 M ionic strength. Increasing the ionic strength to 2.0 M causes partially purified FAS to synthesize primarily C12 and C14 fatty acids. Addition of aliquots of the microsomal pellet and other soluble protein fractions does not alter the pattern of fatty acids synthesized by FAS. The percentage of C12 and C14 fatty acids synthesized at high ionic strength by individual fractions from the FAS peak (Sepharose 6B column) is constant across the peak. None of the soluble protein fractions is able to relieve the inhibition of FAS by phenylmethylsulfonyl fluoride. These results indicate that the FAS of D. melanogaster has the inherent capability to form C12 and C14 fatty acids and that no other soluble protein appears to be involved in their synthesis.     

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 μM) or higher (150 μM) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 μM choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K_m's with different affinities.The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.     

The morphogenesis of cell hairs on Drosophila wings

We describe in this paper details of morphogenesis of wing hairs in Drosophila pupae. The ultimate objective is to relate specific protein components used in hair construction to specific components produced in the rapidly changing patterns of gene expression that are characteristic for the period of hair differentiation in wing cells (H. K. Mitchell and N. S. Petersen, 1981, Dev. Biol. 85, 233-242). Hair extrusion to essentially full size occurs quite suddenly at about 34 hr (postpupariation) and this is followed by deposition of a double-layer of cuticulin during the next 4 to 5 hr. Extreme changes in shape of cells and hairs, probably related to actin synthesis, then occur for the next 5 to 6 hr. Deposition of fibers within the hairs and on hair pedestals follows. Formation of cuticle on the cell surface begins and continues until some time in the 60-hr range. It appears that cuticle is formed only on the cell surface and not in hairs or on the top of hair pedestals. The protein synthesis patterns associated with these events are described.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.