Synapse loss and axon retraction in response to local muscle degeneration

During metamorphosis in the moth, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. As the larval muscle degenerates, its motoneuron loses its end plates and retracts axon branches from the degenerating muscle. Muscle degeneration is under the control of the insect hormones, the ecdysteroids. Topical application of an ecdysteroid mimic resulted in animals that produced a localized patch of pupal cuticle. Muscle fibers underlying the patch showed a gradient of degeneration. The motoneuron showed end-plate loss and axon retraction from degenerating regions of a given fiber but maintained its fine terminal branches and end plates on intact regions. The results suggest that local steroid treatments that result in local muscle degeneration bring about a loss of synaptic contacts from regions of muscle degeneration. © 1996 John Wiley & Sons, Inc.     

Effects of a protein synthesis inhibitor on the hormonally mediated regression and death of motoneurons in the tobacco hornworm,Manduca sexta

The larval–pupal transformation of Manduca sexta is accompanied by the loss of the abdominal prolegs. The proleg muscles degenerate, the dendritic arbors of proleg motoneurons regress, and a subset of the proleg motoneurons dies. The regression and death of proleg motoneurons are triggered by the prepupal peak of ecdysteroids in the hemolymph. To investigate the possible involvement of protein synthesis in these events, we gave insects repeated injections of the protein synthesis inhibitor, cycloheximide (CHX), during the prepupal peak. Examination of insects 3–5 days following CHX treatment showed that CHX inhibited the death of proleg motoneurons and the production of pupal cuticle in a dose-dependent fashion. When insects were allowed to survive for 10 days after the final CHX injection, motoneuron death and pupal cuticle production sometimes occurred belatedly, apparently in response to the ecdysteroid rise that normally triggers adult development. CHX treatments that inhibited motoneuron death were less effective in inhibiting dendritic regression in the same neurons. In another set of experiments, abdomens were isolated from the ecdysteroid-secreting glands prior to the prepupal peak, and infused with 20-hydroxyecdysone (20-HE). Single injections of CHX delivered just prior to the start of the 20-HE infusion inhibited motoneuron death and pupal cuticle production, but in the range of doses tested, did not prevent dendritic regression. Our findings suggest that protein synthesis is a required step in the steroid-mediated death of proleg motoneurons, and that dendritic regression is less susceptible to inhibition by CHX than is motoneuron death. © 1993 John Wiley & Sons, Inc.     

Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat

Satellite cell proliferation was assessed inlow-frequency-stimulated hypothyroid rat fast-twitch muscle by5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining oflabeled muscle nuclei, and by staining for proliferating cell nuclearantigen (PCNA). BrdU labeling and PCNA staining were highly correlatedand increased approximately fourfold at 5 days of stimulation, decayedthereafter, but remained elevated over control in 10- and 20-daystimulated muscles. Myogenin mRNA was ~4-fold elevated at 5 days and1.5-fold at 10 days. Staining for myogenin protein yielded resultssimilar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequencystimulation. By 20 days, myogenin staining was observed intransforming fast fibers that coexpressed embryonic and adult myosinheavy chain isoforms. In the slower fiber populations (i.e., IIA andI), myogenin-positive transforming fibers that coexpressed embryonicmyosin heavy chain, appeared already at 5 days. Thus the satellite cellprogeny on slower fibers seemed to proliferate less and to fuse earlierto their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.

     

Innervation regulates the metamorphic fates of larval abdominal muscles in the moth, Manduca sexta

 With the onset of metamorphosis, the abdominal muscles of the moth, Manduca sexta, follow one of three developmental fates: maintenance, respecification, or death. The maintained muscles retain their larval size and morphology throughout adult development. The respecified and dying muscles dedifferentiate, which involves regression, nuclear degeneration, and myofibril breakdown. Nuclei in both dying and respecified muscles also proliferate. The amount of nuclear degeneration is greater in the dying muscle fibers, and the amount of nuclear proliferation is greater in the respecified muscles. Four to ten days after pupation, the sizes of the respecified muscles stabilize while the dying muscles are lost. During regression, a subset of the respecified muscle fibers die. The surviving respecified muscle fibers grow and differentiate during the last half of adult development. In respecified muscles, denervation triggers an increased amount of nuclear degeneration and a decreased amount of nuclear proliferation. As a result, denervated respecified fibers experience increased muscle regression including an increased loss of muscle fibers and sometimes muscle death. Surviving respecified fibers still grow and differentiate yet are only 5 to 12% of the control size. Denervation triggers dedifferentiation in maintained muscles, resulting in fiber loss and occasionally muscle death. The percentage of fibers which dedifferentiate varies between different muscles. Denervation also triggers nuclear proliferation, with the amount of nuclear proliferation correlated with the extent of dedifferentiation of the individual muscle fibers. The dedifferentiated maintained fibers subsequently undergo differentiation in the absence of muscle growth. Received: 10 July 1997 / Accepted: 21 April 1998     

Endocrine regulation of terminal muscle differentiation: Atrophy and degeneration of the intersegmental muscles of Lepidoptera

The intersegmental muscles of the Lepidoptera pass through three separate, sequential differentiated states during pharate adult development: status quo; atrophy; degeneration. Each of these developmental programs is characterized by a distinct morphology, physiology and endocrine responsiveness. The factors responsible for regulating these differentiative changes are ecdysteroids. In Manduca sexta, the haemolymph ecdysteroid titre declines in a circadian-modified fashion during the last three days of adult development, which parallels the maturation of the intersegmental muscles. Abdomen-ligation, which causes a precipitous decline in the ecdysteroid titre, causes the precocious atrophy and degeneration of these muscles, whereas injection of, or infusion with, 20-hydroxyecdysone greatly delays such changes. While the terminal differentiation of the epidermis and nervous system is also regulated by ecdysteroids, endocrine manipulations have suggested that the development of the intersegmental muscles is independent of these tissues.In the silkmoth Antheraea polyphemus, ecdysteroids are also responsible for regulating intersegmental muscle differentiation, but eclosion hormone (a peptide) acts as the proximal trigger for the activation of the degeneration program. The declining ecdysteroid titre initiates the atrophy program and subsequently determines the timing of both release of eclosion hormone and intersegmental muscle sensitivity to the peptide. Eclosion hormone then acts directly on the muscles, via cGMP, to activate the degeneration program. Ecdysteroids appear to prevent premature muscle degeneration by regulating a biochemical step distal to both the eclosion hormone receptor and the rise in cGMP.     

Labelling of DNA and differential sister chromatid staining after BrdU treatment in vivo

A method of labelling DNA in vivo with 5-bromodeoxyuridine (BrdU) is described. After 6 h permanent subcutaneous infusion of BrdU in rodents (adult Microtus agrestis, pregnant NMRI-mice), cell nuclei which have undergone DNA synthesis during the BrdU treatment can be differentiated from the nuclei of other cycle stages by means of their altered staining behaviour after Giemsa. 24 h after the BrdU treatment, mitoses from both bone marrow of the adult animals and tissues from the fetuses showed a differential sister chromatid staining. In male M. agrestis, sister chromatid exchanges were most frequently found in the euchromatic part of the X and in the constitutive heterochromatin of both sex chromosomes.     

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.     

Neuronal differentiation and long‐term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster,Panulirus argus

The fate of continuously generated cells in the soma clusters of the olfactory midbrain of adult spiny lobsters, Panulirus argus, was investigated by in vivo pulse‐chase experiments with the proliferation marker 5‐bromo‐2′‐deoxyuridine (BrdU) combined with immunostainings for neuropeptides of mature neurons. A BrdU injection after a survival time (ST) of 14 h labeled about 100 nuclei in the lateral soma clusters (LC), comprised of projection neurons, and about 30 nuclei in the medial soma clusters (MC), comprised of local interneurons. The BrdU‐positive nuclei were confined to small regions at the inside of these clusters, which also contain nuclei in different phases of mitosis and thus represent proliferative zones. After STs of 2 weeks or 3 months, the number of BrdU‐positive nuclei was doubled, indicating a mitosis of all originally labeled cells. Dependent on ST, the BrdU‐positive nuclei were translocated from the proliferative zones towards the outside of the clusters, where somata of mature neurons reside. Immunostainings with antibodies to the neuropeptides FMRFamide and substance P, both of which label a large portion of somata in the MC and a pair of giant neurons projecting into the LC, revealed that in both clusters the proliferative zones are surrounded by, but are themselves devoid of, labeling. In the MC, some BrdU‐positive somata were double‐labeled by the FMRFamide antibody after an ST of 3 months, and by the substance P antibody after STs of 6 and 11/14 months, but not after 3 months. In the LC, BrdU‐positive somata after an ST of 3 months partially and after 6 and 11/14 months widely overlapped with the arborizations of the giant neurons, indicating the establishment of synaptic input. The experiments show that cells generated in proliferative zones in the LC and MC of adult spiny lobsters after a final mitosis differentiate into neurons within months, survive for at least 1 year, and are integrated into the circuitry of the olfactory midbrain. A new hypothesis about the mechanism of adult neurogenesis in the central olfactory pathway of decapod crustaceans is developed, linking it to neurogenesis during embryonic and larval development. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 181–203, 2001     

Sexually distinct development of vocal pathways in Xenopus laevis

Deterministic rules, rather than experience, are thought to regulate the development of simple behaviors in vertebrates and invertebrates. We revisited this issue through examination of the sexually distinct vocalizations of African clawed frogs (Xenopus laevis), a reproductive behavior used by sexually mature males and females. We discovered that, as expected for simple behavior, female vocalizations develop through deterministic rules. The rare calls of juvenile females are indistinguishable from those of adult females. The vocal pathways of juvenile females, as measured by the contractile properties of the laryngeal muscles (the vocal muscles) and the laryngeal motoneuron somata (vocal motoneurons) size, are the developmental default and do not differentiate as they mature. Male Xenopus, in contrast, produce extensive vocalizations with rudimentary acoustic structure before reaching sexual maturity. Moreover, the functional properties of the vocal central pattern generator mature before muscle fibers and motoneuron size are fully masculinized. The results suggest that neuronal activity during development may be important in organizing the contractile properties of the muscle fibers in male, but not in female Xenopus. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 862–874, 2010     

Remodeling of an identified motoneuron during metamorphosis: central and peripheral actions of ecdysteroids during regression of dendrites and motor terminals

During metamorphosis of the moth Manduca sexta, an identified leg motoneuron, the femoral depressor motoneuron (FeDe MN), undergoes reorganization of its central and peripheral processes. This remodeling is under the control of two insect hormones: the ecdysteroids and juvenile hormone (JH). Here, we asked whether peripheral or central actions of the ecdysteroids influenced specific regressive aspects of MN remodeling. We used stable hormonal mimics to manipulate the hormonal environment of either the FeDe muscle or the FeDe MN soma. Our results demonstrate that motor-terminal retraction and dendritic regression can be experimentally uncoupled, indicating that central actions of ecdysteroids trigger dendritic regression whereas peripheral actions trigger terminal retraction. Our results further demonstrate that discrete aspects of motor-terminal retraction can also be experimentally uncoupled, suggesting that they also are regulated differently.     

CONNEXIN43 GAP JUNCTION LEVELS DURING DEVELOPMENT OF THE THORACIC AORTA ARE TEMPORALLY CORRELATED WITH ELASTIC LAMINAE DEPOSITION AND INCREASED BLOOD PRESSURE

A characteristic property of the vascular smooth muscle cell is its ability to modulate between a contractile phenotype, responsible for control of vascular tone and tension, through to a synthetic phenotype, capable of migration and synthesis of extracellular matrix molecules. Smooth muscle cells are coupled by gap junctions, the membrane structures which permit direct intercellular passage of ions and small molecules, and which play a role both in electrical coupling and intercellular communication during patterning and development. We have previously found that connexin43 type gap junction expression is upregulated in the synthetic phenotype smooth muscle cellin vitroand during atherosclerotic plaque formation in human coronary arteries. On the basis of immunohistochemical labelling, confocal laser scanning microscopy and digital image analysis, we now report that relatively high levels of connexin43 are expressed during development of the rat thoracic aorta, temporally correlating with reported periods of smooth muscle cell proliferation and secretion of elastic laminae. A major peak in expression occurs at seven days post-natal, with a second less pronounced peak at 72 days post-natal. The principal peak in gap junction levels appears to coincide with increased post-natal blood pressure and aorta media thickening. The amount of gap junction labelling falls off to normal adult levels as the smooth muscle cells modulate towards the contractile phenotype and growth is completed. The results indicate an association between direct cell-to-cell communication and synthetic phenotype smooth muscle cell activity during aortic growth and patterning.     

Remodeling of an identified motoneuron during metamorphosis: hormonal influences on the growth of dendrites and axon terminals

During metamorphosis of the tobacco hawkmoth Manduca sexta, the femoral depressor motoneuron (FeDe MN) undergoes remodeling of its dendrites and motor terminals. Previous studies have established that remodeling of MNs during metamorphosis is mediated by the same hormones that control metamorphosis: the ecdysteroids and juvenile hormone (JH). During the pupal stage, the ecdysteroids promote adult-specific growth of MNs in the absence of JH, but JH or its synthetic mimics can interfere with ecdysteroid-mediated growth if applied during early sensitive periods. Hence, the application of a JH mimic (JHM) either systemically or locally to a target muscle has been used to distinguish those aspects of motor-terminal remodeling that are controlled by ecdysteroid action on the CNS from those that are influenced by ecdysteroid action on the peripheral targets. Here, we have extended the analysis of central and peripheral hormonal influences on MN remodeling by injecting JHM locally into the CNS thus altering the hormonal environment of the FeDe MN soma without altering the hormonal environment of its target muscle. Our results demonstrate that adult dendritic growth and motor-terminal growth can be experimentally uncoupled, suggesting that each is regulated independently. JHM application to the CNS perturbed dendritic growth, but had no measurable impact on motor-terminal growth. Peripheral actions of ecdysteroids, therefore, appear sufficient to promote the development of adult-specific motor terminals but not the development of an adult-specific dendritic arbor.     

Ovarian ecdysteroid biosynthesis and female germline stem cells

The germline stem cells (GSCs) are critical for gametogenesis throughout the adult life. Stem cell identity is maintained by local signals from a specialized microenvironment called the niche. However, it is unclear how systemic signals regulate stem cell activity in response to environmental cues. In our previous article, we reported that mating stimulates GSC proliferation in female Drosophila. The mating-induced GSC proliferation is mediated by ovarian ecdysteroids, whose biosynthesis is positively controlled by Sex peptide signaling. Here, we characterized the post-eclosion and post-mating expression pattern of the genes encoding the ecdysteroidogenic enzymes in the ovary. We further investigated the biosynthetic functions of the ovarian ecdysteroid in GSC maintenance in the mated females. We also briefly discuss the regulation of the ecdysteroidogenic enzyme-encoding genes and the subsequent ecdysteroid biosynthesis in the ovary of the adult Drosophila.     

Evidence of DNA replication in an arbuscular mycorrhizal fungus in the absence of the host plant

Summary This study provides evidence thatGigaspora margarita replicates its nuclear DNA, even in the absence of a host plant. Three experimental approaches were used: (i) static cytofluorimetry to quantify the DNA content, (ii) pulse treatments with bromodeoxyuridine (BrdU), which is an analogue of thymidine, to reveal nuclei undergoing DNA synthesis, and (iii) ultrastructural observations to study changes in chromatin morphology during the fungal cell cycle. A slight second peak of approximately twice the value of a major peak was found by cytofluorimetry, showing that a small number of nuclei had entered in cycle during in vitro development. Nuclei which had incorporated BrdU were observed after pulses of 24 h; nuclei with condensed chromatin were also apparent at this time. The results demonstrate thatG. margarita has all the metabolic pathways needed to replicate its nuclear DNA even in the absence of the host, suggesting that more complex mechanisms inhibit the extended growth in vitro of arbuscular mycorrhizal fungi.Abbreviations AM-fungi arbuscular mycorrhizal fungi - A.U. arbitrary units - BrdU 5-bromo-2-deoxyuridine - DAPI 4,6-diamidino-2-phenylindole - UV ultraviolet light     

Contractile and fatigue properties of the rat diaphragm musculature during the perinatal period.

The following two hypotheses regarding diaphragm contractile properties in the perinatal rat were tested. First, there is a major transformation of contractile and fatigue properties during the period between the inception of inspiratory drive transmission in utero and birth. Second, the diaphragm muscle properties develop to functionally match changes occurring in phrenic motoneuron electrophysiological properties. Muscle force recordings and intracellular recordings of end-plate potentials were measured by using phrenic nerve-diaphragm muscle in vitro preparations isolated from rats on embryonic day 18 and postnatal days 0-1. The following age-dependent changes occurred: 1) twitch contraction and half relaxation times decreased approximately two- and threefold, respectively; 2) the tetanic force levels increased approximately fivefold; 3) the ratio of peak twitch force to maximum tetanic force decreased 2.3-fold; 4) the range of forces generated by the diaphragm in response to graded nerve stimulation increased approximately twofold; 5) the force-frequency curve was shifted to the right; and 6) the propensity for neuromuscular transmission failure decreased. In conclusion, the diaphragm contractile and phrenic motoneuron repetitive firing properties develop in concert so that the full range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.     

Muscle temperature, contractile speed, and motoneuron firing rates during human voluntary contractions.

A study was made of motoneuron firing rates and mechanical contractile parameters during maximum voluntary contraction of human hand muscles. A comparison of muscles that had been fatigued after a 60-s maximum voluntary contraction (MVC) with muscles that were cooled by approximately 5 degrees C showed that the contractile properties, in particular the rates of contraction and relaxation, were similarly affected in both conditions. In contrast, the motoneuron firing rate was affected differently by the two treatments. In the case of the fatigued muscles the motoneuron firing rate was reduced by 36%, as was expected from previous studies, but in the case of the cooled muscles, there was no significant change in the motoneuron firing rate. We conclude that the reflex reduction in the motoneuron firing rate seen in the fatigued muscle is not triggered directly by a change in the mechanical properties of the muscle.     

Myogenesis during holothurian intestinal regeneration

Echinoderms are well known as being able to regenerate body parts and thus provide excellent models for studying regenerative processes in adult organisms. We are interested in intestinal regeneration in the sea cucumber, Holothuria glaberrima, and focus here on the regeneration of intestinal muscle components. We have used immunohistochemical techniques to describe the formation of the intestinal muscle layers. Myoblasts are first observed within the regenerating structure, adjacent to the coelomic epithelia. Within a few days, these cells acquire muscle markers and form a single cell layer that underlies the epithelia. Animals injected with BrdU at various regeneration stages have been subsequently analyzed for the presence of muscle differentiation markers. BrdU-labeled muscle nuclei are observed in myocytes of 3-week regenerates, showing that these cells originate from proliferating precursors. The peak in muscle precursor proliferation appears to occur during the second week of regeneration. Therefore, new muscle cells in the regenerating intestine originate from precursors that have undergone cell division. Our results suggest that the precursor cells arise from the coelomic epithelia. We also provide a comparative view of muscle regeneration in an echinoderm, a topic of interest in view of the many recent studies of muscle regeneration in vertebrate species. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from NIH-RCMI (RRO-3641-01) and the University of Puerto Rico     

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: a cytophysiological analysis of the ring gland

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.     

Segment‐specific muscle degeneration is triggered directly by a steroid hormone during insect metamorphosis

During metamorphosis of the hawkmoth, Manduca sexta, some larval muscles degenerate while others are respecified for new functions. In larvae, accessory planta retractor muscles (APRMs) are present in abdominal segments 1 to 6 (A1 to A6). APRMs serve as proleg retractors in A3 to A6 and body wall muscles in A1 and A2. At pupation, all APRMs degenerate except those in A2 and A3, which are respecified to circulate hemolymph in pupae. The motoneurons that innervate APRMs, the APRs, likewise undergo segment‐specific programmed cell death (PCD), as a direct, cell‐autonomous response to the prepupal peak of ecdysteroids. The segment‐specific patterns of APR and APRM death differ. The present study tested the hypothesis that APRM death is a direct, cell‐autonomous response to the prepupal peak of ecdysteroids. Prevention of the prepupal peak prevented APRM degeneration, and replacement of the peak by infusion of 20‐hydroxyecdysone restored the correct segment‐specific pattern of APRM degeneration. Surgical denervation of APRMs did not perturb their segment‐specific degeneration at pupation, indicating that signals from APRs are not required for the muscles' segment‐specific responses to ecdysteroids. The possibility that instructive signals originate from APRMs' epidermal attachment points was tested by treating the epidermis with a juvenile hormone analog to prevent pupal development. This manipulation likewise did not alter APRM fate. We conclude that both the muscles and motoneurons in this motor system respond directly and cell‐autonomously to prepupal ecdysteroids to produce a segment‐specific pattern of PCD that is matched to the functional requirements of the pupal body. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Determination of the fraction of S-phase cells in root meristems using bromodeoxyuridine labeling

The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.