Plant cell-size control: growing by ploidy?

The size of plant cells is determined by genetic, structural and physical factors as well as by internal and external signals. Our knowledge of the molecular mechanisms of these controls is still rudimentary. Recent studies indicate that ploidy level exerts an important control on cell size. By increasing ploidy, endoreduplication may allow cells to reach extraordinary sizes. This process is widespread in plants and may provide a means to manipulate the cell volume.

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein.

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.

The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants.

Plant organs develop mostly post-embryonically from persistent or newly formed meristems. After cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. Factors controlling transition from mitotic cycles to differentiation programmes have not been identified yet in plants. Here we describe ccs52, a plant homologue of APC activators involved in mitotic cyclin degradation. The ccs52 cDNA clones were isolated from Medicago sativa root nodules, which exhibit the highest degree of endopolyploidy in this plant. ccs52 represents a small multigenic family and appears to be conserved in plants. Overexpression of ccs52 in yeast triggered mitotic cyclin degradation, cell division arrest, endoreduplication and cell enlargement. In Medicago, enhanced expression of ccs52 was found in differentiating cells undergoing endoreduplication. In transgenic M.truncatula plants, overexpression of the ccs52 gene in the antisense orientation resulted in partial suppression of ccs52 expression and decreased the number of endocycles and the volume of the largest cells. Thus, the ccs52 product may switch proliferating cells to differentiation programmes which, in the case of endocycles, result in cell size increments.

Vascularization of fetal cell suspension grafts in the excitotoxically lesioned adult rat thalamus.

Several studies have considered the establishment of vascularization in intracerebral solid transplants of neural tissue. The widely supported interpretation of the results is that the vascular network of the solid grafts is already present before implantation into the host brain. The situation is different when dissociated fetal tissue is transplanted as a cell suspension because in these conditions the fetal vascular network is disrupted. The present study has, therefore, been undertaken to follow the angiogenesis in a transplant of dissociated fetal cells implanted into the excitotoxically neuron-depleted thalamus. The vascular network is compared to that observed in the intact and in the lesioned thalamus both in terms of morphology of the capillaries and of the function of the blood-brain barrier (BBB). In the transplant, capillaries, stained by Indian ink, are very few in number and have very fine calibers during the first 20 days after grafting. Some structures can be identified as immature blood vessels at the electron microscopic level. The blood vessels are progressively more numerous in the graft and they demonstrate mature ultrastructural features 2 months after grafting. Last, there is no leakage of the BBB for peroxidase. The vascularization seems to follow a pattern of maturation comparable to that described during development in the literature. In contrast, in the lesioned area, there is a reactive angiogenesis: 10 days after the excitotoxic injection (shortest time studied), there are many wide caliber vessels with expanded perivascular spaces engorged with mesodermal cells. A microvascularization also develops transiently during the first two months. Capillaries are abnormal from the functional point of view, since there is a leakage of the BBB to macromolecules. The use of an experimental model in which transplant had to grow in a lesioned area permits to determine two types of vascularization: an apparently normal developmental timetable, normal morphological and functional characteristics, in the transplant; a reactive angiogenesis, in the lesioned area.

First month of development of fetal neurons transplanted as a cell suspension into the adult CNS.

It has been demonstrated elsewhere that fetal thalamic tissue, when transplanted as a cell suspension into the excitotoxically neuron-depleted adult somatosensory thalamus, can grow, differentiate, and receive projections from host afferents. In the present study, we used the same paradigm to analyse the transplanted neurons during their morphogenesis, i.e. during the first month after transplantation. Using various anatomical criteria, at the light and electron microscope levels, we compared the development of transplanted neurons with the normal ontogeny of homologous neuronal populations. Confined solely to the mechanically lesioned area during implantation at seven days post-grafting, the transplant increased in size to occupy most of the previously neuron-depleted area by the third week after grafting. The final size of the transplant thus depended upon the size of the lesion. At seven days post-grafting, the neurons were small in size and the cellular density was high. At this immature stage few synaptic contacts were visible and the ultrastructure was characterized by large extracellular spaces. At 10 days post-grafting, the size of the neurons had increased and the cellular density had decreased. Both an extensive dendritic proliferation and a simultaneous active synaptogenesis could also be observed. All these events continued to evolve and during the third week the neuropil progressively acquired more mature ultrastructural characteristics. Synaptic contacts exhibiting characteristics comparable to those observed in the intact thalamus also became more numerous. At 20 days post-grafting, axonal myelination had started, the development of the graft apparently stopped and the various criteria had stabilized. Until that developmental stage, growth of grafted neurons compared to that of normal thalamic ones. At later stages, however, grafted neurons failed to grow larger and did not reach the size of the homologous population in the adult animal. It seems, therefore, that transplants of thalamic fetal neurons can be used as a tool with which to study thalamic neuronal development, within definable limits.

Immunocytochemical study of serotoninergic and noradrenergic innervation of the ventrobasal complex of the rat thalamus.

Whereas the anatomy and function of monoaminergic afferents to the spinal cord areas involved in somesthesia and pain have been widely studied, little is known about the monoaminergic innervation of the primary somatosensory thalamic relay nucleus. The present study demonstrates immunocytochemically at both the light and the electron microscopic levels the presence of noradrenergic and serotoninergic fibers in the ventrobasal complex of the rat thalamus (VB). Despite the presence of numerous immunoreactive varicosities, synaptic differentiation was not observed at the level of apposition of membranes between monoaminergic afferents and VB neuronal profiles. The hypothesis of a non-synaptic modulation of VB neuronal activity by monoaminergic afferents is discussed.

[Possible role of the neuronal environment in the vascularization of an intracerebral graft]. / Rôle possible de l'environnement neuronal dans la vascularisation d'une greffe intracérébrale.

Fetal thalamic cells implanted into the excitotoxically lesioned thalamus of adult rats construct a neo-nucleus. This study shows that the vascular network of this neo-nucleus exhibits normal characteristics considering both the morphology (at the light and ultrastructural levels) and the presence of a blood-brain barrier for macromolecules. A role of the neuronal environment in this reconstruction is suggested.

Propriospinal fibers reaching the lumbar enlargement in the rat.

Propriospinal fibers reaching the lumbar enlargement were investigated in rat by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate coupled or not coupled with gold particles. Unilateral or medial bilateral injections were done. Identification of projection cells was done by tetramethylbenzidine histochemistry or gold-silver intensification procedures. Unilateral injections resulted in bilateral labeling, with patterns and density related to the spinal segments of origin. Sacral, lumbar and thoracic afferents showed identical patterns. Ipsilateral connections originated laterally from dorsal, intermediate and ventral horns. Contralateral connections originated medially from laminae VII and VIII and laterally from the reticular extension of the neck of the dorsal horn. Cervical afferents were symmetrical, arising from both lamina VIII and the reticular extension of the neck of the dorsal horn. Lamina X projection cells were seen at all levels when injection sites involved this area. Laminae III and IV were almost totally devoid of projection cells. Superficial layer cells (laminae I and II) showed some labeling when injections were situated dorsally. The organization of these tracts in rat is similar to that in cat and monkey. Their origin is discussed in relation to those of long ascending pathways reaching supraspinal levels.

[Morphological changes of the terminations of afferent pathways in a neurodegenerative lesion induced by kainic acid]. / Altérations morphologiques des terminaisons de voies afférentes lors d'une lésion neurodégénérative induite par l'acide kaïnique.

30 days after kainic acid injection into the rat ventrobasal thalamus, lemniscal afferents were labeled using wheat-germ agglutinin conjugated to HRP. They appeared considerably swollen in the area where neuronal post-synaptic targets had been eliminated. Electron microscopic analysis of the lesioned tissue revealed the presence of large profiles containing numerous organelles, particularly smooth endoplasmic reticulum, and giving rise to thin excrescences filled with neurofilaments. Since these morphological features are typical of regenerating "growth cones", we conclude that afferent terminals deprived of their post-synaptic targets undergo morphological changes preparing them for new synapses.

Ultrastructural analysis of the terminals of various somatosensory pathways in the ventrobasal complex of the rat thalamus: an electron-microscopic study using wheatgerm agglutinin conjugated to horseradish peroxidase as an axonal tracer.

We used wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) as an anterograde tracer to label the terminals of the lemniscal, spinothalamic, and trigeminothalamic pathways in the ventrobasal complex of the rat thalamus (VB). The use of benzidine dihydrochloride (BDHC) as the chromogen allowed us to view the labeled profiles with the electron microscope and permitted us to compare the morphology of the terminals from the various pathways. We found that all the labeled somatosensory pathways terminate in the VB in the form of large terminals that contain round synaptic vesicles and make numerous asymmetrical synaptic contacts, usually with dendritic protrusions and proximal dendrites. The present results demonstrate that pathways conveying noxious and non-noxious somatosensory information terminate upon thalamic neurons with synaptic terminals having similar morphological features.

Properties of deep spinothalamic tract cells in the rat, with special reference to ventromedial zone of lumbar dorsal horn.

Spinothalamic tract cells lying at the base of the dorsal horn or in deeper areas were identified in the rat at the lumbar spinal cord level by injecting horseradish peroxidase or stimulating posterior thalamic areas. Two groups of neurons were delineated; each constituted a homogeneous population of cells in terms of electrophysiological properties. Ventromedial dorsal horn cells lay at the base of the dorsal horn in its most medial aspect. They constitute a densely packed formation, which in the rat's ascending pathway system is restricted to the spinothalamic tract. Their main characteristics were a) ongoing activity, which consisted of small groups of spikes occurring at regular intervals, with a maximal discharge during ankle extension; b) unilateral receptive fields, which were restricted to the distal parts of the limb and involved both proprioceptive (ankle, joint digits) and exteroceptive (foot skin) structures; c) input of innocuous origin (midrange articular movement, touch, pressure) that was excitatory and input of noxious cutaneous origin (radiant heat) that was inhibitory; the same areas of foot skin were thus potentially either excitatory or inhibitory, depending on the nature of the stimulus applied; d) ankle flexion could differentially modulate both ongoing activity and excitatory responses from distal parts of the limb, inhibiting the former and potentiating the latter. In consequence, the ability of these cells to be driven by both proprioceptive and cutaneous inputs from limb extremities as well as by interactions of these inputs would appear to be very favorable for informing the central nervous system about limb position in both passive postural changes and the step cycle. It is suggested that they supply the thalamus with integrated information about locomotion. Lamina VII cells also constituted a subpopulation of neurons with very homogeneous electrophysiological properties. They were characterized by widespread inhibitory receptive fields, possibly spanning both hindlimbs and the ipsilateral forelimb. Inhibition was most effective when the stimulus (pressure, pinch) was intense and applied to proximal parts of the hindlimb. Ongoing activity consisted of spikes set at regular intervals and sometimes related to midrange movements of the knee. Although the significance of the widespread inhibitory receptive fields remains unclear, it has been suggested that these neurons are involved in the transmission of proprioceptive information.(ABSTRACT TRUNCATED AT 400 WORDS)

Postsynaptic fibers reaching the dorsal column nuclei in the rat.

Postsynaptic fibers reaching the dorsal column nuclei were investigated in rat by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate. Each nucleus received only ipsilateral afferents with most of the labeled cells forming a band which covered the mediolateral extent of the dorsal horn in an area that resembled lamina IV in the cat. The labeling excluded the reticular extension of the neck of the dorsal horn. Lumbosacral afferents were restricted to the gracilis nucleus and cervicothoracic afferents to the cuneatus nucleus. Cervical and anterior lumbar levels showed additional projections coming from their most medial parts. The organization of this second-order pathway in rat is similar to that in cat and monkey.

Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase.

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the lateral reticular nucleus (LRN). Labeled cells were found at all spinal levels and in particular large numbers in cervical and lumbar segments. Various spinal areas gave rise to cells of origin of this tract, which appears to be more prominent than any other tract previously studied with a similar approach. Labeling common to all spinal segments was observed in (1) ventromedial parts of both intermediate zone and ventral horn (laminae VII, VIII and X), mainly contralaterally; (2) the reticular extension of the neck of the dorsal horn, partly bilateral; and (3) superficial layers of the dorsal horn and nucleus of the dorsolateral funiculus (NDLF), mainly contralateral and projecting essentially to the lateral zone of the LRN. Additional labeling was observed at cervical and lumbar levels, each with specific qualities: (1) the cervical enlargement, which displayed labeling in the central part of the ipsilateral intermediate zone (lamina VII); (2) the rostral lumbar levels, which had projections from the contralateral median portion of the neck of the dorsal horn. These latter projections appear to be specific to pathways reaching the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive (except lamina I and NDLF projections) and the differences between these afferents and those projecting to the dorsal reticular formation, i.e., the nucleus reticularis ventralis.

Reticularis thalami afferents to the ventrobasal complex of the rat thalamus: an electron microscope study.

Afferents from the nucleus reticularis thalami (RT) to the thalamic ventrobasal complex were studied in the rat by looking for degenerating terminals after selective neurotoxic lesion of RT using injections of kainic acid. Several lines of evidence are presented indicating that RT afferents terminate in the VB by F type (Gray type II) terminals and that F type terminals in the VB all depend of RT neurons.