Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [³⁵S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.     

Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or II (basic or acidic FGF)

After amputation of a newt limb, a blastema forms on the amputation plane and later differentiates to regenerate all the missing parts of the limb. Proliferation of blastema cells is under the control of severed nerves which deliver a 'neurotrophic factor' (NTF) of unknown nature. In order to characterize this factor we use a primary culture of blastema mesenchymal cells; changes in mitotic index after 48-h colchicine treatment indicate mitogenic activity of potential growth substances. These cells, which are stimulated by nerve extracts (mitotic index X 6), were tested with two purified growth factors extracted from bovine retina or brain (EDGF I = basic FGF and EDGF II = acidic FGF). We show that these two growth factors stimulate proliferation of blastema cell cultures in a dose-dependent manner. Maximal stimulation was obtained at 3 pM for EDGF I (mitotic index X 5.7) or 300 pM for EDGF II (mitotic index X 4.9). So it appears that these two growth factors have a mitogenic activity on blastema mesenchymal cells similar to that obtained with nerve extracts. The fact that two different growth factors can stimulate these cells raises the question of whether both are present in NTF and/or whether there are receptors to both EDGF I and EDGF II on mesenchymal cell membranes.     

Cell Cycle Duration in the Root Meristem of Sonoran Desert Cactaceae as Estimated by Cell-flow and Rate-of-cell-production Methods

Slow rates of cactus growth in the Sonoran Desert and high productivityof some Cactaceae under cultivation suggest that relativelylow growth rates are not the consequence of a long cell divisioncycle but of short optimal periods for growth and adverse environmentalfactors. To verify this hypothesis, the duration of the celldivision cycle (T)in the root apical meristem of seedlings ofthree sympatric species from the Sonoran Desert [Ferocactuspeninsulae(F. A. C. Weber) Britton & Rose ‘Townsendianus’(Britton & Rose) N. P. Taylor, stat. nov.,Stenocereus gummosus(Engelm.)Gibson & Horak andPachycereus pringlei(S. Watson) Britton& Rose] was estimated with the rate-of-cell-production (RCP)and the cell-flow (colchicine) methods. Both methods were appliedduring the steady-state growth phase, which was relatively shortin the first two species because of the determinate patternof root growth. The RCP method permitted estimation ofTin eachroot individually. Durations of the cell division cycle wereinversely proportional to the rate of root growth (r²rangedfrom 0.42 to 0.88,P<0.05).T,determined by the cell-flow method,ranged from 14.4 to 19.3 h in these species and was within thesame range asTdetermined by the RCP method. The averageTdeterminedby the RCP method was 67 to 75% of that determined by the cell-flowmethod. Results obtained with both methods are compared andanalysed. The proposed hypothesis appears to be correct, indicatingthat these species can be more productive under cultivationthan in the wild due to the relatively short duration of thecell division cycle. Adaptive features of these findings arealso considered.Copyright 1998 Annals of Botany Company Cactaceae, cell division cycle, root growth, root meristem, Sonoran Desert     

Partial purification of a low-molecular-weight growth factor from chicken brain

The regenerating amphibian limb serves as a useful model for studying factors influencing cell proliferation and differentiation. In particular, peripheral nerves are thought to provide a stimulus for growth of the blastema, presumably via the elaboration of an as yet unidentified neurotrophic factor. In the present study, pressure ultrafiltration coupled with chromatofocusing have proven to be effective methods of partially purifying a neurotrophic factor from adult chicken brains. This chick brain growth factor (CBGF) appears to be a heat-stable, basic peptide of low molecular weight (less than 6,000). It is a potent mitogen in vitro, at nanomolar concentrations, for both blastema cells and Swiss mouse 3T3 fibroblasts. CBGF is apparently distinct from other peptide mitogens and/or neuromodulators that have been reported to stimulate blastema growth in vivo and in vitro. These include substance P, FGF from bovine brain and pituitary, EGF, transferrin (sciatin), and spinal cord growth factor (SCGF). The possible relationship of CBGF to other neural regulatory molecules is discussed.     

Flowering in Pisum: the Effect of Genotype, Plant Age, Photoperiod, and Number of Inductive Cycles

The genotypes If e Sn hr, Lf e Sn hr, and If e Sn Hr requirefewer inductive cycles as they age. It is suggested that thisresults from a decrease in the activity of the Sn gene in theleaves as they age, resulting in a higher ratio of promoterto inhibitor. Gene Lf does not affect the rate of this agingbut it does increase the number of inductive cycles requiredfor flower induction over the first 5 weeks of growth. The geneHr has no effect until week 4 but thereafter causes a reductionin the effect of age on the Sn gene. The genotype If e Sn Hrcan be induced by a single inductive cycle (32 h of light) fora relatively long period. The length of dark period required for the expression of theSn gene is shown to be less than 4 h providing a relativelylong photoperiod precedes the dark period. It appears that noper manent induction of tissue by photoperiods favourable toflowering occurs in peas. The critical photoperiod for plantsof genotype if e Sn Hr is shown to be between 12 and 14 h atl7·5 °C and the usefulness of the term ‘criticalphotoperiod’ is discussed with respect to quantitativelong-day plants.     

PLASTICITY OF THE LIFE CYCLE OF XEROPICTA DERBENTINA (KRYNICKI, 1836), A RECENTLY INTRODUCED SNAIL IN MEDITERRANEAN FRANCE

Xeropicta derbentina (Krynicki, 1836), a native of Eastern MediterraneanEurope, was introduced to southeastern France during the 1940sand is now widely spread across Provence. In summer it aggregateson plants, making its populations clearly visible. However,its life cycle within the Mediterranean basin is poorly documented.While X. derbentina in its native area exhibits an annual lifecycle, this species has been found in Provence to have a bienniallife cycle. Moreover, in southeastern France, field studieswithin a restricted area show variations in demographic structure.In consequence, the life cycle of X. derbentina and the demographicpatterns observed require clarification. Five populations withvarious demographic structures were studied over 1 year in thesame location, i.e. under the same climatic conditions. Thefield study was complemented by laboratory observations on mating,egg-laying and hatching. Xeropicta derbentina appears to bea semelparous species, with an annual life cycle being foundon four plots. The reproductive period begins at the end ofsummer and lasts until the beginning of winter. First egg-layingoccurs within 1 week after mating and lasts up to 30 days. Hatchingtakes place 15–20 days after egg-laying. Xeropicta derbentinapossesses multiple mating and egg-laying sessions, involvingsuccessive hatching. Populations are mainly characterized bytwo growth stages, the first in spring when newly-hatched snailsevolve into juveniles, and the second in late summer when theyreach maturity. However, on the highest density plot, a bienniallife cycle is observed for some newly-hatched snails that showan interrupted growth during summer and evolve into juvenilesonly in the second autumn. Moreover, this life cycle not onlyvaries among plots but also at a 1-year interval within plots.Hence, the life span of X. derbentina is between 12 and 20 months,but can be extended up to 30 months according to whether hatchingoccurs early or late and whether they survive the first andsecond winters. Xeropicta derbentina is thus able to have variousgrowth speeds and life spans, and appears to switch from anannual life cycle to a biennial cycle in response to populationdensity or climatic conditions. (Received 8 October 2004; accepted 15 December 2004)     

Neural control of RNA synthesis in regenerating limbs of the adult newtTriturus viridescens

Summary Polyacrylamide gel electrophoresis was used to investigate the role of nerves in controlling patterns of RNA synthesis in regenerating limbs of the adult newt,Triturus viridescens. Denervation has the same effect on nerve-dependent and independent stages of regeneration, reducing by approximately 40–50% the synthesis of ribosomal and transfer RNA. Although a differential qualitative response of messenger RNA synthesis to denervation between nerve-dependent and independent stages has not been ruled out, the results would indicate that the effect of the nerve on RNA metabolism in individual blastema cells is the same over the whole process of regeneration. Since the one constant effect of denervation on regeneration is to inhibit regenerate growth in volume, the emancipation of blastemal morphogenetic activity from nerve requirements is more likely to be a function of attaining a critical mass of blastema cells, rather than a change in the metabolic response of blastema cells to the nerve.Research supported by a Biomedical Sciences Grant from the School of Life Sciences, University of Illinois, to D.L.S.     

Nerve-independent DNA synthesis and mitosis in regenerating hindlimbs of larval Xenopus laevis

Summary Xenopus laevis larvae at stage 52–53 (according to Nieuwkoop and Faber 1956) were subjected to amputation of both limbs at the thigh level as well as to repeated denervations of the right limb. Results obtained in larvae sacrificed during wound healing (1 after amputation), blastema formation (3 days) and blastema growth (5 and 7 days) showed that denervated right limbs have undergone the same histological modifications observed in innervated left limbs and have formed a regeneration blastema consisting of mesenchymal cells with a pattern of DNA synthesis and mitosis very similar to that in presence of nerves. Also, the patterns of cellular density in regenerating right and left limbs were very similar. On the whole, the data here reported show a highly remarkable degree of nerve-independence for regeneration in hindlimbs of larval Xenopus laevis at stage 52–53 and lend some substance to the hypothesis that, in early limbs, there would exist trophic factors capable of replacing those released by nerves, promoting DNA synthesis and mitosis in blastemal cells. Offprint requests to: S. Filoni     

Nerves and Proliferation of Progenitor Cells in Limb Regeneration

Nerves, in conjunction with the apical epidermal cap (AEC), play an important role in the proliferation of the mesenchymal progenitor cells comprising the blastema of regenerating urodele amphibian limbs. Reinnervation after amputation requires factors supplied by the forming blastema, and neurotrophic factors must be present at or above a quantitative threshold for mitosis of the blastema cells. The AEC forms independently of nerves, but requires nerves to be maintained. Urodele limb buds are independent of nerves for regeneration, but innervation imposes a regenerative requirement for nerve factors on their cells as they differentiate. There are three main ideas on the functional relationship between nerves, AEC, and blastema cells: (1) nerves and AEC produce factors with different roles in maintaining progenitor status and mitosis; (2) the AEC produces the factors that promote blastema cell mitosis, but requires nerves to express them; (3) blastema cells, nerves, and AEC all produce the same factor(s) that additively attain the required threshold for mitosis.     

Neural Control of Cell Cycle Events in Regenerating Salamander Limbs

Nerves, wound epidermis, and injury are indispensable for salamanderlimb regeneration, but their mechanism of action is not understood.A hypothesis has been presented (Tassava and Mescher, 1975)which suggests that injury is important to dedifferentiationand entry of limb stump cells into the cell cycle, nerves arerequired for one or more G₂ events in order that cells can proceedto mitosis, and the wound epidermis maintains the daughter cellsin the cell cycle. The resultant cells accumulate to form theblastema. Complete and partial denervation experiments, which attemptedto test this hypothesis, are discussed. Blastema cell cycleparameters, measured after complete denervation, did not varygreatly from innervated controls, even though denervated blastemaswere resorbed. Blastema cell cycle parameters of partially denervatedlimbs, which exhibited delayed regeneration, were likewise notlengthened when compared to completely innervated controls.These results are consistent with the view that after eithercomplete or partial denervation, some blastema cells continueto cycle and reach the M phase in the same time as controls.Other blastema cells block completely, never reach M, and arethen removed. A possible mechanism for resorption of denervatedblastemas is presented.     

Nerve-induced ectopic limb blastemas in the Axolotl are equivalent to amputation-induced blastemas

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.     

Promotion of mitosis in cultured newt limb regenerates by a diffusible nerve factor

Summary Regeneration blastemata of adult newt forelimbs were cultured transfilter to dorsal root ganglia on extremely low porosity (0.05 μm) filters. Mitotic index profiles in these blastemata were compared with those obtained using filters of greater porosity (0.45 μm). In the above experiments nerves and blastema tissue were separated by 5 or 25 μm, i.e., the thickness of the respective filters. The results show that the transfilter mitogenic effect of the nerves was retained when the lower pore size filters were used. In addition, sensory ganglia grown at the bottom of a culture well, separated from the blastema explants by a distance of approximately 2 mm, were nevertheless able to promote blastema cell proliferation. The ganglia can thus be considered to be providing a “sustained conditioning” of the medium with neuromitogenic factor(s). This study also shows that nerves can promote blastema cell mitosis, although cell-to-cell contact between nerves and responding cells was precluded. This work was supported by National Science and Engineering Research Council (NSERC) Grants A6933 to M. Globus and A9753 to S. Vethamany-Globus.     

A COMPARISON OF THE LIFE CYCLES OF THE SLUGS DEROCERAS RETICULATUM (MULLER) AND ARION INTERMEDIUS NORMAND ON PERMANENT PASTURE

Life cycles of the slugs Deroceras reticulatum and Arion intermediuswere compared over a four-year period on permanent pasture nearOvingham, Northumberland. Both species were semelparous, theirlife cycles taking about one year. D. reticulatum, unlike A.intermedius and other species, had two overlapping generationsthat laid their eggs in late spring and autumn respectivelyand also had one instead of two immature phases in its lifecycle. The pattern and rates of growth for the immature phasesin the life cycle of A. intermedius differed markedly from thosedescribed under laboratory conditions. Growth in the infantilephase was delayed in the field by low winter temperatures whilesecond stage growth was usually delayed in late spring by dryconditions. The life cycle of A. intermedius appears to be synchronisedby seasonal changes in photoperiod, unlike that of D. reticulatum.Growth in D. reticulatum continued throughout the winter monthsexcept under exceptionally cold conditions and was not usuallydelayed by dry conditions in spring. The size of hermaphroditegland relative to body weight in D. reticulatum reached a maximumin mature-unmated slugs and then became progressively smalleras the slug approached the post-reproductive stage which wasrelatively brief. Exceptionally dry conditions in 1962 delayedthe development of the hermaphrodite gland, the maximum sizereached was significantly reduced and fewer eggs were laid. (Received 17 September 1987; accepted 2 January 1988)     

Effects of the Photoperiod Genes Ppd1 and Ppd2 on Growth and Development of the Shoot Apex in Wheat

Two major genes influencing the photoperiod response in wheat,Ppd1 and Ppd2, have been identified on the group 2 chromosomes.Substitution lines, which had been characterized on the basisof time of ear emergence as carrying either the insensitiveor sensitive alleles of the two Ppd genes, were used to investigatethe effect of these genes on development. They were grown undershort photoperiods, and growth and development of the shootapex was measured. The primary influence of the Ppd genes was on ear growth. Inthe plants carrying the insensitive alleles, Ppd1 and Ppd2,the relative growth rate of the floral apex was faster thanthat of plants with the sensitive alleles, ppd1 and ppd2. Therewere no differences in the rate of spikelet initiation, butthe spikelets of the ppd lines grew and developed more slowly. The Ppd2 material segregated for another gene located on chromosome2B affecting duration of the life cycle. This gene also affectedthe relative growth rate of the ear. It was considered that the major effect of the Ppd1, Ppd2 andthe second genetic factor on chromosome 2B is on floral growthrate. Differences in apex morphology, stem growth and ear emergenceare thought to be due to the differences in floral apex growthand size. Wheat, photoperiod genes, shoot apex development, shoot apex growth     

Genetic Variation in Sorghum for the Inhibition of Maize Pollen Tube Growth

Aniline blue fluorescence was used to study the growth of maizepollen tubes in the stigmas of 13 diverse sorghum accessions.In 12, only short maize pollen tubes were formed, but in thesingle exception (Sorghum nervosum Nr481) maize pollen tubesgrew at least as far as the base of the style. The S. bicolorgenotypes S9B and CMS (a cytoplasmic male sterile line) werehybridized with Nr481, and analysis of maize pollen tube growthin F₁ plants, and BC₁ plants using Nr481 as the recurrent parent,suggested that differences in inhibition of pollen tube growthwere due to variation at a single locus, which we propose todesignate lap (Inhibition of alien pollen tubes). AccessionNr481 appears to be homozygous for a recessive allele permittingmaize pollen tube growth. Attempts were made to produce sorghumx maize hybrids using Nr481 and CMS derivatives which were knownto allow maize pollen tube growth to the base of the style.A putative hybrid endosperm was obtained in one Nr481 x Seneca60 maize cross, but this was not repeatable and no hybrid plantswere produced. A fundamental problem may be the large size ofthe maize pollen tube, which could have difficulty growing throughthe sorghum ovary and in entering the micropyle. Sorghum bicolor spp. bicolor (L.) Moench, Zea mays L, sorghum, maize, pollen tube growth, hybridization barriers     

Transfilter mitogenic effect of dorsal root ganglia on cultured regeneration blastemata,in the newt,Notophthalmus viridescens

Cone stage forelimb blastemata from adult newts were separated into proximal and distal regions and cultured along with dorsal root ganglia in both transfilter and cis (same side) configurations, for a period of 96 h in modified Parker's medium (CMRL-1415) containing insulin and l-thyroxin. Mitotic index and differentiation of cartilage were assessed in ganglionated and nonganglionated, proximal and distal explants after 4 days in vitro. The results show that the nerve influence on the regeneration blastema appears to be mediated by a chemical substance capable of transmission through thin filters of low porosity Moreover the neurotrophic substance has mitogenic properties. The ganglia stimulated blastema cell proliferation transfilter, increasing it from a basal level (mitotic index = 0.339), observed in noninnervated explants to almost threefold values (M.I. = 1.124) in corresponding distal innervated explants. In addition, this transfilter mitogenic effect was manifested in the form of a proximodistal gradient with the highest mitotic index close to the neurons, which diminished with distance from the nerve source. When blastema explants were grown in physical contact with ganglionic neurons (cis configuration), they transcended the proliferation phase within the 4 days of culture and differentiation of cartilage whorls resulted. Presumably, a critical mass of blastema cells is achieved earlier in the presence of a higher concentration of neurotrophic factor.     

Studies on Morphological Changes of the Blue-green Alga Nostoc muscorum A with Special Reference to the Role of Light

Morphological changes of Nostoc muscorum A were studied withspecial reference to growth conditions. According to Lazaroff(1973), N. muscorum A has a life cycle dependent on the lightconditions; cells of a coccoid form grow in the dark (aseriatestage) while cells of a filamentous form grow in light (seriatestage). The conversion from the aseriate to the seriate stageis photocontrolled by red stimulation and green suppression.We reexamined (i) the light effect on the morphological formsof N. muscorum A growing under various conditions, includingN₂-fixing and non-fixing, and (ii) the light effect on the controlof the aseriate to seriate conversion in the dark in relationto the effect on the dark growth. Results for (i) indicatedthat cell forms did not necessarily depend on the light butwere determined by growth conditions; under conditions sufficientfor supporting rapid growth, cells grew at the seriate stage,and under insufficient conditions, at the aseriate stage. Thelight effect to induce and support filamentous growth was explainedas the photosynthetic energy supply for improving the growth.Results for (ii) revealed that red light induced the dark aseriateto seriate conversion and at the same time enhanced the darkgrowth after the illumination. Green light suppressed both theconversion and the dark growth stimulated by red light. Thered-green photocontrol of the dark conversion was explainedby the enhancing effect of red light and the inhibitory effectof green light on the dark growth. Morphological changes betweencoccoid and filamentous forms of N. muscorum A are probablynot obligatory for continued growth. ¹Present address: Department of Botany, Faculty of Science,Kyoto University, Kitashirakawa, Kyoto 606, Japan. (Received September 22, 1980; Accepted December 6, 1980)