bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic acid and pattern formation in the developing chick wing: SEM and quantitative studies of early effects on the apical ectodermal ridge and bud outgrowth
Authors: Lee J. , Tickle C.
Published: April 2021

When retinoic acid is locally applied to the anterior margin of developing chick wing buds on ion-exchange beads, dose-dependent changes in the skeletal pattern result. At low doses, additional digits develop. At high doses, there is thinning of the symmetrical wing. Local application of retinoic acid to the apex of the bud also leads to pattern changes, but in contrast normal wing patterns are almost always obtained following application posteriorly. These effects are manifest at 6–7 days after the operation although only a brief exposure (14–20 h) to retinoic acid is required. Therefore the morphology of wing buds was studied at shorter times after the start of treatment.

The local application of retinoic acid to the wing bud margin leads to changes in extent of the apical ridge that can be detected at 24 h after application. The behaviour of the apical ridge with varying doses and positions of retinoic acid application has been analysed quantitatively and dose response curves obtained. At low doses of retinoic acid, the length of the apical ridge increases or remains constant, but then progressively decreases with higher doses. The progressive obliteration of the ridge starts first near the bead and then involves more distant parts of the bud. Thus the region of the ridge affected depends on the position at which the retinoic acid is applied.

We propose that these effects on the apical ridge reflect dose-dependent responses to the local concentration of retinoic acid that varies with distance from the source. At high doses, the apical ridge disappears but at low doses it is maintained. Since grafts of polarizing region tissue also have a graded effect on ridge morphology, a possible interpretation of the retinoic acid effects is that tissue adjacent to the source is converted into polarizing region tissue. Alternatively, retinoic acid may act directly on the ridge cells.

The changes in the extent of the apical ridge produced by retinoic acid lead to different forms of bud outgrowth. The form of the outgrowth depends on the dose of retinoic acid, the position of application and the interaction between the effects of the local source of retinoic acid and those of the polarizing region of the host bud. These considerations give some insights into why anterior application of retinoic acid leads to the development of additional digits whereas posterior application generally gives normal wings.
Cleavage and gastrulation in the egg-brooding, marsupial frog, Gastrotheca riobambae
Authors: Elinson Richard P. , Pino Eugenia M. Del
Published: April 2021

The marsupial frog Gastrotheca riobambae has several reproductive adaptations, most prominent of which is the incubation of the embryo in a pouch on the mother’s back. We have followed cleavage and gastrulation by microscopical observation and by vital staining, and have found several alterations in these processes which may reflect the reproductive adaptations. The large, yolky egg has a cap of yolk-poor cytoplasm at the animal pole which is incorporated into a translucent blastocoel roof consisting of a single cell layer. The epithelium of the yolk sac is derived from the roof. The inconspicuous blastoporal lips form near the vegetal pole from cells of the marginal region. Gastrulation movements include the epibolic stretching of the surface towards the blastopore and a contraction of the vegetal surface. The blastoporal lips close over a small archenteron, and the cells of the lips become the embryonic disc, a discrete group of small cells which give rise to most of the embryo’s body. The great size difference between animal and vegetal blastomeres during cleavage, the single-celled blastocoel roof, the dissociation in time between archenteron formation and its expansion, the embryonic disc and the slow development distinguish G. riobambae embryos from those of other frogs. The importance of the marginal region which produces the embryonic disc and the unimportance of the most animal region whose fate is primarily yolk sac emphasizes the role of the marginal region in amphibian development.
Interactions between neurites and somite cells: inhibition and stimulation of nerve growth in the chick embryo
Authors: Stern Claudio D. , Sisodiya Sanjay M. , Keynes Roger J.
Published: April 2021

After neural processes emerge from the neural tube in the chick embryo, their growth is restricted to the cranial halves of the neighbouring somites. In this study we have developed an in vitro system to model the interactions between these tissue types. Pioneer neurites display a hierarchy of preferences in terms of the substrates they can grow on. As expected, tissue culture plastic does not support neural outgrowth, but this can be overcome by coating the plastic substrate with either collagen or poly-L-lysine. Neural crest, cranial half somite, and a number of other tissues support growth well, while caudal half somite and tail bud mesenchyme do so to a much smaller extent.

The binding pattern of a variety of lectins was assessed in cryostat sections of embryos and in cultured cells of the above tissues. It was found that peanut agglutinin can discriminate between cranial and caudal sclerotome both in vitro and in the embryo, since it binds preferentially to caudal sclerotome in both cases. This difference is expressed as soon as the sclerotome forms.

The significance of these findings is twofold: first, they show that the interactions that take place during peripheral neural segmentation can be modelled in vitro; second, they represent the first instance of a molecular difference between the cranial and caudal halves of the sclerotome, detectable both in culture and in the embryo.
The effects of local application of retinoic acid on limb development and regeneration in tadpoles of Xenopus laevis
Authors: Scadding S. R. , Maden M.
Published: April 2021

Vitamin A can have different effects on developing and regenerating limbs depending on the mode of administration. Previous work has demonstrated the differential effect of retinol palmitate on limb development and regeneration in Xenopus laevis. The purpose of the present investigation was to determine the effects of vitamin A on limb development and regeneration in Xenopus when administered by a local implantation method. Xenopus tadpoles had both hindlimbs implanted with either a block of silastin carrying retinoic acid or an anion exchange resin bead carrying retinoic acid and then the right hindlimb was amputated and the effect of the retinoic acid on limb development and regeneration was studied. The results showed that in developing hindlimbs the effects of silastin implants carrying retinoic acid was to cause skeletal reductions or deletions similar to those induced by immersion of the tadpole in retinol palmitate. On the other hand, in regenerating hindlimbs, the silastin implants caused a range of skeletal reductions and deletions as well as occasional accessory structures but notably induced no proximodistal (PD) duplications, unlike the effect of immersion in retinol palmitate where PD duplications were a common response. Implantation of anion exchange resin beads carrying retinoic acid had no significant effect on either development or regeneration beyond stage 50, presumably because the dose of the retinoic acid was so low. Thus the results suggest that the mode of administration of vitamin A has a very significant influence on its effects. The significance of this observation for vitamin A experiments on limbs is discussed.
Changes in patterns of protein synthesis in axolotl oocytes during progesterone-induced maturation
Authors: Gautier Jean , Tencer Renée
Published: April 2021

Patterns of protein phosphorylation and synthesis during axolotl (Ambystoma mexicanum) oocyte maturation were studied by incorporation of [32P]orthophosphate and [35S]methionine into polypeptides, followed by two-dimensional gel electrophoresis. Various alterations were observed after progesterone treatment: de novo appearance of [35S]methionine-labelled polypeptides, a quantitative increase in previously synthesized proteins and a quantitative decrease in or disappearance of other previously synthesized proteins. Changes in 32P- and 35S-labelling were observed very early during maturation. Neither prior oocyte enucleation nor α-amanitin treatment had a significant effect on these changes. Stimulation with MPF provided the same final protein pattern as PG treatment. However, cholera toxin inhibited all the changes seen during maturation. Comparisons between the patterns of [35S]methionine- and [32P]phosphate-labelling provide further information on the biochemical events that take place during oocyte maturation.
Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane*
Authors: Maro Bernard , Johnson Martin H. , Webb Michelle , Flach Gin
Published: April 2021

The influence of mouse oocyte chromosomes on their immediate environment has been investigated following their dispersal by dissolution of the metaphase spindle with nocodazole. Small clusters of chromosomes become redistributed around the egg cortex in a microfilamentdependent process. Each cluster has the capacity, on removal from nocodazole, to organize a spindle that rotates to yield a polar body. In this process of spindle formation, the chromosome clusters are able both to promote tubulin polymerization in their vicinity and to recruit microtubule-organizing centres (MTOCs) which organize the polymerized tubulin into spindles. In addition each oocyte chromosome cluster, as well as the non-dispersed sperm-derived haploid group of chromosomes, induces a focal accumulation of subcortical actin (corresponding to a filamentous area devoid of organelles) and a loss of surface Concanavalin A binding activity (corresponding to a loss of surface microvilli) in the overlying cortex. This induction ceases with the formation of pronuclei whether or not the pronuclei migrate centrally. Pronuclear formation is sensitive to the action of nocodazole for up to 2–4h postinsemination, and pronuclear migration is totally sensitive to the drug. If pronuclei are blocked in a peripheral location by nocodazole they are associated with an elevation in Con A binding activity of the overlying membrane which corresponds to an area of the surface rich in blebby microvilli.
Effect of experimentally induced calcium deficiency on development, metabolism and liver morphogenesis of the chick embryo
Authors: Ono Tamao , Tuan Rocky S.
Published: April 2021

To study the effects of systemic calcium deficiency on embryonic development, chick embryos maintained in long-term shell-less cultures were compared to control embryos incubated in ovo with respect to various parameters of metabolism and growth. After incubation day 14, retarded growth and development were apparent in shell-less embryos which exhibited severe hypocalcaemia and hyperphosphataemia. A development-specific necrosis of the liver tissues was observed in both shell-less and control embryos, but the frequency and extent of tissue abnormality were significantly greater in the former. Serum levels of lactate dehydrogenase and alkaline phosphatase were considerably elevated in shell-less embryos. Electrophoretic analysis revealed that the relative levels of two major serum proteins were also altered in shell-less embryos.
Cleavage in vitro following destruction of some blastomeres in the marsupial Antechinus stuartii (Macleay)
Authors: Selwood Lynne
Published: April 2021

The effect of destruction of some blastomeres on subsequent cleavage in vitro was investigated in the marsupial Antechinus stuartii. Blastomeres were killed by puncturing with a fine glass needle. The positions of the punctured and surviving blastomeres were recorded with respect to the yolk mass and defects on the shell membrane. Blastomeres were punctured in 2-cell, 4-cell, 14-cell, 16-cell and 22-cell embryos. Subsequent development of the embryos was followed in vitro. Embryos developed at a normal rate after destruction of some blastomeres. Puncturing of the shell, mucoid layer and zona pellucida, without damage to blastomeres, did not affect subsequent development. The embryos developed in accordance with the proportion of surviving blastomeres. Embryos with half of the blastomeres surviving developed as embryos with half the zona pellucida lined by blastomeres. Embryos with 1/4 of the blastomeres destroyed developed as 3/4 embryos. It is not known, without transfer into surrogate mothers, whether these fractional embryos were capable of developing into complete embryos.
Bud induction in decapitated Hydra attenuata by 5-azacytidine: a morphological study
Authors: Petrocellis L. De , Maharajan V. , Petrocellis B. De , Minei R.
Published: April 2021

The effect of 5-azacytidine (5-azaCR) on head regeneration and budding in hydra are reported. Hydra attenuata were exposed to various doses of 5-azaCR for 48 h and then decapitated and cultured. Head regeneration and bud formation were observed for 12 days after decapitation. Untreated control hydra regenerated heads within 7 to 8 days of decapitation with a budding index of 0·2. Buds invariably arose in the normal budding zone (below the gastric region).

In the group treated with 0·8mM-5-azaCR, 9 days after decapitation head regeneration was seen in only 13 % of animals, and an average of two buds per hydra were formed, most of which were in the vicinity of the distal end. Induction of budding was also seen in the animals that regenerated heads.

In animals exposed to 1 mM-5-azaCR three main types of responses were observed 9 days after decapitation. (1) 44% of the animals regenerated normal heads; about half of them developed at least one bud and these buds originated in the budding zone. (2) 17·5% of the animals developed abnormal, long hypostome-like structures with single or bifurcated tentacles at their tips. There were at least two buds per animal and they were invariably at abnormal sites. (3) 32 % of the animals failed to regenerate heads, although they developed two buds. 87 % of these buds originated in abnormal sites of the body column and a large number (72%) did not detach even by the 12th day after decapitation.

Both 5 and 10 mM of 5-azaCR were toxic to the animals; the survivors formed large globeshaped heads. Bud induction was seen in 60% and 28% of animals in the 5 and 10 mM groups, respectively.

These observations demonstrate that 5-azaCR induces bud formation in hydra at doses that inhibit head regeneration. This bud induction might be due to a specific expression of gene products responsible for bud formation.
Myoblasts and notochord influence the orientation of somitic myoblasts from Xenopus laevis
Authors: Mccaig Colin D.
Published: April 2021

The orientation of developing myoblasts extending a bipolar axis in the presence of explanted myoblasts or whole notochord has been studied in vitro. Myoblasts tended to elongate perpendicular to the lines of diffusion of substances from these tissues. A slow-release source of agar impregnated with medium conditioned by segmented somitic myoblast or notochord also caused myoblasts to elongate perpendicular to the lines of diffusion from the source. Medium conditioned by neural tube cells or unsegmented mesoderm cells did not influence the orientation of myoblasts.

It is concluded that somites and notochord release diffusible substances in vitro which are capable of directing the orientation of developing myoblasts. In vivo, a somite-derived material could play a role in determining the direction of myoblast elongation in the presomitic mesoderm. An interaction between somite and notochord-derived secretions could influence the rotation of presomitic myoblasts to form a segmented somite.
Reversible and irreversible effects of retinol upon the phenotypic properties of embryonal carcinoma cells
Authors: Sherman Michael I. , Eglitis Martin A. , THOMAS RICHARD
Published: April 2021

We have generated an embryonal carcinoma cell line, NR1, which is not growth-inhibited in response to retinol. Although the retinol-treated cells undergo morphological change, show reduced levels of the surface antigen SSEA-1 and possess increased surface reactivity with antifibronectin serum, the extent of phenotypic change of NR1 cells in response to retinol is not so great as that following treatment with retinoic acid. Furthermore, unlike cells from lines such as F9, the retinol-promoted morphological alterations appear to be reversible. The increased adherence of NR1 cells to glass coverslips in the presence of retinol suggests that stronger interaction with the substratum is responsible for the observed alteration in appearance of the cells. It is possible that NR1 cells exposed to retinol progress no further than a reversible, early stage of differentiation. Alternatively, retinol-treated cells might express a group of markers normally associated with, or accompanying, an irreversible, differentiated phenotype even though the cells are not, in fact, undergoing differentiation.
The pigmentary system of developing axolotls
Authors: Frost S. K. , Epp L. G. , Robinson S. J.
Published: April 2021

The axanthic mutant in the Mexican axolotl (Ambystoma mexicanum) was analysed with respect to the differentiation of pigment cells. Transmission electron micrographs revealed the presence of melanophores and cells that are described as unpigmented xanthophores in axanthic skin. Iridophores apparently failed to differentiate in axanthic axolotls (a pattern similar to that observed in melanoid axolotls). Chromatographic analyses of skin extracts confirmed that there are no pteridines (xanthophore pigments) in axanthic skin, suggesting that the axanthic gene may affect pteridine biosynthesis at some point early in the biosynthetic pathway. Why iridophores fail to differentiate in these animals is not known, but this, too, may be related to an inability to synthesize pigments properly. Xanthophore and iridophore pigments both presumably derive from purine precursors.

Finally, all axanthic animals were found to be infected by a virus. Electron microscopic results demonstrated the presence of numerous macrophages in the dermis of the skin, occupying positions typical of pigment cells. The virus was localized primarily in macrophages, but was also observed in pigment cells. The virus is, as yet, uncharacterized but is thought to contribute to the low survivability of axanthic adults.
Fate of brachial muscles of the chick embryo innervated by inappropriate nerves: structural, functional and histochemical analyses
Authors: Butler Jane , Cauwenbergs Peter , Cosmos Ethel
Published: April 2021

The extent of interaction between brachial muscles and foreign (thoracic) nerves of the chick embryo was determined during an extended period of development in ovo from the perspectives of innervation pattern, function (motility analyses), muscle growth (quantitative analyses of muscle volume) and fibre-type expression (myosin-ATPase profiles). Results indicated that according to all parameters analysed, initially a compatible union existed between the foreign nerves and their muscle targets. During subsequent stages of development, deterioration of the once compatible relationship emerged, until eventually denervation of muscles, i.e. an actual loss of intramuscular nerve branches, was observed. The process of denervation, which proceeded at a differential rate among individual muscles, however was restricted to brachial muscles derived from the premuscle masses of the wing bud. In contrast, brachial muscles of myotomal origin were spared the fate of wing-bud-derived muscles and maintained a successful union with the foreign nerves.
The generation of cell surface polarity in mouse 8-cell blastomeres: the role of cortical microfilaments analysed using cytochalasin D
Authors: Fleming T. P. , Pickering S. J. , Qasim F. , Maro B.
Published: April 2021

The mechanism by which a surface pole of microvilli is generated in mouse 8-cell blastomeres has been investigated. 4-cell and 8-cell embryos (or cell couplets) were incubated for precise times during their respective cell cycles in medium containing cytochalasin D (CCD) to disrupt the microfilament system. The blastomeres were analysed immediately for the distribution and state of organization of their microvilli, using three morphological techniques. The results indicate that the surface pole, characterized by microvilli containing CCD-resistant core filaments, is not generated by the gradual segregation of stable microvilli to the apical surface. An alternative model is proposed, based upon (a) the stabilization of the apical cytocortex prior to the elongation of apical microvilli and (b) the destabilization of cytocortical elements in contact areas and the shortening and loss of basolateral microvilli.
The timing of compaction: control of a major developmental transition in mouse early embryogenesis
Authors: Levy Jeremy B. , Johnson Martin H. , Goodall Harry , Maro’ Bernard
Published: April 2021

The effect of protein synthesis inhibitors on compaction of the 8-cell mouse embryo has been investigated. The effects observed depended upon the duration and time of drug application and on the features of compaction scored. Continuous application from the late 2-cell or early 4-cell stages allowed cell flattening and surface polarization to occur in most embryos and advanced development of these features in many of them. Cell coupling developed only when drug addition was delayed until the mid 4-cell stage, and cytoplasmic polarization developed only when drug addition was delayed until the late 4-cell stage. We suggest that control over the timing of compaction is achieved at a post-translational level via a global permissive change within the blastomeres of the embryo.
Regeneration from half lower arms in the axolotl
Authors: Wigmore Peter
Published: April 2021

A technique involving grafting of pieces of skin from the head onto the limb in order to isolate halves of the limb is described. This technique was used to isolate posterior, anterior, dorsal and ventral halves of the lower arm. All halves produced regenerates but no part of the limb was able to produce a high proportion of regenerates with a complete pattern of skeletal structures. Posterior half stumps regenerated limbs with a mean digit number of 2·7 and had a normal dorsoventral muscle pattern. Anterior half stumps produced a high proportion of single-digit regenerates and had a mean digit number of 1·3. Dorsal and ventral half stumps regenerated limbs with a mean digit number of 2·8 and 2·3 respectively. Hypomorphic regenerates from dorsal and ventral half stumps often had only dorsal or ventral muscle. These results are in contrast to those from the upper arm (Wigmore & Holder, 1985) where a complete skeletal and muscular pattern regenerated from posterior and dorsal halves and hypomorphic regenerates were obtained from anterior and ventral half limbs.
The clonal model of vertebral column development: a reinvestigation of vertebral shape using Fourier analysis
Authors: O’Higgins P. , Johnson D. R. , McAndrew T. J.
Published: April 2021

Little work has been done on the skeletal system of allophenic mice (chimaeras). In this paper we re-examine the evidence presented for the clonal theory of vertebral development using quantitative methods which take account of variation. We conclude that the clonal theory is either an overly simplistic approximation of the real situation, or that the evidence so far given in its support is unsubstantiated.
Whisker growth induced by implantation of cultured vibrissa dermal papilla cells in the adult rat
Authors: Horne Kenneth A. , Jahoda Colin A. B. , Oliver Roy F.
Published: April 2021

Retention of the capacity to induce the growth of hair by cultured adult rat vibrissa dermal papilla cells has been investigated. Small pellets of serially cultured papilla cells were implanted into the bases of the exposed follicular epidermis of amputated adult rat vibrissa follicles. Amputated follicles that received no cell implants or implants of cultured dorsal skin fibroblasts were used as controls. Over 50 % of follicles implanted with cultured papilla cells in the passage range 1 –3 grew hairs. In contrast none of the follicles that received late passage cells (range 6 –15) produced hairs; and spontaneous regeneration of hair occurred in only 3 % of the control follicles. These results demonstrate that cultured papilla cells of early passage numbers retain their ability to induce hair growth. Histological examination confirmed that the implanted papilla cells interacted with follicular epidermis to organize the development of new, hairproducing bulbs, each containing a discrete dermal papilla. An important observation was that aggregative behaviour leading to papilla formation was only manifested by early passage papilla cell implants. This persisting embryonic characteristic appears to be an essential functional component of papilla cell activity which operates to regulate the profound morphogenetic changes that occur during the hair growth cycle.
The differentiation of mouse gonads in vitro : a light and electron microscopical study
Authors: Mackay Sarah , Smith Robert A.
Published: April 2021

Indifferent urogenital complexes were excised from mouse foetuses assessed by developmental criteria as day 10·5 or 11. After 4 or 6–7 days in culture, complexes were fixed and examined by light and electron microscopy. The effect of culturing sexed complexes in mixed sex groups was investigated. The effect of the presence or absence of foetal calf serum in the culture medium was considered. No evidence of inhibition of one sex by the other was found. Ovaries developed further in cultures than testes.
Factors modulating mouse lens epithelial cell morphology with differentiation and development of a lentoid structure in vitro
Authors: Muggleton-Harris A. L. , Higbee N.
Published: April 2021

The morphological and cellular changes that occur with differentiation and development of a lentoid structure from cultured mouse lens epithelial cells have been found to be dependent on the presence of lens capsule in association with the cells. The development of the ‘lentoid body’ is a multiphase process involving cell replication, synthesis of mucosubstances and a basement collagen membrane, cell aggregation and differentiation. Stage-specific synthesis of lens proteins confirms that the genes regulating normal differentiation in vivo are operating in the in vitro system.

The hydrated collagen gel studies described in this report demonstrate that the cuboidal morphology and apical-basal polarity of the lens epithelial cells are dependent on their relationship with the lens capsule. Following a replicative phase the cells assume a mesenchyme-like morphology and migrate into the gel. Trypsinized cells freed from the lens capsule replicate but form colonies on the surface of the gel. The implications of these results are discussed with respect to previous observations made on normal lens development and the abnormalities associated with the congenital cataractous embryonic lens.
Differentiation and grafting of haemopoietic stem cells from early postimplantation mouse embryos
Authors: Hollands P.
Published: April 2021

Haemopoietic stem cells evidently arise in early postimplantation mouse embryos at day 6 of gestation, a day earlier than previously thought (Moore & Metcalf, 1970). Disaggregated embryonic cells were injected into mice given a lethal dose of X-irradiation. The presence of donor haemoglobin (Whitney, 1978) and donor lymphocytic glucose phosphate isomerase (GPI) (Siciliano & Shaw, 1976) to detect donor erythrocytes and lymphocytes, respectively, were monitored by starch gel electrophoresis. The presence of donor cells was also assessed by using donor embryos carrying the T6 marker chromosomes. Decidual cells dissected free of embryos did not colonize any recipients. Disaggregated cells from early mouse embryos first colonized the liver and then repopulated the haemopoietic systems of recipients, producing adult donor haemoglobin within 2–3 days and donor GPI within 3–5 days. 80 % of grafted X-irradiated recipients survived and donor markers were found in each of them. All nongrafted controls died within 14 days of X-irradiation and none of them showed donor markers. Disaggregated embryonic cells could be grafted across major histocompatibility barriers unlike adult bone marrow. Haemopoietic stem cells could not be identified in disaggregated cells from embryos aged less than 6 days gestation.
Dorsal root ganglia grafts stimulate regeneration of denervated urodele forelimbs: timing of graft implantation with respect to denervation
Authors: Tomlinson Bruce L. , Tassava Roy A.
Published: April 2021

Amphibian forelimb regeneration is a nervedependent process; nerves presumably release one or more neurotrophic factors that stimulate blastema cell division. To date several candidate molecules/factors have been shown to stimulate macromolecular synthesis and/or mitosis but sustained cell cycle activity and blastema development have not been achieved. Because dorsal root ganglia (DRG) implants are capable of promoting regeneration of denervated adult newt limbs (Kamrin & Singer, 1959), we have evaluated the DRG stimulation of regeneration in denervated limbs of adult newts and larval axolotls; two alternative timing strategies were tested as a step toward defining bioassay parameters that best reflect neurotrophic activity. The frequency of regeneration in denervated adult newt limbs was compared after providing DRG before or at the time of denervation (to maintain neurotrophic and cell cycle activity) versus DRG implantation at various postdenervation times (to resupply neurotrophic activity and restimulate suppressed cell cycle activity).

The results show that denervated adult newt limbs regenerated most frequently using the maintenance strategy, but as the denervation interval was extended in the restimulation strategy, the frequency of regeneration declined. Larval axolotl limbs responded positively in both maintenance and restimulation DRG-grafting protocols. These results suggest that the efficacy of DRG stimulation of regeneration in adult newts was related to the relative number of blastema cells present at the time of denervation and the proliferative status of the blastema cells; bioassays with denervated adult newt limbs should be designed with these constraints in mind. Because such constraints are not as problematic with the larval axolotl, this species may provide the best opportunity for further defining bioassay parameters related to the neurotrophic stimulation of regeneration.
Homarine (N-methylpicolinic acid) and trigonelline (N-methylnicotinic acid) appear to be involved in pattern control in a marine hydroid
Authors: Berking S.
Published: April 2021

A morphogenetically active compound has been isolated from tissue extract of Hydractinia echinata and identified to be N-methylpicolinic acid (homarine). When applied to whole animals, homarine prevents metamorphosis from larval to adult stage and alters the pattern of adult structures. The concentration of homarine in oocytes is about 25 mM. During embryogenesis, metamorphosis and early colony development the overall homarine content does not change. Adult colonies contain a fourfold lower homarine concentration than larvae. The polyp’s head contains twofold more homarine than the gastric region and the stolons. A second, similarly active compound, N-methylnic-otinic acid (trigonelline), has also been identified in Hydractinia tissue at concentrations about one-third that of homarine.

Incubation of larvae in 10 to 20 μM-homarine or trigonelline prevents head as well as stolon formation. If the compounds are applied in a pulse during metamorphosis, a large part of the available tissue forms stolons. Since μM concentrations of homarine and trigonelline are morphogenetically active, whereas mM concentrations are present in the tissue it appears that both substances are stored within the tissue.
Reformation of the pattern of neuromuscular connections in the regenerated axolotl hindlimb
Authors: Stephens Nigel , Holder Nigel
Published: April 2021

Retrograde neuronal tracing with horseradish peroxidase (HRP) was used to determine the position in the spinal cord of motor neurone pools innervating muscles in the regenerated axolotl hindlimb. This method allows a detailed analysis of the accuracy of reformation of neuromuscular connections. The results show that regenerated distal limb muscles are reinnervated by motor neurones in the same region of the cord as those that innervate normal control distal limb muscles but that proximal muscles are innervated by a mixture of motor neurones in a normal position and motor neurones in a region of the spinal cord that normally supplies innervation to distal limb muscles. This difference between the reinnervation of proximal and distal limb muscles suggests that axons destined for proximal muscles may not enter distal limb territory during reinnervation of the regenerated limb.
Cytoskeletal and DNA structure abnormalities result from bypass of requirement for the cdc10 start gene in the fission yeast Schizosaccharomyces pombe
Authors: Marks John , Fankhauser Christian , Reymond Alexandre , Simanis Viesturs
Published: April 2021

The cdc10 gene of the fission yeast S. pombe is required for traverse of the start control in late G1 and commitment to the mitotic cell cycle. To increase our understanding of the events which occur at start, a pseudoreversion analysis was undertaken to identify genes whose products may interact with cdc10 or bypass the requirement for it. A single gene, sctl+ (suppressor of cdc ten), has been identified, mutation of which suppresses all conditional alleles and a null allele of cdc10. Bypass of the requirement for cdc10+ function by sctl-1 mutations leads to pleiotropic defects, including microtubule, microfilament and nuclear structural abnormalities. Our data suggest that sct1 encodes a protein that is dependent upon cdc10+ either for its normal function or expression, or is a component of a checkpoint that monitors execution of p85cdc10 function.
A quantitative electron microscopic study of changes in microtubule arrays and wall microfibril orientation during in vitro cotton fiber development
Authors: Seagull Robert W.
Published: April 2021

A quantitative electron microscopic (E/M) study of the changes in microtubule arrays and wall microfibril orientation has been done on in vitro grown cotton fibers. Microtubules change orientation during cotton fiber development. During fiber initiation and early elongation, microtubules have a generally random orientation. Microtubules re-orient into shallow pitched helices as elongation and primary wall deposition continue, and into steeply pitched helices during secondary wall deposition. Accompanying the changes in orientation are increases in microtubule length, number, proximity to the plasmalemma and a decreased variability in orientation of the microtubules. Based on these observations, three pivotal stages in microtubule patterns were identified during fiber development: (1) the transition between fiber initiation and elongation, where microtubules develop a shallow pitched helical orientation; (2) the transition between primary and secondary wall synthesis, where microtubules abruptly shift orientation to a steeply pitched helical pattern; and (3) early in secondary wall synthesis, where there is a four fold increase in microtubule number. Microfibrils exhibit changes in orientation similar to the microtubules; however significant differences were found when the precise orientations of microtubules and microfibrils were compared. During secondary wall synthesis, wall microfibrils exhibit some variability in orientation due to inter-fibril bundling, thus indicating that components of the wall may also influence final microfibril orientation.
The contribution of the extracellular matrix to gravisensing in characean cells
Authors: Wayne Randy , Staves Mark P. , Leopold A. Carl
Published: April 2021

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However, when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing Is sensitive to protease treatment; Proteinase K, tbermolysin and collagenase, but not trypsin, "-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.
The 14 kDa β-galactoside binding lectin in myoblast and myotube cultures: localization by confocal microscopy
Authors: Harrison F. Lynne , Wilson T. J. Greer
Published: April 2021

The endogenous 14 kDa β-galactoside-specific lectin has been localised in myoblasts and myotubes by indirect immunofluorescence using confocal microscopy. The antibodies used in these experiments suggest that in myoblast cultures the lectin is abundant on the cell surface and concentrated at ruffled edges of migrating cells. The quantity and distribution of cell surface lectin is independent of the nature of the substratum and undiminished by brief trypsinisation or lactose-washing of the cell suspension. The lectin is also abundant intracellularly, apparently relatively free in the cytoplasm until the cells approach confluency, when the lectin is concentrated into particular areas of cytoplasm resulting in a ‘patchy’ appearance of stained cells. After fusion to form multinucleate myotubes, intracellular lectin is less abundant and concentrated at the periphery of myotubes, from where lectin appears to be released in vesicles packed with lectin which ‘bud off from the myotube surface. In recolonising cultures, lectin is apparently more abundant and lectin-packed vesicles can also be seen ‘budding off from myoblasts. In differentiated cultures, extracellular lectin is detected both on the myotube surface and in fibrillar arrays of extracellular material.
Genes for intermediate filament proteins and the draft sequence of the human genome
Authors: Hesse Michael , Magin Thomas M. , Weber Klaus
Published: April 2021

We screened the draft sequence of the human genome for genes that encode intermediate filament (IF) proteins in general, and keratins in particular. The draft covers nearly all previously established IF genes including the recent cDNA and gene additions, such as pancreatic keratin 23, synemin and the novel muscle protein syncoilin. In the draft, seven novel type II keratins were identified, presumably expressed in the hair follicle/epidermal appendages. In summary, 65 IF genes were detected, placing IF among the 100 largest gene families in humans. All functional keratin genes map to the two known keratin clusters on chromosomes 12 (type II plus keratin 18) and 17 (type I), whereas other IF genes are not clustered. Of the 208 keratin-related DNA sequences, only 49 reflect true keratin genes, whereas the majority describe inactive gene fragments and processed pseudogenes. Surprisingly, nearly 90% of these inactive genes relate specifically to the genes of keratins 8 and 18. Other keratin genes, as well as those that encode non-keratin IF proteins, lack either gene fragments/pseudogenes or have only a few derivatives. As parasitic derivatives of mature mRNAs, the processed pseudogenes of keratins 8 and 18 have invaded most chromosomes, often at several positions. We describe the limits of our analysis and discuss the striking unevenness of pseudogene derivation in the IF multigene family. Finally, we propose to extend the nomenclature of Moll and colleagues to any novel keratin.
mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope
Authors: Kapiloff Michael S. , Jackson Nicole , Airhart Nathan
Published: April 2021

The physical association of regulatory enzymes and ion channels at relevant intracellular sites contributes to the diversity and specificity of second messenger-mediated signal transduction in cells. mAKAP is a scaffolding protein that targets the cAMP-dependent protein kinase A and phosphodiesterase type 4D3 to the nuclear envelope of differentiated cardiac myocytes. Here we present data that the mAKAP signaling complex also includes nuclear envelope-resident ryanodine receptors and protein phosphatase 2A. The ryanodine receptor is the major cardiac ion channel responsible for calcium-induced calcium release from intracellular calcium ion stores. As demonstrated by a combination of immunohistochemistry and tissue fractionation, mAKAP is targeted specifically to the nuclear envelope, whereas the ryanodine receptor is present at both the sarcoplasmic reticulum and nuclear envelope intracellular membrane compartments. At the nuclear envelope, a subset of cardiac ryanodine receptor is bound to mAKAP and via the association with mAKAP may be regulated by protein kinase A-mediated phosphorylation. By binding protein kinase A and ryanodine receptor, mAKAP may serve as the scaffold for a cAMP- and calcium ion-sensitive signaling complex.
Sequential degradation of proteins from the nuclear envelope during apoptosis
Authors: Kihlmark Madeleine , Imreh Gabriela , Hallberg Einar
Published: April 2021

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.
Internalization of cholera toxin by different endocytic mechanisms
Authors: Torgersen Maria L. , Skretting Grethe , van Deurs Bo , Sandvig Kirsten
Published: April 2021

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-β-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.
Addition and correction: the NF-κB-like DNA binding activity observed inDictyosteliumnuclear extracts is due to the GBF transcription factor
Authors: Traincard François , Ponte Eleonora , Pun Jason , Coukell Barrie , Veron Michel
Published: April 2021

We have previously reported that a NF-κB transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide κB sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-κB-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-κB pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-κB signal transduction pathway in Dictyostelium.