bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of temporary ischaemia on rat muscle spindles
Authors: Diwan F. H. , Milburn Alice
Published: April 2021

Soleus muscle of adult rat is revascularized 5–8 days after sectioning the supplying blood vessels. The temporary ischaemia thus produced results in the rapid concomitant degeneration of extra- and intrafusal muscle fibres along with their nerve terminals and supplying axons.

The basal lamina of all muscle fibres usually remains intact throughout the degenerative phase. Necrotic sarcoplasm is removed by phagocytic cells. Satellite cells survive the temporary ischaemia and give rise to presumptive myoblasts which fill the basal-lamina tubes. These myoblasts fuse to form myotubes which, by the 14th day after devascularization, are maturing into muscle fibres in the absence of any innervation. Within the spindle, nuclear-bag fibres degenerate more rapidly than nuclear-chain fibres. Regeneration proceeds more rapidly within the basal-lamina tubes of the original bag fibres than within those of the chain fibres.

Reinnervation of regenerating extra- and intrafusal fibres begins 21 days after devascularization and is completed some 7 days later, during which time further equatorial differentiation of some reinnervated intrafusal fibres may occur.

Regenerated spindles vary considerably with respect to their innervation and equatorial nucleation. Most contain short, thin, additional muscle fibres as well as those that have regenerated within the basal-lamina tubes of the original fibres.
The pigmentary system of developing axolotls: III. An analysis of the albino phenotype
Authors: Frost S. K. , EPP L. G. , Robinson S. J.
Published: April 2021

The albino mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analyses of pteridine pigments were also carried out, and the pattern of pteridines in albino animals was found to be more complex than, and quantitatively enhanced (at all developmental stages examined) over, the pattern observed in comparable wild-type axolotls. The golden colour of albino axolotls is due primarily to sepiapterin (a yellow pteridine) and secondarily to riboflavin (and other flavins).

Coincident with enhanced levels of yellow pigments, xanthophore pigment organelles (pterinosomes) in albino skin reach a mature state earlier than they do in wild-type axolotl skin. This morphology is conserved throughout development in albino animals whereas it is gradually lost in the wild type. Unpigmented melanophores from albino axolotls are illustrated for the first time, and in larval albino axolotls the morphology of these cells is shown to be very similar to xanthophore morphology. In older albino animals xanthophores are easily distinguished from unpigmented melanophores. Iridophores seem to appear in albino skin at an earlier stage than they have been observed in wild-type skin. Morphologically, wild-type and albino iridophores are identical.
Albumin and transferrin synthesis in whole rat embryo cultures
Authors: Williams Carolyn L. , Priscott Paul K. , Oliver I. T. , Yeoh George C. T.
Published: April 2021

The uptake of [3H]leucine by the rat yolk sac and embryo and the subsequent synthesis of albumin and transferrin have been studied in whole embryo culture. Rat embryos of 12 days gestation were used in all experiments. Isotopically labelled transferrin was detectable in yolk-sac and embryo tissue extracts. In contrast, [3H]albumin could not be found in either tissue extract. Levels of radioactive transferrin in the yolk sac of cultured whole conceptuses decreased during 12 h in cold media. Embryonic transferrin showed an opposite trend in that it increased over 12 h by nearly 30-fold. In view of these results experiments were conducted in embryos and yolk sacs cultured in separate bottles. Radioimmunoprecipitation for transferrin revealed that there was synthesized protein in the yolk sac which then decreased by approximately 30 % after 2h in normal cultured medium. There was no evidence of transferrin synthesis in embryo extracts over a 12 h period. These results present evidence that the visceral yolk sac is the primary site of transferrin synthesis in the rat and that the protein is thereafter transported, intact, to the embryo.
Pathways of regenerated retinotectal axons in goldfish I. Optic nerve, tract and tectal fascicle layer
Authors: Stuermer Claudia A. O.
Published: April 2021

This study investigates the order of regenerating retinal axons in the goldfish. The spatiotemporal pattern of axon regrowth was assessed by applying horseradish peroxidase (HRP) to regenerating axons in the optic tract at various times after optic nerve section and by analysing the distribution of retrogradely labelled ganglion cells in retina. At all regeneration stages labelled ganglion cells were widely distributed over the retina. There was no hint that axons from central (older) ganglion cells might regrow earlier, and peripheral (younger) ganglion cells later, as occurs in normal development.

The absence of an age-related ordering in the regenerated optic nerve was demonstrated by labelling a few axon bundles intraorbitally with HRP (Easter, Rusoff & Kish, 1981) caudal to the previous cut. The retrogradely labelled cells in retina were randomly distributed in regenerates and not clustered in annuli as in normals. Tracing regenerating axons which were stained anterogradely from intraretinal HRP applications or retrogradely from single labelled tectal fascicles illustrated the fact that the regenerating axons coursed in abnormal routes in the optic nerve and tract.

On the surface of the tectum regenerated fibres re-established a fascicle fan. The retinal origin of tectal fascicles was assessed by labelling individual peripheral, intermediate and rostral fascicles with HRP. The retrogradely labelled ganglion cells in the retina were often more widely distributed than in normals, but were mostly found in peripheral, intermediate and central retina, respectively.

The order of fibre departure from each tectal fascicle was revealed by placing HRP either on the fascicle’s proximal or on its distal half. With proximal labelling sites labelled ganglion cells were found in the temporal and nasal retina, and with distal labelling sites labelled ganglion cells were confined to nasal retina only. Further, the axonal trajectories of anterogradely labelled dorsotemporal retinal ganglion cells were compared to those of dorsonasal retinal ganglion cells in tectal whole mounts. Dorsotemporal axons were confined to the rostral tectal half, whereas dorsonasal axons followed fascicular routes into the fascicles’ distal end and reached into caudal tectum. This suggests that the fibres exited along their fascicle’s course in a temporonasal sequence.

Thus in the tectum, fibres in fascicles restore a gross spatial and age-related order and tend to follow their normal temporonasal sequence of exit.
The preliminary characterization of mitogens secreted by embryonic chick wing bud tissues in vitro
Authors: Bell K. M.
Published: April 2021

Embryonic chick wing bud tissues secrete diffusible mitogens when cultured in vitro (Bell & McLachlan, 1985). These molecules may play an important role in limb development since media conditioned by morphogenetically active regions of the wing bud possess greater mitogenic activity than media conditioned by non-morphogenetic regions. These studies show that while the chick-derived growth factors were mitogenic for mouse-derived NIH 3T3,10T1/2 and NR6 cells and chick limb bud cells, they did not stimulate DNA synthesis in 3B11, PC13 END, normal rat kidney or bovine endothelial cells. Furthermore, the effects of the chick-derived mitogens were synergistically enhanced by insulin and PGF2α but remained unaffected by ECDGF, EGF, FGF and MSA. These findings indicate that embryonic chick limb bud cells synthesize and secrete growth factors which resemble in function other well-characterized growth factors and in particular PDGF.
Clonal analysis of chimaeric patterns in aortic endothelium
Authors: Schmidt Günter H. , Wilkinson Maureen M. , Ponder Bruce A. J.
Published: April 2021

The mosaicism in aortic endothelium of mouse aggregation chimaeras is demonstrated using the lectin Dolichos biflorus agglutinin as a strain-specific marker. The general fragmentary appearance and considerable size range of patches suggests that endothelial cells do not proliferate in a highly coherent manner. The developmental significance of the observed patterns is investigated by means of a quantitative statistical analysis -the Greig-Smith analysis of variance. This method examines the spatial distribution of patches and is able to detect and characterize pattern at various scales. The results show that (1) patches are non-randomly distributed at all scales examined and (2) ‘clusters of clusters’ occur at one small and one large scale, defining territories of ‘primary’ and ‘secondary’ descendent clones, which arose respectively during early and late periods in the development of the endothelium. We conclude from this analysis that (1) cell mixing is never complete, even in the early embryo and (2) cell mingling is not uniform during development. A different pattern was previously demonstrated for intestinal epithelium (Schmidt, Wilkinson & Ponder, 1985c) indicating the potential value of the method for quantitative comparison of mosaicism between tissues and also different developmental stages. Our results suggest that the analysis of patch sizes is likely to be less informative in terms of developmental mechanisms, than the analysis of the spatial arrangement of patches.
Comparison of microfilament patterns in nurse cells of different insects with polytrophic and telotrophic ovarioles
Authors: Gutzeit Herwig O. , Huebner Erwin
Published: April 2021

The localization of F-actin (microfilaments) in the nurse cells of ovarian follicles has been studied in 12 different insect species by fluorescence microscopy after specifically staining F-actin with rhodamine-conjugated phalloidin. In the analysed species with polytrophic ovaries (Apis mellifica, Pimpla turionellae, Bradysia tritici, Ephestia kuehniella, Protophormia terraenovae) a dense F-actin network was found to be associated with the nurse cell membranes. Only in Protophormia were microfilament bundles seen to extend from the cell membrane into the nurse cell cytoplasm and in a few cases appeared to make contact with the nuclear membrane. In the analysed coleopteran species with telotrophic ovarioles (Strangalia melanura, Leptinotarsa decemlineata, Oryzaephilus surinamensis) the fluorescence was also concentrated at the nurse cell membranes only. However, in all analysed hemipteran species (Lyguspratensis, Calocoris affinis, Graphosoma lineatum, Euscelis plebejus) the microfilament pattern was very different: while the nurse cells stained only weakly, we always found a characteristic (in some species massive) microfilament network surrounding the trophic core, a central area in the germarium from where material is transported through the trophic cords into the oocytes. The observed differences in the microfilament patterns are likely to reflect different mechanisms for transporting macromolecules and organelles within the ovariole.
Pattern discontinuity, polarity and directional intercalation in axolotl limbs
Authors: Muneoka Ken , Holler-Dinsmore Gregory V. , Bryant Susan V.
Published: April 2021

Axolotl limb stumps with dorsal-ventral confrontations between digits 2 and 3 but with a normal anterior-posterior pattern were created by grafting between contralateral limbs. Graft and host differed in ploidy to permit a determination of the origin of cells in the regenerated limb. After regeneration, limbs were analysed for skeletal and muscle patterns and for the distribution of marked cells in the regenerate. Regenerated limbs showed varying degrees of abnormality in their dorsal-ventral organization. Following regeneration, the original dorsalventral discontinuities were in some cases maintained and in others resolved. The maintenance or resolution of pattern discontinuities occurred in a position-dependent manner. Cell marker analysis indicates a relationship between the resolution of discontinuities and the extent to which cells become displaced across the original graft-host interface. These data lend support to the suggestion that circumferential intercalation is directionally biased.
Developmentally regulated expression of insulin-like growth factors by differentiated murine teratocarcinomas and extraembryonic mesoderm
Authors: Heath John K. , Shi Wai-Kang
Published: April 2021

The expression of plasma membrane receptors for insulin-like growth factors (IGFs) by PC13 embryonal carcinoma (EC) cells, and their immediate differentiated progeny PC13END was examined by binding radiolabelled IGF-I to cell monolayers. Both cell types express high-affinity IGF receptors, but the apparent number of unoccupied receptor sites falls by about 60 % upon differentiation. Crosslinking studies reveal that both type 1 and type 2 IGF receptors are expressed by PC13EC cells.

PC13END-cell-conditioned medium contains developmentally regulated, separable activities, one of which reacts directly with IGF-II, and the other with IGF for plasma membrane receptors. The former activity represents a soluble secreted IGF-binding protein. The latter activity is structurally and functionally similar to rat IGF-II.

Polyclonal antibodies raised against purified rat IGF-II specifically recognize multiple forms of IGF in radiolabelled culture supernatants and material which closely resembles the soluble IGF-binding protein. Immunoprecipitation of radiolabelled culture supernatants with anti-rat IGF-II reveals that the differentiation of PC13EC cells is accompanied by the coexpression of IGF-like molecules and the soluble binding protein, and that IGF-like molecules are expressed by extraembryonic tissues of mesodermal origin in the early postimplantation mouse embryo.

These findings show that IGF-like molecules are expressed in early mammalian development and may act in an autocrine fashion in vivo.
Estimation of the primordial pool size of the mouse liver using a histochemically demonstrable X-linked enzyme in the adult female mouse
Authors: Wareham Kathryn A. , Williams E. D.
Published: April 2021

We have used a histochemical technique to study cellular mosaicism in the livers of female mice heterozygous for an abnormal form of the X-linked enzyme ornithine carbamoyltransferase. The proportion of cells expressing the normal enzyme was found to be consistent within a liver lobe, but discordant between lobes, with as few as 4 % or as many as 91 % of cells being enzyme positive. Systematic observation of variation of the proportion of positive cells in different lobes of 12 mice was used to calculate the primordial pool size for each lobe and for the liver as a whole. Although confidence limits for this small study are wide, the data suggest that the liver pool is about 15 cells, and that it enlarges to at least 50 cells before subdividing into six lobe pools ranging in size from a minimum of 4 to a maximum of at least 14 cells.
The cellular origin of fibronectin in the basement membrane zone of developing tooth
Authors: Hurmerinta Kirsti , Kuusela Pentti , Thesleff Irma
Published: April 2021

The cellular source of fibronectin in the dental epitheliomesenchymal interface was studied in interspecies combinations of mouse and quail tissue. Species-specific fibronectin antibodies were produced by immunizing rabbits with purified mouse or chicken fibronectin and by absorbing both antisera with purified heterologous fibronectin and insoluble tissue extract. The absorbed antisera to mouse and chicken fibronectin showed fluorescent staining only in mouse and chicken tissue sections, respectively, but not vice versa. When the mouse mesenchymal dental papilla was combined and cultured either with the mouse enamel organ or with the quail pharyngeal epithelium, mesenchymal cell differentiation was initiated and typical alignment of mesenchymal cells along the basement membrane was seen. Examination with transmission electron microscope revealed a typical bilaminar basal lamina with adherent fibrillar matrix on its mesenchymal aspect. Immunofluorescent localization of fibronectin with the mouse-specific fibronectin antiserum showed a brilliant staining in the mesenchymal tissue and in the basement membrane zone. When the chicken-specific fibronectin antiserum was used, no staining was detected in either tissue recombinations. We have suggested earlier that fibronectin in the dental basement membrane plays an important role during the differentiation of mesenchymal cells into odontoblasts. The present study demonstrates that fibronectin in the basement membrane of the developing tooth is produced exclusively by the differentiating mesenchymal cells.
Selforganization of ependyma in regenerating teleost spinal cord: evidence from serial section reconstructions
Authors: Anderson Marilyn J. , Choy Chi Y. , Waxman Stephen G.
Published: April 2021

Multiple ependymal structures have been observed in regenerating spinal cord of the teleost Apteronotus albifrons. Evidence is presented for two modes of formation of the secondary ependymas: budding off from the primary ependyma, and de novo origin of a tube-like ependymal structure within a group of undifferentiated cells. Serial sections of regenerated cord provide evidence that undifferentiated cells not in immediate contact with the main ependymal layer can organize and differentiate into an ependymal structure in the regenerating spinal cord. These findings suggest that a significant amount of morphological organization can take place independent of the normal developmental sequence and environment.
Cell distribution during mouse secondary palate closure
Authors: Brinkley Linda L. , Bookstein Fred L.
Published: April 2021

The patterns of distribution of both total mesenchymal cells and the ratios of [3H] thymidine-labelled to total cells were mapped during secondary palatal shelf reorientation in vivo and in vitro. Smoothed spatial averaging, a computer-assisted method which takes into account the positions of all cells across an entire histological section of the shelf, was employed. Changes in shelf cross-sectional area and cell size were also measured. Three shelf regions, anterior and posterior presumptive hard and presumptive soft palate, were studied at developmental stages which were 30, 24 and 18 h prior to expected in vivo elevation, after in vivo reorientation and during the course of in vitro reorientation. Region-specific patterns of cell distribution change with shelf reorientation. These changes were observable within 6h. Increases in cell number by cell division may enhance some high local cell densities, but cannot account for decreases in cell density. Increase in cell size is not a factor in decreasing cell density, nor is cell death. Displacement of cells by expansion of the extracellular matrix may be involved.
The histogenetic potential of neural plate cells of early-somite-stage mouse embryos
Authors: Chan W. Y. , Tam P. P. L.
Published: April 2021

The mesencephalic neural plate of early-somite-stage mouse embryos differentiated underneath the renal capsule to form mostly neural tissues together with other tissues some of which were probably of neural crest cell origin. The capacity to form non-neural tissues such as skeletal tissues and melanocytes was lost at about the 5-somite stage. The lateral areas of the plate tended to form non-neural tissues more than the medial areas. The cephalic neural plate of presomite head-fold-stage embryos differentiated extensively to form both ectodermal and mesodermal tissues. However, upon completion of neurulation, the mesencephalic neuro-epithelium of forelimb-bud-stage embryos gave rise to neural tissues only. Therefore there is a progressive restriction in the histogenetic capacity of the mesencephalic neural plate during neurulation and this could be attributed to the cellular commitment for neural differentiation and the loss of the neural crest cells.
Disruption of segmentation in a short germ insect embryo
Authors: Mee Jane E. , French Vernon
Published: April 2021

A heat shock (of 15 min at 48°C) given to early embryos of the locust, Schistocerca gregaria, results in localized abnormalities in the segment pattern subsequently formed. Most defects involve two consecutive segments of the thorax or abdomen, and these are analysed in detail. The abdominal defects fall into three main classes each of which involves the absence of a particular region of the segment pair and, in one class, duplication of the region which remains. The thoracic defects similarly involve absence of parts of the segments and the formation of a single limb base from which one, two, or three limbs develop.

Heat shock may result in the absence of parts of segments in two distinct ways. It may interfere with the process of segmentation or it may delete parts of already formed segment primordia. These possibilities are discussed although, at present, neither can be excluded.

The duplication observed in some abdominal disruptions and the formation of triple limbs indicates that the absence of parts of embryonic segments is followed by pattern regulation similar to that occurring in regeneration studies on larval segments and appendages of other insects. Two out of the three classes of abnormality can be explained in terms of intercalary regeneration restoring pattern continuity, but it is possible that discontinuities persist in the remaining class.
Genetic activity at the albino locus in Cattanach’s insertion in the mouse
Authors: Deol M. S. , Truslove Gillian M. , McLaren Anne
Published: April 2021

Cattanach’s insertion (Is(In7;X)lCt or XCt) includes the normal allele at the albino locus (c+), which is subject to inactivation of the X chromosome carrying it, so that XCtX; cc mice have albino and pigmented patches. The X-autosome translocation T(X;16)16H or XT16H leads to preferential inactivation of the other X chromosome in female cells, so that XCtXT16H; cc mice are almost entirely white. However, they grow darker with age, as if reversal of inactivation of the c+ allele were taking place in increasing numbers of melanocytes. To test whether this is dependent only on age or whether it is related to the number of times the animal has moulted, hair was repeatedly plucked from selected areas at the early telogen stage when the follicles are also removed, assuming that the melanocytes or melanoblasts in that region of the skin would be forced to undergo further divisions to colonize the new follicles. The plucked areas grew darker at the same rate as the rest of the coat, suggesting that the progressive reversal of inactivation is dependent only on age.

As direct examination of melanocytes in the follicles is difficult, they were examined in the choroid and the retinal pigment epithelium (RPE) of the eye. The frequency of the pigmented cells was lower in the choroid than in the RPE. Since the melanocytes in these structures are different in origin as well as in physical characteristics, it appears that cell type influences either reversal of inactivation, or the frequency with which the influence of the X chromosome extends to the albino locus.
Ciliary band formation in the doliolaria larva of Florometra
Authors: Lacalli T. C. , West J. E.
Published: April 2021

The development of ciliary band pattern in the doliolaria larva of Florometra serratissima is described based on scanning and transmission electron microscopy. The uniformly ciliated epithelium of the post-hatching larva develops four regularly spaced bands over a period of approx. 20 h generating an epithelial pattern that is, essentially, a series of stripes. The first visible events of pattern formation progress over the larval surface in a posterior-to-anterior and dorsal-to-ventral sequence, but the initial pattern is not, in fact, striped. It instead consists of a close-packed array of oval interband domains separated and surrounded by belts of band cells. Secondarily the interband domains expand laterally and coalesce to form continuous, broad stripes, while the bands remain as narrow stripes between them. Two possible explanations for this unusual sequence of events are discussed: (1) that it can be understood in evolutionary terms with reference to band pattern in other echinoderm larvae, and (2) that it is a morphogenetic necessity because limitations inherent in the patterning mechanism prevent the direct formation of regular stripes.
The control of pigment cell pattern formation in the California newt, Taricha torosa
Authors: Tucker R. P. , Erickson C. A.
Published: April 2021

Neural crest-derived pigment cells form species-specific patterns of pigmentation in amphibian embryos. We have characterized the appearance and changes in pigment cell distribution in the embryos of the California newt, Taricha torosa. Black melanophores first appear scattered over the surface of the somites intermingled with yellow xanthophores in stage 34/35 embryos. The melanophores then migrate either dorsally to form a dorsal stripe at the apex of the somites or ventrally along the intersomitic furrows to form a midbody stripe at the somite-lateral plate mesoderm border. Xanthophores remain between the two melanophore stripes and are also found in the dorsal fin and head. The formation of the dorsal stripe coincides with a change in melanophore tissue affinity from the surface of the somites to the subectodermal extracellular matrix (ECM). The latter substratum is the location of the cue used to organize the dorsal stripe. In addition, melanophores become elongate and highly arborized, which would allow them to extend to the region where the dorsal stripe forms. In contrast, xanthophores do not form long processes in vitro. This suggests that the ability of melanophores but not xanthophores to search for a cue at the apex of the somites may account in part for the segregation of these cells types. Melanophores and xanthophores are trapped to form the midbody stripe by the pronephric duct, which is located just beneath the ectoderm at the bases of the intersomitic furrows. Ablation of the duct prevents formation of the midbody stripe, although melanophores and xanthophores still fail to migrate ventrally over the lateral plate mesoderm. Melanophores grafted to the ventral midline fail to leave the confines of the donor tissue. This suggests that a factor in the lateral plate mesoderm in addition to the pronephric duct is inhibiting further ventral migration. There is no gross morphological difference in the organization of the subectodermal ECM dorsal and ventral to the pronephric duct as revealed by alcian blue, ruthenium red and staining with antibodies to fibronectin. We also conclude that the directed dispersal of the neural crest into the space between the somites and ectoderm is due to contact inhibition of cell movement, since T. torosa neural crest cells demonstrate contact inhibition in vitro and there are enough cells in the lateral migratory spaces to make contact events likely during dispersal.
Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs
Authors: Etkin Laurence D. , Pearman Bradley
Published: April 2021

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6-to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60 % of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50 % of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.
Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the ascidianHalocynthia roretzi*
Authors: Mita-Miyazawa Izumi , Nishikata Takahito , Satoh Noriyuki
Published: April 2021

To obtain specific immunological probes for studying molecular mechanisms involved in the early embryonic development of ascidians, we have produced monoclonal antibodies directed against a homogenate of larvae of the ascidian Halocynthia roretzi. Among these, we have screened monoclonal antibodies that specifically recognize cells and/or tissues of the embryo. Characterization of six epidermis-specific monoclonal antibodies (including larval tunic-specific and larval fin-specific), three muscle-specific antibodies, two endoderm-specific antibodies, one notochord-specific antibody and two monoclonal antibodies that specifically recognize trunk-lateral cells suggests that these monoclonal antibodies may be useful as markers for analysing molecular mechanisms involved in specification of these cells. Seven monoclonal antibodies characteristically stain intercellular materials of the developing embryo and may therefore be valid for studying cellular construction of the embryo. Furthermore, monoclonal antibodies that recognize components of follicle cells, perivitelline space and sperm have also been established.
Muscle cell differentiation in ascidian embryos analysed with a tissue specific monoclonal antibody
Authors: Nishikata Takahito , Mita-Miyazawa Izumi , Deno Takuya , Satoh Nori Yuki
Published: April 2021

Utilizing a muscle-specific monoclonal antibody (Mu-2) as a probe, we analysed developmental mechanisms involved in muscle cell differentiation in ascidian embryos. The antigen recognized by Mu-2 was a single polypeptide with a relative molecular mass of about 220 ×103. It first appeared at the early tailbud stage and continued to be expressed until the swimming larva stage. There were distinct and separate puromycin and actinomycin D sensitivity periods during the occurrence of the antigen, suggesting the new synthesis of the polypeptide by developing muscle cells. Embryos that had been permanently arrested with aphidicolin in the early cleavage stages up to the 32-cell stage did not express the antigen. DNA replications may be required for the antigen expression.

Embryos that had been arrested with cytochalasin B in the 8-cell and later stages developed the antigen, and the number and position of the arrested blastomeres exhibiting the differentiation marker almost corresponded to those of the B4.1-line muscle lineage. Furthermore, in quarter embryos developed from each blastomere pair isolated from the 8-cell embryo, all the B4.1 as well as a part of b4.2 partial embryos expressed the antigen, while the a4.2 and A4.1 partial embryos did not show the antigen expression. These results may provide further support for the existence of cytoplasmic determinants for muscle cell differentiation in this mosaic egg.
Ethanol-induced inhibition of pinocytosis and proteolysis in rat yolk sacin vitro
Authors: Steventon G. B. , Williams K. E.
Published: April 2021

Pinocytic capture of 12SI-labelled polyvinylpyrrolidone and of formaldehyde-denatured l25I-labelled bovine serum albumin by 17 · 5-day rat visceral yolk sacs incubated in vitro was rapidly and strongly inhibited by low concentrations (0 · 01 and 0 · 05%, v/v) of ethanol. The induced inhibition of pinocytosis was readily reversible, but a marked lag was observed before ethanol-exposed tissue regained its full proteolytic capacity towards the exogenous protein.

These observations suggest that the acute administration of ethanol to a pregnant rat may give rise to concentrations of ethanol in the maternal blood and/or uterine fluid that induce dysfunction of the yolk sac. In late gestation such inhibition of yolk-sac function may interfere with the transfer of passive immunity across the yolk sac. If similar dysfunction is induced earlier in gestation, in the period before the chorioallantoic placenta is functional, this could cause a transient period of inhibition of histiotrophic nutrition that may be important to the pathogenic mechanism of action of ethanol as a teratogen.
Ciliary band formation in the doliolaria larva ofFlorometra: II. Development of anterior and posterior half-embryos and the role of the mesentoderm
Authors: Lacalli T. C. , West J. E.
Published: April 2021

We report the results of cutting experiments on embryos of the crinoid Florometra serradísima, which produce, in the doliolaria stage, a striped pattern of ciliary bands. Embryos at gastrula and post-hatching elongation stages were divided into anterior and posterior fragments. Complementary fragments express parts of the total pattern without adding extra pattern elements, i.e. the pattern is a mosaic. Some fragments elongate which, from an examination of internal structures, we interpret as due to the elongation and displacement of the mesenteric sac. The number of pattern elements expressed correlates with degree of elongation and internal landmarks correlate with certain external pattern features. This suggests that the pattern mosaic may reside in the internal tissues, i.e. in the mesentoderm, but we are as yet unable to prove this. The results are discussed with reference to the roles of tissues of different germ layer origin in related embryos, including vertebrates, in which the mesentoderm has a significant instructive role.
Behavioral responses of streamer F mutants of Dictyostelium discoideum: Effects of cyclic GMP on cell motility
Authors: Segall Jeffrey E.
Published: April 2021

Streamer F (stmF) mutants have a prolonged increase in intracellular cGMP in response to addition of the chemoattractant cAMP. The speed of movement and area of stmF cells were quantitated as the cells were stimulated with a rapid, uniform increase in extracellular cAMP. The speed of stmF cells rapidly drops as does that of the wild-type, but then requires about 300 seconds to recover. In contrast, the speed of the parental strain, XP55, recovers within 60-70 seconds. This prolonged drop in speed correlates with the time during which intracellular cGMP remains high, suggesting that intracellular cGMP induces this prolonged reduction in speed. Mutants from other streamer complementation groups do not show this altered response. Area measurements indicate that stmF cells do not cringe or round up as XP55 does, but spread with the same kinetics as XP55. Chemotactic orientation of the stmF cells in stable spatial gradients is similar to or slightly greater than that of the wild-type. Tracking of cells moving during aggregation indicates that the stmF cells show large drops in speed between pulses, resulting in the banding pattern seen in streams. The cells can still respond to new pulses, resulting in an aggregation time that is similar to that of XP55.
Tumour infiltrating macrophages are capable of bone resorption
Authors: Quinn J. M. W. , Athanasou N. A.
Published: April 2021

The cell types responsible for osteolysis associated with skeletal metastasis of solid malignancies are unclear. Tumour infiltrating macrophages (TIMs) isolated from primary mammary carcinomas of C3H/Avy mice were cultured on bone slices to assess their ability to resorb bone. After 14 days in co-culture with murine marrow stromal cell line ST2 and added 1,25-dihydroxyvitamin D3 and dexamethasone, TIMs showed increased tartrate-resistant acid phosphatase activity and formed numerous lacunar resorption pits. In the absence of ST2 cells, TIMs did not form lacunar resorption pits but produced roughening of the bone surface with exposure of mineralized collagen fibres. Normal alveolar macrophages, in both the presence and absence of ST2 cells similarly produced only surface resorption. TIMs are thus capable of both low-grade (surface) and high-grade (lacunar) pathological bone resorption, a specific interaction with stromal cells being necessary for the latter to occur. TIM-mediated bone resorption could account for different clinical and pathological patterns of tumour osteolysis.
Stretch-activated Ca2+ and Ca2+-activated K+ channels in the hyphal tip plasma membrane of the oomycete Saprolegnia ferax
Authors: Garrill Ashley , Lew Roger R. , Heath I. Brent
Published: April 2021

We have used the patch clamp technique to study ion channels involved in hyphal tip growth in the oomycete Saprolegnia ferax. The development of a technique in which protoplasts are produced in distinct linear arrays has permitted the study of transport proteins in protoplasts derived from different regions of the hypha. Using the cell-attached mode we find two K+ channels of different amplitudes, both of which are activated by Ca2+, as shown by the addition of the Ca2+ ionophore A23187. In nearly all the recordings (95%) there was a characteristic but variable oscillatory nature to the activity of these channels. K+ fluxes through these channels are inward and are of a sufficient magnitude to have a significant impact on cell turgor. Hyphal tip, but rarely distal protoplasts, also contain two stretch-activated channels, one displaying larger amplitudes and permeable to both Ca2+ and K+ and a smaller channel permeable to Mg2+. Stretch-activated ion channels permeable to calcium are of particular interest in relation to tip growth and their concentration at the hyphal tip supports a direct role in the tip growth process. This represents a significant advance toward understanding the mechanisms of regulation of this mode of cellular growth.
Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors
Authors: Längst Gernot , Becker Peter B.
Published: April 2021

ATP-dependent chromatin-remodeling machines of the SWI/SNF family are involved in many cellular processes in eukaryotic nuclei, such as transcription, replication, repair and recombination. Remodeling factors driven by the ATPase ISWI make up a subgroup of this family that exhibits defined mechanistic and functional characteristics. ISWI-induced nucleosome mobility endows nucleosomal arrays with dynamic properties and recent results suggest that ISWI-type remodelers have diverse functions that range from transcriptional regulation to chromatin assembly and maintenance of chromosome structure.
Podosomes in osteoclast-like cells
Authors: Pfaff Martin , Jurdic Pierre
Published: April 2021

Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin αVβ3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin αVβ3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region of the integrin β3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the β3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-dependent tyrosine-phosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with β3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to β3 integrin-dependent cell contacts.
Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells
Authors: Gao Wenhua , Pu Yongmei , Luo Kathy Q. , Chang Donald C.
Published: April 2021

During apoptosis, cytochrome c is released from mitochondria to the cytosol to activate a caspase cascade, which commits the cell to the death process. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. To test this theory, we measured directly the dynamic re-distribution of green fluorescence protein (GFP)-tagged cytochrome c and morphological change of mitochondria within living HeLa cells during u.v.-induced apoptosis. We observed that mitochondria did not swell when cytochrome c was released from mitochondria to cytosol during apoptosis. Instead, mitochondria swelled to spherical shapes within 10 minutes of cytochrome c release. This finding strongly suggests that cytochrome c release in apoptosis was not caused by mitochondrial swelling. This conclusion was further supported in two separated experiments using an immunostaining method and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) treatment. In addition, we found evidence that cytochrome c was also released before mitochondrial swelling in apoptosis induced by other cell death-inducing treatments, including tumor necrosis factor (TNF) and actinomycin D.
Steps of nuclear pore complex disassembly and reassembly during mitosis in earlyDrosophilaembryos
Authors: Kiseleva Elena , Rutherford Sandra , Cotter Laura M. , Allen Terence D. , Goldberg Martin W.
Published: April 2021

The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional ‘spindle envelope’. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.
Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses
Authors: Mizutani Yukiko , Tamiya-Koizumi Keiko , Nakamura Noriko , Kobayashi Miya , Hirabayashi Yoshio , Yoshida Shonen
Published: April 2021

To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E. coli. It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm. The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974. The present study was therefore focused on the nuclear localization of this enzyme. Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes. Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not. The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane. On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1. Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e. nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix. Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm. Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974.
Phospholipase C activation by anesthetics decreases membrane-cytoskeleton adhesion
Authors: Raucher Drazen , Sheetz Michael P.
Published: April 2021

Many different amphiphilic compounds cause an increase in the fluid-phase endocytosis rates of cells in parallel with a decrease in membrane-cytoskeleton adhesion. These compounds, however, do not share a common chemical structure, which leaves the mechanism and even site of action unknown. One possible mechanism of action is through an alteration of inositol lipid metabolism by modifying the cytoplasmic surface of the plasma membrane bilayer. By comparing permeable amphiphilic amines used as local anesthetics with their impermeable analogs, we find that access to the cytoplasmic surface is necessary to increase endocytosis rate and decrease membrane-cytoskeleton adhesion. In parallel, we find that the level of phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane is decreased and cytoplasmic Ca2+ is increased only by permeable amines. The time course of both the decrease in plasma membrane PIP2 and the rise in Ca2+ parallels the decrease in cytoskeleton-membrane adhesion. Inositol labeling shows that phosphatidylinositol-4-phosphate levels are increased by the permeable anesthetics, indicating that lipid turnover is increased. Consistent with previous observations, phospholipase C (PLC) inhibitors block anesthetic effects on the PIP2 and cytoplasmic Ca2+ levels, as well as the drop in adhesion. Therefore, we suggest that PLC activity is increased by amine anesthetics at the cytoplasmic surface of the plasma membrane, which results in a decrease in membrane-cytoskeleton adhesion.