bluuebunny/crossref_metadata_2025 huggingface dataset:
Further studies on the melanophores of periodic albino mutant of Xenopus laevis
Authors: Fukuzawa T. , Ide H.
Published: April 2021

It is still unknown why dermal melanophores disappear during larval development, and why no or very few epidermal melanophores appear during and after metamorphosis, in Xenopus laevis showing periodic albinism (ap). To elucidate these points, we investigated (1) the occurrence of depigmentation in mutant (ap/ap) melanophores during in vitro proliferation and (2) the incidence of melanophore differentiation from mutant melanoblasts in the skin in vitro.

During in vitro proliferation of mutant melanophores, ap-type melanosomes decreased in number gradually and instead the number of premelanosomes increased in the cells, which caused depigmentation at the light microscopic level in the culture. Depigmentation was observed only in mutant melanophores, and not in wild-type (+/+) melanophores. These results suggest that autonomous depigmentation of mutant dermal melanophores is the cause of the disappearance of these cells in vivo.

Dopa-positive melanoblasts were demonstrated in both wild-type and mutant skins. However, the melanoblasts of metamorphosed mutant froglets did not differentiate in vitro, while those of wild-type froglets did. These results suggest that mutant melanoblasts in the skin of froglets lose the potency to differentiate into melanophores, and that this causes the lack of mutant melanophores in the froglets. The site of action of the ap gene is also discussed.
The role of movement and tissue interactions in the development and growth of bone and secondary cartilage in the clavicle of the embryonic chick
Authors: Hall Brian K.
Published: April 2021

There has been debate in the literature concerning whether the clavicle arises by intramembranous ossification, i.e. is a membrane bone, and whether secondary cartilage develops from its periosteal cells. A histological study of carefully staged embryos revealed that preclavicular mesenchyme undergoes condensation at H. H. stage 31–32, bone forms by H. H. stage 33 and that a transitory secondary cartilage appears late in H. H. stage 35, only to disappear by H. H. stage 36. Except for the transitory nature of the secondary cartilage, this histogenetic sequence is as seen in craniofacial membrane bones. Enzymic removal of the epithelium overlying clavicular mesenchyme from embryos of H. H. stages 26–34 and chorioallantoic grafting of the isolated mesenchyme, revealed an epithelial requirement for initiation of intramembranous ossification during H.H. stages 26–29, again similar to initiation of craniofacial osteogenesis. Secondary chondrogenesis was initiated neither in embryos paralysed with decamethonium iodide nor when clavicular mesenchyme (H.H. stages 29–33·5) was grafted to the chorioallantoic membranes of paralysed embryos, but did form in a small percentage (16–23 %) of clavicles grafted to the membranes of mobile embryos. Failure of chondrogenesis in the former was attributed to a requirement for movement as a proximate chondrogenic stimulus and the low incidence of chondrogenesis in the latter to the stimulus provided by amniotic movements which persist in paralysed embryos. Secondary cartilage did form when clavicles were organ cultured, either submerged, or at the air-medium interface. This stands in contrast to craniofacial membrane bone such as the quadratojugal, which only forms secondary cartilage in vitro when cultured submerged. Growth of the clavicle was shown to increase 53-fold between 10 and 11 days of incubation, an increase which was diminished but not eliminated in paralysed embryos, and which correlated closely with the dramatic increase in embryonic movement which occurs between 10 and 11 days of incubation. Thus, the clavicle of the embryonic chick shares all of the features and epigenetic requirements of the craniofacial membrane bones, but is more dependent upon biomechanical factors for its growth.
The effect of lethal mutations and deletions within the bithorax complex upon the identity of caudal metameres in the Drosophila embryo
Authors: Whittle J. R. S. , Tiong S. Y. K. , Sunkel C. E.
Published: April 2021

Mutations and deletions of the abdA and AbdB functions in the bithorax complex of Drosophila melanogaster have been examined for their effect upon the hypodermal derivatives of the caudal segments of the embryo, employing light- and scanning electron microscopy. No cuticular structures located posterior to the denticle belt of abdominal segment 8 are affected in abdA- embryos. Embryos of AbdB- genotype no longer have six of the seven pairs of sense organs present in this region, lack posterior spiracles but instead have sclerotized cuticle and sense organs typical of the head region and a rudimentary extra ventral denticle belt. The anal pads, tuft and sense organ 1 do not require BX-C functions for their specification. We discuss the provenance of these cuticular structures and the domain of function of elements within the bi thorax complex in terms of parasegmental metameric units.
Biochemical characterization of the adhesion-related differentiation antigen XT-1
Authors: Bechtol Kathleen B. , Ho Wai Chang , Vaupel Steven
Published: April 2021

The XT-l-molecule, an adhesion-related differentiation antigen of male mouse germ cells, is a 34000 Mr glycoprotein with major charge isomer at pl 5·1 and is an integral component of the cell membrane. On large late pachytene spermatocyte, the molecule is present at a concentration of 2·5×103 moleculesμm-2, which approximates HLA/ABC concentration on lymphocytes. By comparing the reactivity of four anti-XT-1 monoclonal antibodies, three of which elicit germ cell-germ cell adhesion, we have defined two distinct surface regions of the XT-l-molecule. The relationship of the XT-l-molecule with other known adhesion-related molecules and testicular antigens is discussed.
The effect of reducing ATP levels on reorientation of the secondary palate
Authors: Bulleit Robert F. , Zimmerman Ernest F.
Published: April 2021

The force for directing palate shelf reorientation appears to be associated with elements of the presumptive hard palate (Brinkley & Vickerman, 1979; Bulleit & Zimmerman, 1985). The palatal elements that mediate this process do not require palate cells to be metabolically active for expression of the force. This contention was demonstrated using an in vitro system that allows substantial reorientation of the hard palate to occur. ATP levels were reduced by treatment with metabolic inhibitors and the degree of reorientation was measured 1 h following pretreatment with inhibitors. Treatment of cultured embryonic heads under anoxic conditions with 2,4-dinitrophenol or KCN had no effect on the degree of reorientation occurring in vitro. These agents reduced ATP levels by 71 % and 62 %, respectively. Treatment of cultured heads with 2-deoxy-D-glucose under anoxia also had no effect on reorientation. This treatment reduced ATP levels in embryonic heads by 92–94 %. A similar reduction was observed if ATP levels were measured in palate tissue alone. The treatment of cultured heads with 2-deoxy-D-glucose and anoxia not only reduced levels of ATP but also reduced CTP, GTP and UTP. These results indicate that palate shelf reorientation is independent of cellular metabolic activity and supports the hypothesis that reorientation is dependent on a pre-existing infrastructure within the palate shelves.
Modifications of size and pattern of microtubular organelles in overfed cells of a ciliate Dileptus
Authors: Golinska Krystyna
Published: April 2021

The size of mouthparts and their constituent organelles was studied in cells of Dileptus anser, enlarged by overfeeding. The oral structures of large cells were either of normal dimensions and appearance, or they were enlarged and deformed in shape. The increase in size of mouthparts was accompanied by an increase in number of their microtubular organelles. There was, however, no net increase in size of the organelles, as defined by length and number of microtubules they contained.

It is concluded that there exists an upward regulation in the size of the mouthparts as a whole, whilst the size of their constituent organelles is probably not so regulated. It is supposed that the patterning of normal-size organelles into large oral structure may lead to the observed deformations in the shape of the mouthparts.
Neurulation and the cortical tractor model for epithelial folding
Authors: Jacobson Antone G. , Oster George F. , Odell Garrett M. , Cheng Louis Y.
Published: April 2021

We present here a new model for epithelial morphogenesis, which we call the ‘cortical tractor model’. This model assumes that the motile activities of epithelial cells are similar to those of mesenchymal cells, with the added constraint that the cells in an epithelial sheet remain attached at their apical circumference. In particular, we assert that there is a time-averaged motion of cortical cytoplasm which flows from the basal and lateral surfaces to the apical region. This cortical flow carries with it membrane and adhesive structures that are inserted basally and resorbed apically. Thus the apical seal that characterizes epithelial sheets is a dynamic structure: it is continuously created by the cortical flow which piles up components near where they are recycled in the apical region. By use of mechanical analyses and computer simulations we demonstrate that the cortical tractor motion can reproduce a variety of epithelial motions, including columnarization (placode formation), invagination and rolling. It also provides a mechanism for driving active cell rearrangements within an epithelial sheet, while maintaining the integrity of the apical seal. Active repacking of epithelial cells appears to drive a number of morphogenetic processes. Neurulation in amphibians provides an example of a process in which all four of the above morphogenetic movements appear to play a role. Here we reexamine the process of neurulation in amphibians in light of the cortical tractor model, and find that it provides an integrated view of this important morphogenetic process.
Effects of electric field on fusion rate and survival of 2-cell rabbit embryos
Authors: Ozil Jean-Pierre , Modlinski Jacek A.
Published: April 2021

Electric-field-induced blastomere fusion was studied in 2-cell rabbit embryos. Field strengths (1 to 3kVcm−1) and durations (35 to 1000 μs) were chosen so as to provide the right balance between fusion rate, viability and developmental capacity of embryonic cells. Maximum plasma membrane tolerance of 2-cell rabbit embryos was observed at about 3 kV cm−1 for 1000 ps. All surviving ‘fused’ embryos were able to develop in vitro and most of them formed expanded blastocysts. Observation of ‘fused’ embryos immediately after fusion and during the whole cell cycle showed that 27·7 % of the two diploid nuclei remained separated in the hybrid cell. More than one metaphase plate was formed at the onset of mitosis causing direct cleavage into three or four ‘cells’. In the remaining embryos the two diploid nuclei seemed to form a common metaphase plate and cleaved into two equal blastomeres. After transfer to recipient does, 54·4% of these tetraploid embryos developed beyond implantation. Between day 11 and 20, ten live and morphologically fully normal embryos were recovered. Nine embryos were uniformly tetraploid and one recovered on day 18 was a diploid/tetr aploid mosaic. The remaining implantation sites contained either abnormal, very retarded embryos or indefinable embryo remnants. After transfer of ‘nonfused’ embryos treated with 3kVcm−1, 49% gave birth to normal live young. These results suggest that the electric field can be applied successfully in a relatively wide strength and duration range without causing any visible teratogenic effect on treated embryos. Thus, tetraploid embryos can develop normally at least until two-thirds of pregnancy, but the question whether they are able to survive till term remains open.
The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study
Authors: Francavilla Sandro , Moscardelli Sergio , Bruno Bernardino , Barcellona Patrizio Scorza , Martino Cesare De
Published: April 2021

The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct.

At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall.

The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis.

The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.
Scanning electron microscopic observation of mouse embryonic submandibular glands during initial branching: preferential localization of fibrillar structures at the mesenchymal ridges participating in cleft formation
Authors: Nakanishi Yasuo , Sugiura Fukashi , Kishi Jun-Ichi , Hayakawa Taro
Published: April 2021

Branching submandibular glands of 12-day mouse embryos and those cultured in the presence and absence of a collagenase inhibitor from the culture medium of bovine dental pulp or a Clostridial collagenase were examined with the scanning electron microscope. Fracturing of fixed and dried glands with the tip of a fine needle succeeded in exposing the surfaces of the lobules and of their mesenchymal replicas at different stages of branching. At the beginning of branching, corresponding parts of the mesenchyme formed ridges on or in which the fibrillar structures were often found. At the stage forming deeper clefts thicker fibres, 0·5–2·5 μm in diameter, were observed between two adjacent lobules. On the contrary, no apparent differences in the fibrillar structures on the epithelial surfaces were detected between the shallow cleft and noncleft regions at the initial phase of branching. These fibrillar structures were very abundant in glands cultured with collagenase inhibitor and were completely lost in glands cultured with bacterial collagenase, strongly indicating that these materials consisted of collagen. The possible involvement of mesenchyme in epithelial branching is discussed with special reference to mesenchymal traction forces that would be elicited by fibrillar collagens.
Organ distribution of apolipoprotein gene transcripts in 6–12 week postfertilization human embryos
Authors: Hopkins B. , Sharpe C. R. , Baralle F. E. , Graham C. F.
Published: April 2021

In the liver and the yolk sack of 6–12 week postfertilization human embryos, we have detected RNA transcripts from the following apolipoprotein genes: AI, AII, B, CII, CIII and E. The mRNA from the apolipoprotein CIII gene was relatively more abundant in the total RNA from the yolk sack than in that from the liver. The gut and adrenals contained transcripts of all these apolipoprotein genes apart from apolipoprotein AIL The kidneys and heart contained some apolipoprotein transcripts. In conjunction with previous studies, these results suggest that in the human embryo apolipoprotein genes are transcribed in a much larger range of organs than is the case in the adult. Many of these organs lack endoderm tissues.
The appearance and distribution of intermediate filament proteins during differentiation of the central nervous system, skin and notochord of Xenopus laevis
Authors: Godsave S. F. , Anderton B. H. , Wylie C. C.
Published: April 2021

Antibodies against various intermediate filament proteins have been used to follow cell differentiation in the early Xenopus embryo. Three new monoclonal antibodies against Xenopus cytokeratins raised against Triton-insoluble material from tadpoles (RD35/2a, RD35/3a and D3/3a), two antibodies against mammalian cytokeratins (LE65 and LP3K), monoclonal anti-(rat 200K neurofilament protein), rabbit anti-(rat glial filament acidic protein), and rabbit antibodies to hamster and calf vimentin were used. We show that cytokeratins are present in the early central nervous system (CNS) and persist in the ependymal cells of the adult CNS. We also show that the notochord contains cytokeratin. The ontogeny of intermediate filament protein appearance in the CNS, skin and notochord between neural fold stage and swimming tadpole stage are described. These results are discussed in particular with regard to the use of the antibodies as differentiation markers.
Evaluation of the roles of intrinsic and extrinsic factors in occlusion of the spinal neurocoel during rapid brain enlargement in the chick embryo
Authors: Desmond Mary E. , Schoenwolf Gary C.
Published: April 2021

The spinal neurocoel normally occludes during the second day of chick embryogenesis as the lateral walls of the spinal cord become apposed closely in the midline. Concomitantly, the brain initiates its rapid and substantial enlargement. Occlusion, although short-lived, might play a major role in brain enlargement. As a result of occlusion, the brain ventricles are sealed off from the external milieu prior to closure of the posterior neuropore, establishing a closed fluid-filled system. The present study focuses on the mechanisms of occlusion of the spinal neurocoel. We tested two postulated intrinsic factors (microtubule-mediated neuroepithelial cell elongation and microfilament-mediated apical neuroepithelial cell constriction) and five extrinsic factors (three mediad pushing forces generated by the somites, perineural extracellular matrix and expanding surface ectoderm; and two stretching forces generated either vertically by pulling of the elongated notochord or longitudinally by elongation of the embryo) in maintaining occlusion. Our results suggest that occlusion is maintained by other, untested intrinsic factors and/or by forces generated within a perineural collar, composed of cellular and extracellular materials, intimately associated with the basal aspects of the spinal cord. Cytoskeletal-mediated changes in cell shapes, pushing forces and vertical and longitudinal tensions are not involved. Further studies are needed to examine the intrinsic properties of the neuroepithelium and the factors initiating occlusion and reopening.
The role of ultimobranchial bodies in the modulation of the response of chick embryos to 1,25-dihydroxy-cholecalciferol
Authors: Narbaitz Roberto , Rad Jaffar Soleimani
Published: April 2021

Ultimobranchial bodies (UBBs) were dissected from 17-day-old chick embryos and grafted onto the chorioallantoic membrane of 8-day-old embryos. The embryos with UBB grafts as well as sham-grafted controls were injected on the 10th day of incubation with 100 ng 1,25(OH)2D3 dissolved in ethyl alcohol or with an equal volume of ethyl alcohol alone; embryos were sacrificed on the 13th day. Grafted UBBs showed ultrastructural characteristics typical of actively secreting glands. A histological study of the tibiae from all embryos showed that while the grafted embryos responded to the injection of 1,25(OH)2D3 with a peripheral rim of undermineralized bone trabeculae, sham-grafted embryos never did so. These results confirm the original hypothesis that the presence of differentiated UBBs is a precondition for the production of undermineralized bone (osteoid) by 1,25(OH)2D3.

In a second series of experiments, similarly treated embryos were sacrificed on the 10th, 11th, 12th and 13th day; the levels of calcium and inorganic phosphate were determined in their blood. The injection of 1,25(OH)2D3 produced in all embryos hypercalcaemia and hypophos-phataemia. However, the hypophosphataemic response was more prolonged in the embryos with UBB grafts than in sham-grafted ones. These results suggest that the grafted UBBs prolonged the hypophosphataemic response, probably by secreting calcitonin and thus reducing the rate of bone resorption. It is also probable that the prolonged hypophosphataemia produced or contributed to the undermineralization of the peripheral (subperiosteal) trabeculae.
Is maternal mRNA a determinant of tissue-specific proteins in ascidian embryos?
Authors: Jeffery W. R. , Bates W. R. , Beach R. L. , Tomlinson C. R.
Published: April 2021

The generation of different cell types during embryonic development is thought to be mediated by the combined activity of cytoplasmic factors (determinants), which are localized in the egg, and inductive interactions, which occur between different embryonic cells and tissues. Ascidians, animals that exhibit rapid and exceptionally autonomous development (reviewed by Jeffery, 1985), appear to employ cytoplasmic determinants to generate embryonic cell diversity. Although determinants have not been identified in ascidians or other animals, it is hypothesized that they function in at least two different ways. First, as initially pointed out by Morgan (1934), determinants may be regulatory factors which promote differential gene expression in specific cell lineages. Consistent with this possibility, inhibitors of transcription, added prior to gastrulation, block the appearance of some ascidian tissue-specific enzymes and morphological markers whose expression is regulated by the activity of cytoplasmic determinants (Whittaker, 1973; Crowther & Whittaker, 1984). Second, determinants may be localized factors which promote cell diversification independent of zygotic gene expression. This class of determinants is implicated in specifying crucial embryonic events that occur before the onset of zygotic transcription. Evidence for the second class of determinants in ascidians was first obtained from interspecific hybridization studies. These studies showed that haploid andromerogones, which developed from anucleate eggs of one ascidian species fertilized with sperm of another species, exhibited larval adhesive papillae that were morphologically identical to those of the maternal species (Minganti, 1959).
Manipulating the anteroposterior pattern of the Drosophila embryo
Authors: Frohnhófer Hans Georg , Lehmann Ruth , Nüsslein-Volhard Christiane
Published: April 2021

The establishment of pattern and polarity in the insect egg has been studied intensively using a variety of experimental approaches. Drosophila, while the system of choice for genetic analysis of pattern formation has been rather neglected as an experimental organism and species with longer developmental time and larger eggs were preferred in classical studies. Among the dipteran insects, midges such as Chironomous and Smittia with their transparent chorion and synchronous development were found more rewarding. The classical methods of ligation, puncture, transplantation, destruction or removal of material, and centrifugation were applied to eggs of a variety of insect species. Although the degree of response to experimental manipulation was found to be widely different, there were similarities in the type of abnormal patterns produced by the various treatments which suggested more general conclusions:-the anteroposterior pattern is probably controlled by two centres of activity, localized at the anterior and posterior egg pole respectively, with a long-range effect on the entire egg axis (reviewed by Sander, 1976).
Effects of zinc deficiency on morphogenesis of the fetal rat eye
Authors: Rogers John M. , Hurley Lucille S.
Published: April 2021

Maternal zinc deficiency during pregnancy results in a high frequency of fetal eye malformations in the Long-Evans rat. In this study we examine the development of the eye from days 12 through 21 of gestation in conceptuses of dams fed deficient or adequate levels of zinc and also examine maternal plasma and conceptus zinc concentrations during this period. Dams were fed diets containing 0·5 (0·5 Zn group), 4·5 (4·5 Zn group), or 100 (100 Zn AL group) μg zinc per gram diet ad libitum, or 100 μg zinc g−1 diet in amounts restricted on a daily basis to the intake of matched animals from the 0-5 Zn group (100 Zn RI group). Conceptuses were removed and maternal plasma was collected on days 12, 14, 16, 19 and 21 of gestation. Maternal plasma and conceptus zinc concentrations reflected maternal dietary zinc level, with dam plasma Zn concentrations in the order of 0·5 Zn group < 4·5 Zn group < 100 Zn group on all days. A similar pattern held for embryo/fetus zinc, except for days 19 and 21, at which times the 0·5 Zn and 4·5 Zn fetuses had similar zinc concentrations. Histological examination of the developing eye of 0·5 Zn fetuses on days 12 and 14 revealed that invagination of the optic cup was often deficient, and that closure of the choroid fissure did not occur, resulting in colobottiata and retinal folding visible at term. A very few fetuses were found at term to be anophthalmic or have only remnants of ocular tissue. In the absence of coloboma, retinal histogenesis evaluated on day 19 appeared normal in zinc-deficient fetuses, and in fact, the eye was relatively spared the effects of zinc deficiency in these fetuses. No eye malformations were seen in 4·5 Zn fetuses. Thus, low maternal plasma and embryonic zinc concentrations during organogenesis adversely affect morphogenesis of the optic vesicle and closure of the choroid fissure, leading to anophthalmia or colobomatous microphthalmia with retinal folding at term. A possible mechanism for the latter malformation involving lack of vitreous pressure necessary for normal expansion of the developing eye is discussed. When the choroid fissure does close normally, later prenatal histogenesis of the retina does not seem to be adversely affected by zinc deficiency.
The upright position of brush border-type microvilli depends on myosin filaments
Authors: Temm-Grove Constance , Helbing Dirk , Wiegand Christiane , Höner Bernd , Jockusch Brigitte M.
Published: April 2021

We have studied the correlation between the actomyosin organization and microvillar position in an epithelial cell line derived from the proximal pig kidney tubule (LLC-PK1). When grown on glass, these cells are approximately 5-6 μm in height and develop numerous microvilli that project from the dorsal membrane. A fairly homogeneous distribution of microvilli was achieved by synchronization of the cell cycle. These microvilli are of the brush border type, as defined by their content of villin and their anchorage in a myosin-rich terminal web-like structure. When LLC-PK1 cells were injected with two monoclonal antibodies against pig brain nonmuscle myosin, in concentrations yielding a 1:1 ratio of antibody to myosin, neither microvillar number nor length was affected. However, when we examined the cells by scanning electron microscopy 1-3 h after microinjection, we found that one of the antibodies (a-PBM 4) had a profound effect on microvillar position: more than 50% were seen tilted or lying prone on the plasma membrane. The microvilli of cells injected with the other antibody (a-PBM 9) were not significantly different from those of cells injected with control antibodies. This difference correlates with in vitro properties of the antibodies: a-PBM 4 decreases the actin-activated Mg 2+ -ATPase of pig brain nonmuscle myosin quite substantially, while a-PBM 9 affects it only moderately. These differential effects are probably a consequence of the different epitope location as determined for both antibodies, not of differences in antibody affinity. Our data are compatible with the hypothesis that a-PBM 4 also interferes with the actomyosin interaction in situ, thus decreasing the effective crosslinking of microvillar rootlets by myosin filaments in the terminal web. On the basis of this model, we suggest that myosin filaments are essential for the upright position of brush-border type microvilli.
The interendothelial junction in myocardial capillaries: Evidence for the existence of regularly spaced, cleft-spanning structures
Authors: Schulze Charlotte , Firth J. Anthony
Published: April 2021

Water and hydrophilic solutes cross the endothelium of continuous capillaries via the paracellular cleft and possibly other routes. This pathway shows a selectivity to molecule size and charge. However, it is not yet known which systems confer this selectivity.

Isolated rat hearts were perfusion-fixed through the coronary circulation, stained with lanthanum or tannic acid, and further processed for transmission electron microscopy. Thin sections viewed at X 160,000 magnification revealed regularly spaced, cleft-spanning structures in the wider zone of a small percentage of clefts in addition to at least one zonula occludens. Goniometric tilting of the specimen in steps of 5° perpendicular to the plane of the wide zone showed that such “linkers” can be revealed in at least 40% of all clefts. They become visible at some tilt angles, although the same area of the cleft is featureless at other angles. Single linker spacing measurements were obtained using a computerized image analysis system, and compiled in a frequency distribution chart. On the basis of these data, two models of a regular linker distribution within the cleft are illustrated. Our results provide evidence for the presence of regularly spaced, cleft-spanning structures within the interendothelial cleft which may have implications for endothelial cell-cell adhesion and permeability.
The 9E3 protein: immunolocalization in vivo and evidence for multiple forms in culture
Authors: Martins-Green M. , Aotaki-Keen A. , Hjelmeland L. M. , Bissell M. J.
Published: April 2021

The avian gene 9E3/CEF4 belongs to a group of genes whose products are highly conserved and are homologous to inflammatory mediators. These genes, sometimes referred to as the gro family, are also expressed upon wounding or serum-stimulation of quiescent cells, suggesting that they may be important in aspects of growth and/or wound healing. We have used an antibody to the product of the 9E3 gene to show for the first time the distribution in vivo of the protein of one of these genes. The polyclonal antibody was produced against a synthetic peptide, [Cys76], 9E3, (77-103), located at the carboxy end of the molecule. The specificity of the antibody was determined by transfection of the 9E3 cDNA into Cos 7 cells, which do not express this gene. Moreover, despite the high homology between 9E3 and IL-8, the antibody did not crossreact with this molecule. The antibody was used to immunoprecipitate the protein from cultured normal and RSV-transformed chick embryo fibroblasts (CEFs) and to determine its distribution in tissues of newly hatched chicks. The staining was abundant in the cells and extracellular matrix (ECM) of connective tissue and other tissues of mesenchymal origin, such as bone and tendon. Most cells in the granulation tissue of wounds stained, some more intensely than others; the ECM also stained, expecially in areas of scar tissue where collagen is abundant. In RSV-induced tumors, the protein was absent except in necrotic areas where a few cells -potentially macrophages -stained. In general, as expected, the protein was present in the cells and tissues that expressed the mRNA, but there were exceptions. In the smooth muscle layer of arteries and the epidermis of the skin, where the levels of mRNA were too low to be detected by in situ hybridization with a radioactively labeled probe, the protein was present. The antibody immunoprecipated a 14 kDa molecule from the cell extracts of normal and transformed CEFs, and two forms (9 kDa and 6 kDa) from the supernatant of RSV-transformed CEFs. The results presented here suggest that this protein could play a role in tissue remodeling and wound healing.
Hyaluronan-dependent cell migration can be blocked by a CD44 cytoplasmic domain peptide containing a phosphoserine at position 325
Authors: Peck David , Isacke Clare M.
Published: April 2021

CD44 is the principle transmembrane receptor for the extracellular matrix glycosaminoglycan hyaluronan. This receptor:ligand interaction plays an essential role in a number of physiological events including tumour progression, lymphocyte homing into inflammatory sites and tissue morphogenesis during development. In previous studies we have shown that serine phosphorylation is a critical control mechanism for CD44-dependent cell migration. Here we have investigated the target phosphorylation residues by mutating them individually or in combination. These studies demonstrate that Ser325 is the principle CD44 phosphorylation site and that mutation of this residue blocks CD44-mediated cell migration but not hyaluronan binding. In addition, we show that an upstream Ser323 residue is required as part of the kinase consensus site. To further characterize the role of CD44 phosphorylation, phosphorylated and non-phosphorylated peptides spanning the Ser325 region were synthesised and linked to a 16 amino acid Penetratin sequence to mediate efficient plasma membrane translocation. Peptides containing a phosphoserine at residue 325 are efficient blockers of CD44-mediated cell migration but do not reduce CD44 expression or its ability to bind hyaluronan. These data strongly argue that CD44 adhesion and migration are regulated by distinct mechanisms and that migration requires the specific interaction of intracellular component(s) with phosphorylated CD44 receptors.
Initiation of skin basement membrane formation at the epidermo-dermal interface involves assembly of laminins through binding to cell membrane receptors
Authors: Fleischmajer Raul , Utani Atsushi , MacDonald E. Douglas , Perlish Jerome S. , Pan Te-Cheng , Chu Mon-Li , Nomizu Motoyoshi , Ninomiya Yoshifumi , Yamada Yoshihiko
Published: April 2021

To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and α3, β1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of α2, α3, α6, β1, and β4 integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the β1 (AIIB2) and α6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin γ1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin γ2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process.
Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus
Authors: Swan David G. , Stern Rowena , McKellar Sue , Phillips Kirsten , Oura Chris A. L. , Karagenc Tülin Ilhan , Stadler Laura , Shiels Brian R.
Published: April 2021

Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.
ADrosophilamodel of HIV-Tat-related pathogenicity
Authors: Battaglia Piero A. , Zito Santina , Macchini Antonella , Gigliani Franca
Published: April 2021

To analyze the mechanism of Tat-mediated HIV pathogenicity, we produced a Drosophila melanogaster strain transgenic for HIV-tat gene and induced the expression of the protein during Drosophila development. By in vitro and in vivo experiments, we demonstrated that Tat specifically binds to tubulin via the MAP-binding domain of tubulin, and that this interaction delays the polymerization of tubulin and induces a premature stop to microtubule-dependent cytoplasmic streaming. The delay in the polymerization of microtubules, the tracks for the transport of the axes determinants, alters the positioning of the dorso-ventral axis as shown by the mislocalization of Gurken and Kinesin in oocyte of Drosophila after Tat induction. These results validate the use of Drosophila as a tool to study the molecular mechanism of viral gene products and suggest that Tat-tubulin interaction is responsible for neurodegenerative diseases associated with AIDS.
The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore
Authors: Scaërou Frédéric , Starr Daniel A. , Piano Fabio , Papoulas Ophelia , Karess Roger E. , Goldberg Michael L.
Published: April 2021

The zeste-white 10 (zw10) and rough deal (rod) genes of Drosophila both encode kinetochore components, and mutations in either gene greatly increase the missegregation of sister chromatids during mitosis. Here, we present genetic, cytological and biochemical evidence for a close, evolutionarily conserved relationship between the ROD and ZW10 proteins. We show that the phenotypes caused by disruption of either gene’s function are similar in Drosophila and in C. elegans. No additive effects are observed in zw10; rod double null mutants. In flies, the two proteins always colocalize and, moreover, require each other for their recruitment to the mitotic apparatus. The human ROD and ZW10 homologs also colocalize on HeLa cell kinetochores or kinetochore microtubules throughout most but not all of mitosis. Finally, we show that in both Drosophila and human cells, ROD and ZW10 are in fact physically associated, and in Drosophila these proteins are together constituents of a large (700-900 kDa), soluble macromolecular complex.
Inscuteable-dependent apical localization of the microtubule-binding protein Cornetto suggests a role in asymmetric cell division
Authors: Bulgheresi Silvia , Kleiner Elke , Knoblich Juergen A.
Published: April 2021

Drosophila neuroblasts divide asymmetrically along the apical-basal axis. The Inscuteable protein localizes to the apical cell cortex in neuroblasts from interphase to metaphase, but disappears in anaphase. Inscuteable is required for correct spindle orientation and for asymmetric localization of cell fate determinants to the opposite (basal) cell cortex. Here, we show that Inscuteable also directs asymmetric protein localization to the apical cell cortex during later stages of mitosis. In a two-hybrid screen for Inscuteable-binding proteins, we have identified the coiled-coil protein Cornetto, which shows a highly unusual subcellular distribution in neuroblasts. Although the protein is uniformly distributed in the cytoplasm during metaphase, it concentrates apically in anaphase and forms an apical crescent during telophase in an inscuteable-dependent manner. Upon overexpression, Cornetto localizes to astral microtubules and microtubule spin-down experiments demonstrate that Cornetto is a microtubule-binding protein. After disruption of the actin cytoskeleton, Cornetto localizes with microtubules throughout the cell cycle and decorates the mitotic spindle during metaphase. Our results reveal a novel pattern of asymmetric protein localization in Drosophila neuroblasts and are consistent with a function of Cornetto in anchoring the mitotic spindle during late phases of mitosis, even though our cornetto mutant analysis suggests that this function might be obscured by genetic redundancy.
Dis1/TOG universal microtubule adaptors - one MAP for all?
Authors: Ohkura Hiroyuki , Garcia Miguel A. , Toda Takashi
Published: April 2021

Microtubules play central roles in various cellular processes in eukaryotes. The dynamics and organisation of interphase microtubules and mitotic spindles are dramatically altered during the cell cycle and development. However, the molecular mechanisms underlying this dynamic behaviour remain to be understood. In recent years, a novel family of microtubule-associated proteins (MAPs), the Dis1/TOG family, has emerged as a versatile regulator of microtubule function. These MAPs are highly conserved in eukaryotes from yeasts and plants to humans. The localisation and function of these MAPs are not determined simply by their intrinsic microtubule-binding activity. Instead this family executes its diverse roles by interacting with other regulatory molecules, including microtubule motors and centrosomal proteins. The modular structure of these MAPs may allow them to interact with multiple proteins and thereby be involved in a wide variety of microtubule and spindle functions.

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FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments
Authors: Ridley S. H. , Ktistakis N. , Davidson K. , Anderson K. E. , Manifava M. , Ellson C. D. , Lipp P. , Bootman M. , Coadwell J. , Nazarian A. , Erdjument-Bromage H. , Tempst P. , Cooper M. A. , Thuring J. W. J. F. , Lim Z.-Y. , Holmes A. B. , Stephens L. R. , Hawkins P. T.
Published: April 2021

FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal.

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Rapid and reversible changes in dendrite morphology and synaptic efficacy following NMDA receptor activation: implication for a cellular defense against excitotoxicity
Authors: Ikegaya Yuji , Kim Jeong-Ah , Baba Minami , Iwatsubo Takeshi , Nishiyama Nobuyoshi , Matsuki Norio
Published: April 2021

Postsynaptic neuronal dendrites undergo functional and morphological changes in response to pathologically excessive synaptic activation. Although rapid formation of segmental focal swelling (varicosity) is the most prominent hallmark in such excitotoxic injury, little is known about the pathophysiological function of these structural alterations. We used cultured rat hippocampal slices to evaluate the relationship between the formation of varicosities and subsequent neuronal death. Substantial numbers of segmental dendritic varicosities were observed all over the hippocampus within 5 minutes of exposure to 30 μM NMDA, although neuronal death was detected only in the CA1 region 24 hours after NMDA exposure. Sublethal NMDA concentrations (1-10 μM) induced reversible focal swelling in all hippocampal subregions. NMDA-induced neuronal death was prevented either by NMDA receptor antagonists or by the use of Ca2+-free medium, whereas varicosity formation was virtually independent of Ca2+ influx. Rather, the Ca2+-free conditions per se produced dendritic focal swelling. Also, NMDA-induced varicosity formation was dependent on extracellular Na+ concentration. Thus, we believe that varicosity formation is not causally related to neuronal injury and that the two phenomena are separable and involve distinct mechanisms. Interestingly, dendrite swelling was accompanied by AMPA receptor internalization and a rapid, long-lasting depression in synaptic transmission. Moreover, low Na+ conditions or treatment with ethacrynic acid or proteinase inhibitors, which effectively prevent varicosity formation, aggravated NMDA-induced excitotoxicity, and eliminated the regional specificity of the toxicity. Therefore, the pathological changes in dendrite morphology and function may be associated with an early, self-protective response against excitotoxicity.
The peroxisome proliferator-activated receptor γ is an inhibitor of ErbBs activity in human breast cancer cells
Authors: Pignatelli Miguel , Cortés-Canteli Marta , Lai Cary , Santos Angel , Perez-Castillo Ana
Published: April 2021

One of the most interesting recent developments in the nuclear receptor field has been the identification of natural and synthetic agonists of the peroxisome proliferator-activated receptor (PPAR) family, coupled with a growing recognition that the γ isoform (PPARγ) affects pathways important in a variety of human diseases. Here we show that the activation of PPARγ through the 15-deoxy-Δ-12,14-prostaglandin J2 (PG-J2) ligand causes a dramatic inhibition of ErbB-2 and ErbB-3 tyrosine phosphorylation caused by neuregulin 1 (NRG1) and neuregulin 2 (NRG2) in MCF-7 cells. This effect is accompanied by a very efficient blocking of ErbBs effects upon proliferation, differentiation and cell death in these cells. Preincubation of MCF-7 cells with PG-J2 before addition of NRG1 and NRG2 had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 compartment of the cell cycle, and a marked increase in apoptosis. NRG1 and NRG2 induce G1 progression, which was associated with stimulation of the phosphatidylinositol-3 kinase (PI 3-K) pathway, whereas survival was dependent on ERK1/ERK2 activation. Both pathways were inhibited by PG-J2. Furthermore, PG-J2 can abolish the NRG1 and NRG2-induced increase in anchorage-independent growth of these cells. PG-J2 also blocks phosphorylation of other receptor tyrosine kinases, such as IGF-IR, in MCF-7 cells, and suppress proliferation of other breast cancer cell lines. In summary, our data show a specific inhibitory action of PG-J2 on the activity of the ErbB receptors in breast cancer cells.