Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Suppression of the endoplasmic reticulum calcium pump during zebrafish gastrulation affects left-right asymmetry of the heart and brain

Vertebrate embryos generate striking Ca²⁺ patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca²⁺ during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca²⁺ along the dorsal–ventral axis, with higher Ca²⁺ concentrations in the ventral margin and lower Ca²⁺ concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca²⁺ pump with 0.5 μM thapsigargin elevates cytosolic Ca²⁺ in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left–right asymmetry. Brain defects include a left–right reversal of pitx2 expression in the dorsal diencephalon and a left–right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca²⁺ pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca²⁺ pump during gastrulation inhibits expression of no tail (ntl) and left–right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer’s vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca²⁺ plays a role in Kupffer’s vesicle function, influencing ciliary motility and translating the vesicle’s counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca²⁺ plays an additional role in the formation of Kupffer’s vesicle.     

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit

Summary In the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique.In the uterine epithelium of oestrous females the zonula occludens belongs to the tight type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG.Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase.The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.Supported by grant Kü 210/9 from the Deutsche Forschungsgemeinschaft     

斑马鱼Z-OTU蛋白在卵子发生和胚胎发育早期的表达(英文)

斑马鱼z-otu基因编码的蛋白可能具有DUBs活性,它包含OTU和TUDOR结构域,属于OTU相关蛋白酶家族的成员。该研究将原核表达的融合蛋白(OTU结构域和GST)纯化后免疫新西兰兔,获得多克隆抗体anti-Z-OTU,并利用该抗体对Z-OTU蛋白质在斑马鱼卵子发生和早期胚胎发育过程中的表达进行了分析。根据原位和整体免疫组织化学检测结果并结合以前的研究结论,分析并比较了z-otu基因的mRNA和蛋白质的分布,发现在卵子发生和早期胚胎发育过程中,z-otu基因的mRNA和蛋白质表达模式存在明显差异:mRNA仅在卵子发生早期表达,卵母细胞受精后才重新开始表达,而其蛋白在卵子发生过程中均表达;在卵子发生过程中,mRNA分布于细胞质中,而蛋白质先分布于细胞核中,然后向细胞质迁移,接着又向卵母细胞生发泡(germinal vesicle,GV)集中。推测Z-OTU蛋白类似于其他具有去泛素化酶活性的OTU相关蛋白酶,对于卵母细胞减数分裂过程中生发泡破裂、生发泡迁移及维持胚胎的分裂是必需的。     

Gap junctions and intercellular communication in the hepatopancreas of the crayfish (Orconectes propinquus) during molt

Summary The crustacean hepatopancreas is a major metabolic center intimately involved in molting and vitellogenesis. Cells of the hepatopancreas exhibit one of the richest endowments of gap junctions known and are thus presumed to be linked for intercellular communication. In order to monitor hepatopancreatic activity during the molt cycle of crayfish (Orconectes propinquus), the electrical coupling between cells of the hepatopancreatic tubules was measured during postmolt, intermolt and premolt. Samples of hepatopancreas from each of these stages were fixed and freeze-fractured to correlate morphologic features of gap junctions with electrophysiological data. Analysis of the data revealed that ionic coupling was greater in postmolt and premolt tubule cells than in cells of intermolt animals. Platinum replicas of hepatopancreatocyte plasmalemmata revealed that in postmolt, gap junction plaques were smaller and more numerous than those in intermolt and premolt; however, the total area of gap junction plaques per unit membrane area analyzed was approximately the same for hepatopancreatocytes from all molt stages. Although the hepatopancreatic gap junctions exhibited no quantitative differences, those from post- and premolt animals were rounded with tightly packed particles, while plaques from intermolt animals were generally pleomorphic with loosely packed particles. Results of this study suggest that cells of the crayfish hepatopancreas are more coupled in pre- and postmolt, with macular plaques of tightly packed particles, perhaps as a response to the increased metabolic demands of molt, and less well coupled, with irregular plaques of loosely packed junctional particles, during intermolt. The only recognizable morphological correlates of increased cell coupling were tight packing of junctional particles into rounded plaques, while decreased coupling corresponded to junctions with loosely packed irregular aggregates of particles.Supported by the Natural Sciences and Engineering Research Council of Canada (RRS)     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers     

Segment-specific expression of the gap junction gene connexin31 during hindbrain development

 During segmentation of the mouse hindbrain (d8.0–8.5 pc), expression of the gap junction gene connexin31 (cx31) is precisely restricted to rhombomeres (r) 3 and 5. Shortly afterwards, during the turning process, cx31 expression in rhombomere 3 decreases and is no longer detectable at d9.5 pc, whereas expression in rhombomere 5 is maintained until about d10.0 pc. So far, cx31 is the first gap junction gene found to be expressed in rhombomeres. Its precise segmental and temporal expression pattern may reflect a critical requirement of cx31 channels for these odd numbered rhombomeres to acquire distinct cell identities. Received: 9 June 1997 / Accepted: 30 July 1997     

Ontogeny of intestinal motility in correlation to neuronal development in zebrafish embryos and larvae

For the first time, spontaneous intestinal activity was demonstrated and quantified before the onset of exogenous feeding in zebrafish Danio rerio embryos and larvae in vivo , using digital motion analysis. At 3 days post fertilization (dpf), erratic and spontaneous contraction waves were observed in the gut. Later (4–7 dpf), more distinct contraction patterns were distinguished, and anterograde and retrograde contraction waves projecting anally and orally along the intestine, respectively, as well as local rectal contraction waves could be identified and quantified. The frequency of both anterograde intestinal and local rectal contractions increased significantly during the first days of development. There was a tendency towards shorter anterograde contraction waves in the first dpf stage investigated, but the velocity of the waves did not differ significantly between the different dpf stages. The presence of developing neurones in the gut of zebrafish was established using immunohistochemistry, staining for a suite of marker proteins (Hu C/D, HNK-1 and acetylated tubulin). Structural neurones were present in the developing gut from the first dpf stage investigated (2 dpf). In conclusion, during the period (3–7 dpf) when erratic contraction waves turn into a more organized pattern of motility there is also a pronounced development of the innervation, suggesting a correlation in time of the development of gut motility and its neuronal control.     

The carp homeobox gene Ovx1 shows early expression during gastrulation and subsequently in the vagal lobe, the facial lobe and the ventral telencephalon

 The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections. Received: 29 October 1997 / Accepted: 12 November 1997     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.     

Mutations affecting pigmentation and shape of the adult zebrafish

 Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish. Received: 17 June 1996 / Accepted: 15 July 1996     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Neuritic growth cone and ependymal gap junctions in the feline subfornical organ during early development

Summary Intercellular contacts in the subfornical organ (SFO) of kittens 3, 16, and 29 days old were studied in thin sections and by the freeze-etch method. Gap junctions appeared between growing nerve processes and target cells. The junctions were interspersed between immature synapses lacking mitochondria as well as full preand postsynaptic membrane specializations. Gap junctions were seen on filopodia as well as on more mature processes. The morphology of these junctions was typical of those described earlier but they were of small size (0.2–0.3 m).Gap junctions of peculiar form were also seen between ependymal elements in the SFO at 16 days. These were of large size (0.5–0.8 m) and were often of segmented character. This segmentation consisted of bands 3–4 particles in width with a center-to-center spacing of 90 nm with particle free corridors between corresponding to the width of about two rows of particles. The margin of the group might be circumscribed by a row of particles. Although gap junctions of large size were seen between ependymal cells in thin section, features corresponding to the particle free corridors have not been observed to date.On leave of absence from the National Institute of Neurological and Communicative Disorders and Stroke, Section of Functional Neurosurgery, Branch of Clinical Neuroscience, Bethesda, Maryland 20014, USAThis work was supported by grants from the Swiss National Foundation for Scientific Research Nos. 3.636.76 and 3.611.0.75, the EMDO Stiftung and the Dr. Eric Slack-Gyr Stiftung     

Polar effects of lithium in the heart of the zebrafish Danio rerio

 The molecular signalling mechanisms that are believed to govern the patterning of the heart early in embryonic development are not well understood. We have investigated the events which occur during patterning of the vertebrate heart by exposing gastrula stage zebrafish embryos to lithium, which is known to affect the phosphoinositol signalling pathway. Treatment of embryos at 50% epiboly (5.25 h after fertilization at 28.5°C) with 0.3 m LiCl for 5–15 min, results in embryos with defects which range from mild to severe, depending on the length of time the embryos are exposed to lithium. In the heart, defects appear progressively in the inflow tract, the sinus venosus and atrium. By using an antibody that recognizes an atrium-specific isoform of myosin, our results show that lithium treatment at gastrulation specifically affects the atrium and sinus venosus, and has little obvious effect on the ventricle. Defects induced by lithium differ from those induced by retinoic acid (RA) treatment of similarly staged embryos, and suggest that lithium and RA may affect the patterning signals important for establishment of the vertebrate heart by acting on different populations of cells or by influencing different patterning pathways. Received: 8 December 1995 / Accepted: 11 April 1996     

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant’s layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant’s cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell’s trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar ”embryoid” shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events. Received: 26 April 1999 / Accepted: 30 August 1999     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997