Combined toxicity of dissolved mercury with copper, lead and cadmium on embryogenesis and early larval growth of the Paracentrotus lividus sea-urchin

The individual and combined toxicity of dissolved mercury, copper, lead and cadmium has been investigated by using the Paracentrotus lividus sea-urchin embryo-larval bioassay. Embryogenesis success and early larval growth have been recorded after incubation of fertilised eggs in seawater, both with single metals and binary combinations of Hg with every other metal. For individual metals the ranking of toxicity was Hg > Cu > Pb > Cd, with EC₅₀ values of 21.9, 66.8, 509 and 9240g/l, respectively. Lowest observed effect concentrations (LOEC) for early larval growth were approximately three times lower than the EC₅₀ values for Hg, Cu and Pb, and more than two orders of magnitude lower for Cd, emphasizing the danger of underestimating toxicity when only lethal effects are recorded. Marking & Dawson's additive indices ranged from 0.10 to 0.19, indicating additive effects with a slight trend to synergism, which was statistically significant for the Hg–Pb combination only. Hayes' additive indices were within the margins considered acceptable to describe additive interactions.     

酪氨酸激酶受体EphA5及其配体ephrin-A5在小鼠脊髓发育过程中的表达

目的研究两个Eph家族分子,EphA5受体及其配体ephrin—A5,在脊髓发育过程中的表达方式。方法β-半乳糖苷酶基因敲入小鼠和配体亲和探针分析受体的表达,两种不同的亲和探针分析配体的表达,基因敲除小鼠作为对照。结果在发育过程中,EphA5表达于脊髓腹侧,而ephrin—A5表达于脊髓背侧。结论EphA5和ephrin—A5在多个脊髓发育阶段都有表达,他们可能在脊髓背腹侧组织结构的建立过程中发挥重要作用。     

Contribution to the 3D computer assisted reconstruction of pancreatic buds in the rat embryos

The aim of this work was to reconstruct, in the rat embryos, stage 12-23, the three dimensional (3D) distribution of the dorsal and ventral pancreatic buds by of a computer assisted method. Ninety-six rat embryos, CRL 3-16 mm, fixed, dehydrated, and paraffin embedded, were submitted to serial histological sections and stained by hematoxylin-eosin and Heidenhain's azan techniques. The images were digitalized by Canon Camera 350 EOS D. The serial views were aligned anatomically by software and the data were analyzed following segmentation and thresholding. The dorsal pancreas developed from the dorsal wall of the duodenum in stage 12, while the ventral pancreas arose from the ventral wall of the hepatic diverticulum in stage 13 and 14. The rotation of ventral pancreas started in stage 15 and was completed in stage 16. The fusion of both buds was evident in stage 17. In stage 23 the limit between dorsal and ventral bud was still marked by the pathway of superior mesenteric vein.     

NPY genes and AGC kinases define two key steps in auxin-mediated organogenesis in Arabidopsis

Auxin is an essential regulator of plant organogenesis. Most key genes in auxin biosynthesis, transport, and signaling belong to gene families, making it difficult to conduct genetic analysis of auxin action in plant development. Herein we report the functional analysis of several members of 2 gene families (NPY/ENP/MAB4 genes and AGC kinases) in auxin-mediated organogenesis and their relationships with the YUC family of flavin monooxygenases that are essential for auxin biosynthesis. We show that 5 NPY genes (NPY1 to NPY5) and 4 AGC kinases (PID, PID2, WAG1, and WAG2) have distinct, yet overlapping, expression patterns. Disruption of NPY1 does not cause obvious defects in organogenesis, but npy1 npy3 npy5 triple mutants failed to make flower primordia, a phenotype that is also observed when AGC kinase PID is compromised. Inactivation of YUC1 and YUC4 in npy1 background also phenocopies npy1 npy3 npy5 and pid. Simultaneous disruption of PID and its 3 closest homologs (PID2, WAG1, and WAG2) completely abolishes the formation of cotyledons, which phenocopies npy1 pid double mutants and yuc1 yuc4 pid triple mutants. Our results demonstrate that NPY genes and AGC kinases define 2 key steps in a pathway that controls YUC-mediated organogenesis in Arabidopsis.     

Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical–basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.The phytohormone auxin plays critical roles in almost every aspect of plant development including embryogenesis, architecture formation, and tropic growth. One of the most fascinating topics in plant biology is how auxin can have so many diverse and context-specific functions (1). Dynamic auxin gradients, which are regulated mainly by local auxin synthesis, catabolism, conjugation, and polar auxin transport, are essential for integration of various environmental and endogenous signals (2, 3). Auxin perception and signaling systems are responsible for a read-out of the auxin gradients (1).Local auxin biosynthesis is important for leaf development, shade avoidance, root-specific ethylene sensitivity, vascular patterning, flower patterning, and embryogenesis (4). Indole-3-acetic acid (IAA), the naturally occurring principal auxin in plants, is biosynthesized from tryptophan (Trp) through four proposed routes according to their key intermediates, namely indole-3-acetaldoxime (IAOx), indole-3-pyruvic acid (IPyA), indole-3-acetamide (IAM), and tryptamine (TAM) (5). The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)/YUCCA (YUC) linear pathway has been considered as a predominant Trp-dependent auxin biosynthetic pathway (4, 6–8). However, labeling studies and analyses of Trp auxotrophic mutants have long predicted the existence of a Trp-independent IAA biosynthetic pathway (9–13). When the Arabidopsis and maize seedlings were fed with isotope-labeled precursor, IAA was more enriched than Trp, and the incorporation of label into IAA from Trp is low, suggesting that IAA can be produced de novo without Trp as an intermediate (11, 12). The Trp biosynthetic mutant trp1, defective in phosphoribosylanthranilate transferase (PAT), and indole-3-glycerol phosphate synthase (IGS) antisense plants are deficient in steps earlier than indole-3-glycerol phosphate (IGP) formation and display decreased levels of both IAA and Trp (10, 14). However, the trp3 and trp2 mutants, defective in tryptophan synthase α (TSA) and tryptophan synthase β (TSB) subunits, respectively, accumulate higher levels of IAA than the wild type despite containing lower Trp levels (10, 11, 15, 16). These data strongly suggested the existence of the Trp-independent IAA biosynthetic pathway that might branch from IGP and/or indole (10). Further studies suggested that a cytosol-localized indole synthase (INS) may be involved in the Trp-independent biosynthesis of indole-containing metabolite (17). However, the molecular basis, biological functions, and spatiotemporal regulation of the Trp-independent IAA biosynthetic pathway have remained a mystery.In higher eukaryotes, embryogenesis initiates the generation of the species-specific body plan. During embryogenesis, a single-celled zygote develops into a functional multicellular organism with cells adopting specific fates according to their relative positions. In higher plants, essential architecture features, such as body axes and major tissue layers, are established in embryogenesis, and auxin plays a vital role in apical-basal patterning and embryo axis formation (18, 19). Local auxin biosynthesis, polar auxin transport facilitated by PIN-FORMED1/3/4/7 (PIN1/3/4/7), and auxin response coordinately regulate apical–basal pattern formation during embryogenesis (7, 20–22). The TAA and YUC families in Trp-dependent IAA biosynthesis predominantly regulate embryogenesis at or after the globular stage (7, 22). However, the auxin source for embryogenesis before the globular stage remains elusive. In this study, we provide, to our knowledge, the first genetic and biochemical evidence that the cytosol-localized INS is a key component in the long predicted Trp-independent auxin biosynthetic pathway and is critical for apical–basal pattern formation during early embryogenesis in Arabidopsis.     

Effects of hyperglycaemia on sorbitol and myo-inositol contents of cultured embryos: treatment with aldose reductase inhibitor and myo-inositol supplementation

Summary To demonstrate the myo-inositol depletion hypothesis in hyperglycaemia-induced embryopathy, rat conceptuses of 9.5 days of gestation in the early head-fold stage were grown in vitro during neural tube formation for 48 h with increasing amounts of glucose. The effects of an aldose reductase inhibitor and the myo-inositol supplementation were also investigated. Sorbitol and myo-inositol contents were measured in separated embryos and extra-embryonic membranes including yolk sac and amnion at the end of culture. After addition of 33.3 mmol/l and 66.7 mmol/l glucose to the culture media, the myo-inositol content of the embryos was significantly decreased by 43.1% (p<0.05) and 64.6% (p < 0.01) of the control group, while a marked accumulation of sorbitol was observed (25 and 41 times that of the control). Although the addition of an aldose reductase inhibitor (0.7 mmol/l) to the hyperglycaemic culture media containing an additional 66.7 mmol/l glucose significantly reduced the sorbitol content of embryos to approximately one-eighth, the myo-inositol content of embryos remained decreased and the frequency of neural lesions was unchanged (23.1% vs 23.9%, NS). Supplementation of the myo-inositol (0.28 mmol/l) completely restored the myo-inositol content of the embryos and resulted in a significant decrease in the frequency of neural lesions (7.1% vs 23.9%, p < 0.01) and a significant increase in crown-rump length and somite numbers. Much less significantly, sorbitol accumulation was also observed in the extra-embryonic membrane in response to hyperglycaemia, neither hyperglycaemia nor the myo-inositol supplementation modified the myo-inositol contents of the extra-embryonic membrane. We conclude that the mechanism of hyperglycaemia-induced teratogenicity was mediated by the myo-inositol depletion of the embryo at a critical stage of organogenesis.     

ETS1对非洲爪蛙早期胚胎发育的影响

目的:通过在非洲爪蛙胚胎中过表达ETS1探讨该基因对爪蛙胚胎早期发育的影响?方法:将克隆PCR获得的ETS1全长片段连接到质粒上,获得pCS2+-ETS1质粒;利用RT-PCR和Western blot检测ETS1 mRNA和蛋白在胚胎发育不同时期的表达情况;显微注射ETS1 mRNA入爪蛙胚胎,利用整胚原位杂交?定量PCR检测胚层标志性基因的表达;通过Western blot检测过表达ETS1对细胞凋亡和增殖的影响?结果:ETS1从受精卵时期即已开始表达,在器官形成期表达增强,过表达ETS1抑制了细胞分化以及神经胚期中胚层和外胚层标志基因表达,凋亡相关蛋白表达增加,增殖相关蛋白无明显变化?结论:ETS1表达于非洲爪蛙胚胎发育各阶段,过表达ETS1抑制了中胚层和外胚层发育,促进细胞凋亡但不影响细胞增殖?     

Implantation, early placentation, and the chronology of embryogenesis in Tupaia belangeri

Summary Ten developmental stages of Tupaia belangeri are described. On day 6 post conception expanded bilaminar blastocysts were found in the uterine lumen. Implantation occurs on day 7 p.c.: the blastocyst lies in an antimesometrial implantation chamber. Implantation, therefore, is eccentric and not centric as previously described. The implantation chamber is sealed off from the rest of the uterine lumen by the two gland-free longitudinal endometrial pads apposed to each other in the midline. These pads form a decidua reflexa (Hubrecht). Orientation of the embryoblast is antimesometrial. The blastocyst is firmly attached by its abembryonic pole to the smooth surface epithelium of the two apposed endometrial pads. The initial attachment only involves the epithelium of the antimesometrial surface of the endometrial pads. Thus implantation is initially abembryonic and not bilateral. In the next stage the two endometrial pads have separated again, so that the primary site of attachment on them has also been divided. Inside the implantation chamber the trophoblast develops plugs which penetrate into the openings of the uterine glands. Endometrial crypts develop on the surface of the endometrial pads where these are free of the trophoblast. Late on day 9 p. c. the abembryonic pole of the blastocyst has further expanded towards the mesometrium and now fills the entire lumen of the uterus. The attachment plaques have expanded over the entire surface of the endometrial pads due to giant cell activity. Thus implantation has become centric and para-embryonic (bilateral). This condition, previously described for Tupaia in the literature, is only reached secondarily. The giant cells disappear when the maternal basement membrane has broken down. The multilayered cytotrophoblast and the primary villi come into contact with maternal vessels, as has been previously described by Luckett (1968). There is an antimesometrial to mesometrial gradient in the development of the placental discs; the antimesometrial side is always slightly more advanced.Concurrent lactation does not influence the duration of pregnancy in Tupaia belangeri. Implantation is not delayed by lactation. Martin's (1968) hypothesis of a true developmental period of only three weeks in Tupaia is not substantiated: embryogenesis progresses rapidly after implantation. Embryos in an advanced stage of development corresponding to Streeter's horizon XXII (Homo) have been recovered on day 24 p. c. of the 44-day gestation period.

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Zusammenfassung Zehn Entwicklungsstadien von Tupaia belangeri werden beschrieben. Am 6. Tag post conceptionem finden sich freie bilaminäre Blastocysten im Uteruslumen. Die Implantation erfolgt am 7. Tag p. c. in einer antimesometrialen Implantationskammer, also excentrisch und nicht zentral wie bisher angenommen. Die Implantationskammer wird von den beiden drüsenfreien, im Uterus orthomesometral längsverlaufenden Endometrialpolstern abgeschlossen, die sich in der Mitte des Uteruslumens aneinanderlegen. Diese Polster bilden auf diese Weise eine Decidua reflexa (Hubrecht). Der Embryoblast ist antimesometral orientiert. Der abembryonale Pol der Blastocyste ist am glatten Oberflächenepithel der beiden Endometrialpolster fest verankert. Die Implantation erfolgt also zunächst abembryonal und nicht bilateral. Primär erfolgt diese Anheftung nur an der antimesometralen, der Implantationskammer zugekehrten Seite der Endometrialpolster. Beim nächsten untersuchten Entwicklungsstadium beginnen sich die beiden Endometrialpolster voneinander zu trennen. Die Implantationskammer wird eröffnet. Dadurch wird die primäre Anheftungsstelle der Blastocyste in der Mitte geteilt. In der Implantationskammer hat der Trophoblast Zapfen gebildet, die in die Öffnungen der Endometrialdrüsen reichen und der Resorption von Histiotrophe dienen. Wo die Endometrialpolster nicht vom Trophoblasten bedeckt werden, haben sich auf ihrer Oberfläche Krypten gebildet. Trophoblast-Riesenzellen haben das mütterliche Epithel an der primären Anheftungsstelle zerstört. Später am 9. Tag p. c. hat sich die Blastocyste weiter gegen das Mesometrium ausgedehnt und füllt nun das gesamte Uteruslumen aus. Durch die Aktivität der Trophoblast-Riesenzellen hat sich die Anheftungsstelle bis an den mesometralen Rand der Endometrialpolster ausgedehnt. So ist das Bild der zentralen und bilateralen Anheftung entstanden, das in der Literatur für Tupaia beschrieben wird. Es ist also erst sekundär zustande gekommen. Die Riesenzellen verschwinden, wenn die mütterliche Basalmembran durchbrochen ist. Der nun vielschichtige Trophoblast und primäre Zotten nehmen Kontakt mit mütterlichen Gefäßen auf (Luckett, 1968). Von der ersten Anheftung bis zur fertigen allanto-chorialen Placenta ist bei Tupaia ein antimesometral- mesometraler Gradient der Placentation festzustellen: die antimesometrale Seite der Anheftungsstellen an den Endometrialpolstern ist immer etwas weiter entwickelt als die mesometrale Seite.Durch gleichzeitige Lactation wird die Tragzeit von Tupaia belangeri nicht beeinflußt, die Implantation wird nicht verzögert. Martin's (1968) Hypothese, wonach die true developmental period bei Tupaia nur drei Wochen beträgt, können wir nicht stützen: nach der Implantation vollzieht sich die Embryogenese rasch weiter. Am 24. der insgesamt 44 Tage der Tragzeit fanden wir weit entwickelte Embryonen, die Streeters horizon XXII von Homo entsprechen.