Matrilin-1 Is Essential for Zebrafish Development by Facilitating Collagen II Secretion

Matrilin-1 is the prototypical member of the matrilin protein family and is highly expressed in cartilage. However, gene targeting of matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen IIα1a, indicating that the functional loss of this domain occurs as a consequence of matrilin-1 deficiency. The results show that matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events.     

Cell Adhesion in Zebrafish Embryos Is Modulated by March8

March8 is a member of a family of transmembrane E3 ubiquitin ligases that have been studied mostly for their role in the immune system. We find that March8 is expressed in the zebrafish egg and early embryo, suggesting a role in development. Both knock-down and overexpression of March8 leads to abnormal development. The phenotype of zebrafish embryos and Xenopus animal explants overexpressing March8 implicates impairment of cell adhesion as a cause of the effect. In zebrafish embryos and in cultured cells, overexpression of March8 leads to a reduction in the surface levels of E-cadherin, a major cell-cell adhesion molecule. Experiments in cell culture further show that E-cadherin can be ubiquitinated by March8. On the basis of these observations we suggest that March8 functions in the embryo to modulate the strength of cell adhesion by regulating the localization of E-cadherin.     

Rereplication in emi1-Deficient Zebrafish Embryos Occurs through a Cdh1-Mediated Pathway

Disruption of early mitotic inhibitor 1 (Emi1) interferes with normal cell cycle progression and results in early embryonic lethality in vertebrates. During S and G2 phases the ubiquitin ligase complex APC/C is inhibited by Emi1 protein, thereby enabling the accumulation of Cyclins A and B so they can regulate replication and promote the transition from G2 phase to mitosis, respectively. Depletion of Emi1 prevents mitotic entry and causes rereplication and an increase in cell size. In this study, we show that the developmental and cell cycle defects caused by inactivation of zebrafish emi1 are due to inappropriate activation of APC/C through its cofactor Cdh1. Inhibiting/slowing progression into S-phase by depleting Cdt1, an essential replication licensing factor, partially rescued emi1 deficiency-induced rereplication and the increased cell size. The cell size effect was enhanced by co-depletion of cell survival regulator p53. These data suggest that the increased size of emi1-deficient cells is either directly or indirectly caused by the rereplication defects. Moreover, enforced expression of Cyclin A partially ablated the rereplicating population in emi1-deficient zebrafish embryos, consistent with the role of Cyclin A in origin licensing. Forced expression of Cyclin B partially restored the G1 population, in agreement with the established role of Cyclin B in mitotic progression and exit. However, expression of Cyclin B also partially inhibited rereplication in emi1-deficient embryos, suggesting a role for Cyclin B in regulating replication in this cellular context. As Cyclin A and B are substrates for APC/C-Cdh1 - mediated degradation, and Cdt1 is under control of Cyclin A, these data indicate that emi1 deficiency-induced defects in vivo are due to the dysregulation of an APC/C-Cdh1 molecular axis.     

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes

Vacuolar-Type H⁺-ATPase (V-ATPase) takes the central role in pumping H⁺ through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H⁺ secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H⁺ secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H⁺ via V-ATPase at the cellular level.     

3-Pyridinylboronic Acid Ameliorates Rotenone-Induced Oxidative Stress Through Nrf2 Target Genes in Zebrafish Embryos

Neurochemical Research - Parkinson’s disease (PD) is one of the most common forms of neurodegenerative diseases and research on potential therapeutic agents for PD continues. Rotenone is a...     

The Large GTPase Mx1 Is Involved in Apical Transport in MDCK Cells

In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin‐related large GTPase Mx1 is a component of post‐Golgi vesicles carrying the neurotrophin receptor p75^NTR. Moreover, siRNA‐mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.      

马兜铃酸对斑马鱼胚胎肾毒性作用

选用肾发育完成(受精后3天)的斑马鱼(Danio rerio)胚胎,用马兜铃酸进行染毒处理,观察胚胎的表型变化及死亡情况,分析马兜铃酸对胚胎的毒性作用及规律;利用肾荧光观察及肾组织切片,观察马兜铃酸处理后胚胎肾形态和肾组织结构的改变情况;利用qPCR检测马兜铃酸处理前后nephrin的表达变化,初步探讨足细胞在马兜铃酸毒性作用中的功能状态。20μmol/L马兜铃酸处理24 h后,胚胎出现明显眼周水肿表现;在马兜铃酸高浓度组(40~80μmol/L),除眼周水肿外,胚胎血循环系统功能出现异常,表现为心率降低、血流缓慢甚至停滞;荧光显微镜下观察发现,马兜铃酸处理组胚胎肾出现肾小球囊性膨胀、前肾管囊性扩张和形态异常;切片显示马兜铃酸处理组胚胎肾组织结构受到损害,表现为肾小球结构疏松、囊性扩张,前肾管上皮细胞细胞排列松散、紊乱及管腔扩张样改变;qPCR结果,马兜铃酸处理组斑马鱼胚胎nephrin的表达水平比对照组显著降低(P0.01)。研究表明,马兜铃酸能损害斑马鱼胚胎肾结构和功能,其毒性作用与肾小球足细胞的功能改变有关。     

The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice

Iron is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire iron in the soil. Phytosiderophores chelate and solubilize insoluble iron hydroxide in the soil. Subsequently, plants take up iron-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals, including iron. In this study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-β-glucuronidase revealed that TOM2 was expressed in tissues involved in metal translocation, whereas TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, whereas TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared with non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released ¹⁴C-labeled deoxymugineic acid, the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of deoxymugineic acid, which is required for normal plant growth.     

迷迭香酸对硫酸铜致斑马鱼胚胎毒性的抑制作用

本文探讨硫酸铜(CuSO_4)对斑马鱼(Danio rerio)胚胎发育的毒性效应,使用迷迭香酸(RA)抑制CuSO_4对斑马鱼胚胎发育的毒性并探讨其作用机制。收集受精后1 h(1 hpf)的斑马鱼胚胎暴露于不同浓度的CuSO_4溶液,或含有不同浓度迷迭香酸的CuSO_4溶液,对照组培养在E3培养液中,观察胚胎死亡、孵化及畸形情况,计算胚胎死亡率、孵化率和畸形率;以活性氧(ROS)荧光探针DCFH-DA染色法检测迷迭香酸保护下胚胎的活性氧水平。对实验数据进行方差分析。结果显示:(1)CuSO_4浓度超过一定量时能诱导斑马鱼胚胎死亡和畸形,胚胎孵化率也降低。CuSO_4对96 hpf斑马鱼胚胎的半致死浓度(LC50)为7.7μmol/L,半致畸浓度(EC50)为1.9μmol/L。(2)在96 hpf,迷迭香酸与8μmol/L CuSO_4共同处理组斑马鱼胚胎的死亡率明显降低,孵化率升高。迷迭香酸与1.6μmol/LCuSO_4共同处理组斑马鱼胚胎的畸形率降低。(3)CuSO_4单独处理组的活性氧含量明显高于迷迭香酸与CuSO_4共同处理组和对照组。结果表明,CuSO_4暴露对斑马鱼胚胎发育的毒性效应可能与活性氧升高导致的氧化应激相关;迷迭香酸抑制CuSO_4对斑马鱼胚胎发育的毒性作用,可能与减少活性氧生成有关。     

Phenotype Classification of Zebrafish Embryos by Supervised Learning

Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification.     

视黄酸缺乏对斑马鱼心脏房室分化的影响

目的 通过化学遗传学方法建立视黄酸缺乏的斑马鱼模型,探讨视黄酸缺乏对斑马鱼胚胎心脏前后轴发育即房室分化的影响.方法 在斑马鱼胚胎孵育的5 hpf,用不同浓度梯度的视黄醛脱氢酶2抑制剂DEAB(1×10-6、5×10-6、10×10-6、25×10-6 mol/L)处理斑马鱼胚胎,实时观察斑马鱼胚胎发育的全过程.通过给予斑马鱼胚胎外源性视黄酸,观察其对DEAB的拮抗作用.应用胚胎整体原位杂交观察视黄酸缺乏对心脏特异基因vmhc和amhc表达的影响.结果 斑马鱼胚胎的生存率随着DEAB处理浓度的增加而降低,当DEAB浓度≥5×10-6 mol/L时,斑马鱼的畸胎率达100%.5×10-6 mol/L DEAB的致畸作用能够被1×10-9mol/L外源性视黄酸所拮抗.整体原位杂交结果显示视黄酸缺乏会导致斑马鱼胚胎心脏房室分化异常,表现为vmhc表达细胞的范围增大,amhc表达细胞的范围缩小.结论 通过外源性DEAB处理能有效地建立视黄酸缺乏的斑马鱼模型,DEAB影响胚胎发育存在剂量依赖性.视黄酸在斑马鱼心脏前后轴发育过程中起重要调控作用,心脏发育早期视黄酸缺乏会抑制心房的发育而支持心室的发育,出现房室分化异常.     

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca²⁺ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2^−/−/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb. Immunohistochemistry and western blotting confirmed this pattern and revealed expression of EFhd2 during neuronal maturation. In cortical neurons, EFhd2 was detected in neurites marked by MAP2 and co-localized with pre- and post-synaptic markers. Approximately one third of EFhd2 associated with a biochemically isolated synaptosome preparation. There, EFhd2 was mostly confined to the cytosolic and plasma membrane fractions. Both synaptic endocytosis and exocytosis in primary hippocampal EFhd2^−/− neurons were unaltered but transport of synaptophysin-GFP containing vesicles was enhanced in EFhd2^−/− primary hippocampal neurons, and notably, EFhd2 inhibited kinesin mediated microtubule gliding. Therefore, we found that EFhd2 is a neuronal protein that interferes with kinesin-mediated transport.     

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp⁴²⁷, Glu⁴⁴⁸, Glu⁴⁵⁶, Asp⁴⁷⁵, and Glu⁵¹⁶. In addition, Glu⁵²⁴ and Glu⁵³⁰ were involved in putrescine and spermidine uptake activity, and Glu⁵²⁸ and Glu⁵⁴⁰ were weakly involved in putrescine uptake activity. Furthermore, Asp⁵⁵¹ was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.     

外源性视黄酸对斑马鱼心血管系统发育的影响

目的观察不同浓度外源性视黄酸对斑马鱼早期胚胎和心血管系统发育的影响,为进一步研究视黄酸影响斑马鱼心脏前后轴(A-P轴)发育的分子机制提供形态学依据。方法选择斑马鱼胚胎孵育的3,6,9·5,12h四个时间点,用不同浓度视黄酸(1×10-6,1×10-7,4×10-8,1×10-8mol/L)处理斑马鱼胚胎,在解剖显微镜下实时观察斑马鱼胚胎心脏发育的全过程和视黄酸对斑马鱼心脏发育的影响。并采用胚胎整体原位杂交技术观察flk-1mRNA在斑马鱼胚胎的表达。结果1×10-6mol/L视黄酸可导致斑马鱼胚胎表现出多系统的严重畸形,胚胎很快死亡。在胚胎孵育的9·5、12h给与10-7~10-8mol/L浓度的视黄酸,胚胎只表现出心血管系统的畸形,其他系统无明显异常。胚胎整体原位杂交显示视黄酸对flk-1mRNA在斑马鱼胚胎血管的表达没有影响。结论视黄酸影响斑马鱼胚胎心脏发育有剂量依赖性和严格的时间窗,视黄酸影响心脏前后轴发育的关键时间是原肠胚晚期。视黄酸处理组胚胎的循环缺陷主要为心脏发育异常所致。10-7~10-8mol/L浓度视黄酸在9·5、12h处理斑马鱼胚胎可以作为研究心脏发育调控机制的动物模型。     

RP1 Is a Phosphorylation Target of CK2 and Is Involved in Cell Adhesion

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser²³⁶ in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP²³⁶ show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser²³⁶ by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.     

Arabidopsis PCR2 Is a Zinc Exporter Involved in Both Zinc Extrusion and Long-Distance Zinc Transport

Plants strictly regulate the uptake and distribution of Zn, which is essential for plant growth and development. Here, we show that Arabidopsis thaliana PCR2 is essential for Zn redistribution and Zn detoxification. The pcr2 loss-of-function mutant was compromised in growth, both in Zn-excessive and -deficient conditions. The roots of pcr2 accumulated more Zn than did control plants, whereas the roots of plants overexpressing PCR2 contained less Zn, indicating that PCR2 removes Zn from the roots. Consistent with a role for PCR2 as a Zn-efflux transporter, PCR2 reduced the intracellular concentration of Zn when expressed in yeast cells. PCR2 is located mainly in epidermal cells and in the xylem of young roots, while it is expressed in epidermal cells in fully developed roots. Zn accumulated in the epidermis of the roots of pcr2 grown under Zn-limiting conditions, whereas it was found in the stele of wild-type roots. The transport pathway mediated by PCR2 does not seem to overlap with that mediated by the described Zn translocators (HMA2 and HMA4) since the growth of pcr2 hma4 double and pcr2 hma2 hma4 triple loss-of-function mutants was more severely inhibited than the individual single knockout mutants, both under conditions of excess or deficient Zn. We propose that PCR2 functions as a Zn transporter essential for maintaining an optimal Zn level in Arabidopsis.