非肌性肌球蛋白II-C在斑马鱼胚胎发育过程中的表达*

目的：分析NM II-C在斑马鱼胚胎发育过程中表达的时空变化。方法：收集不同发育阶段的斑马鱼胚胎，应用整体原位杂交技术研究NM II-C在斑马鱼胚胎发育过程中的表达变化情况。结果：受精后17和22小时，NM II-C由原先的广泛表达渐渐集中于前脑、中脑和后脑；受精后26和36小时，NM II-C表达分布到整个中枢神经系统；受精后48小时，NM II-C表达在脊髓变得很弱，主要集中于大脑。结论：揭示了NM II-C基因在斑马鱼胚胎发育过程中的时间和空间上的表达进程，为进一步探讨其在发育中的调控作用提供了实验基础。     

Oxytocin receptor signalling modulates novelty recognition but not social preference in zebrafish

Sociality is a complex phenomenon that involves the individual´s motivation to approach their conspecifics, along with social cognitive functions that enable individuals to interact and survive. The nonapeptide oxytocin (OXT) is known to regulate sociality in many species. However, the role of OXT in specific aspects of sociality is still not well understood. In the present study, we investigated the contribution of the OXT receptor (OXTR) signalling in two different aspects of zebrafish social behaviour: social preference, by measuring their motivation to approach a shoal of conspecifics, and social recognition, by measuring their ability to discriminate between a novel and familiar fish, using a mutant zebrafish lacking a functional OXTR. Although oxtr mutant zebrafish displayed normal attraction to a shoal of conspecifics, they exhibited reduced social recognition. We further investigated whether this effect would be social‐domain specific by replacing conspecific fish by objects. Although no differences were observed in object approach, oxtr mutant fish also exhibited impaired object recognition. Our findings suggest that OXTR signalling regulates a more general memory recognition of familiar vs novel entities, not only in social but also in a non‐social domain, in zebrafish.     

How Zebrafish Can Drive the Future of Genetic-based Hearing and Balance Research

Over the last several decades, studies in humans and animal models have successfully identified numerous molecules required for hearing and balance. Many of these studies relied on unbiased forward genetic screens based on behavior or morphology to identify these molecules. Alongside forward genetic screens, reverse genetics has further driven the exploration of candidate molecules. This review provides an overview of the genetic studies that have established zebrafish as a genetic model for hearing and balance research. Further, we discuss how the unique advantages of zebrafish can be leveraged in future genetic studies. We explore strategies to design novel forward genetic screens based on morphological alterations using transgenic lines or behavioral changes following mechanical or acoustic damage. We also outline how recent advances in CRISPR-Cas9 can be applied to perform reverse genetic screens to validate large sequencing datasets. Overall, this review describes how future genetic studies in zebrafish can continue to advance our understanding of inherited and acquired hearing and balance disorders.     

去水卫矛醇对斑马鱼神经发育毒性评价

目的 探讨去水卫矛醇(dianhydrogalactitol,DAG)诱导斑马鱼胚胎及幼鱼神经发育毒性作用及其机制。方法 在一般毒性评价的基础上,对斑马鱼胚胎进行相应的分组暴露给药,采用幼鱼的自主运动反应、光照刺激反应等试验观察DAG对斑马鱼神经及行为的影响;通过脑部组织病理学检查、吖啶橙染色,观察DAG对斑马鱼脑部的组织影响;利用实时荧光定量PCR方法测定斑马鱼幼鱼体内多巴胺神经元相关基因(DAT、TH、GCH1)及神经抑/促凋亡相关基因(Bax、Bcl-2)的相对表达量。结果 DAG在20,40,75 mg·mL^-1下能明显抑制斑马鱼的自主运动,且自主运动抑制率呈现明显的浓度相关性;DAG在20,40,75 mg·mL^-1下对斑马鱼反应速度有较明显的抑制作用,反应能力下降率呈现浓度相关性;DAG各浓度组斑马鱼脑组织形态变小,但组织结构均未见显著异常;采用吖啶橙染色检测斑马鱼整体胚胎细胞凋亡情况,发现给药组头部绿色荧光比对照组明显,说明细胞凋亡增多,且细胞凋亡呈剂量依赖性增加,与表观一致;DAG在75,150,300,425,600 mg·L^-1下可导致斑马鱼幼鱼多巴胺能神经元相关基因DAT、TH、GCH1的mRNA相对表达量下调,Bax/Bcl-2的RNA相对表达量随着给药浓度增加而上升。结论 高浓度DAG对斑马鱼胚胎和幼鱼具有神经发育毒性作用,可能与多巴胺能神经元的抑制作用有关。     

补骨脂酚的提取纯化工艺优选及其对骨质疏松症的治疗作用分析

目的:优选补骨脂酚的提取纯化工艺,并考察其对骨质疏松症的治疗作用。方法:使用乙醇回流法提取,经D101型大孔树脂纯化,通过单因素试验优选工艺条件,冷冻干燥后得到补骨脂酚;采用斑马鱼体内试验评价补骨脂酚对骨密度和骨骼发育钙化的影响。结果:优选的工艺条件为加8倍量85%乙醇浸泡过夜,回流提取3次,每次2 h,边搅拌边往补骨脂酚粗提物中加入0.2%氢氧化钠水溶液至完全溶解,过预处理好的D101型大孔树脂柱处理,上样流速1 BV·h~(-1),静置4 h,加水6 BV洗脱,用60%乙醇2 BV洗脱,加95%乙醇4 BV洗脱,收集95%乙醇的洗脱液;制备的补骨脂酚质量分数80%,收率10%。斑马鱼体内活性研究表明补骨脂酚能提高斑马鱼骨质疏松症的骨密度,且能促进正常斑马鱼骨骼发育钙化。结论:此工艺具有绿色环保、操作简便等优点,所得补骨脂酚的纯度和收率均比较理想,可开发成预防或治疗骨质疏松症的药品和食品。     

Alu‐Alu mediated intragenic duplications in IFT81 and MATN3 are associated with skeletal dysplasias

Skeletal dysplasias are a diverse group of rare Mendelian disorders with clinical and genetic heterogeneity. Here, we used targeted copy number variant (CNV) screening and identified intragenic exonic duplications, formed through Alu‐Alu fusion events, in two individuals with skeletal dysplasia and negative exome sequencing results. First, we detected a homozygous tandem duplication of exon 9 and 10 in IFT81 in a boy with Jeune syndrome, or short‐rib thoracic dysplasia (SRTD) (MIM# 208500). Western blot analysis did not detect any wild‐type IFT81 protein in fibroblasts from the patient with the IFT81 duplication, but only a shorter isoform of IFT81 that was also present in the normal control samples. Complementary zebrafish studies suggested that loss of full‐length IFT81 protein but expression of a shorter form of IFT81 protein affects the phenotype while being compatible with life. Second, a de novo tandem duplication of exons 2 to 5 in MATN3 was identified in a girl with multiple epiphyseal dysplasia (MED) type 5 (MIM# 607078). Our data highlights the importance of detection and careful characterization of intragenic duplication CNVs, presenting them as a novel and very rare genetic mechanism in IFT81‐related Jeune syndrome and MATN3‐related MED.     

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre‐loxP‐controlled lentiviral vectors expressing CRIPTO in cell line‐derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft‐derived ex vivo tumour slices. CRIPTO‐overexpressing patient‐derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial‐to‐mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2‐CRIPTO cells formed tumours when injected into immune‐compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High‐level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO‐expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.