Zebrafish Jak2a plays a crucial role in definitive hematopoiesis and blood vessel formation

We examined the role of zebrafish (Danio rerio) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24 hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2^−/− transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36 hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72 hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.     

Plakoglobin has both structural and signalling roles in zebrafish development

Plakoglobin, or gamma-catenin, is found in both desmosomes and adherens junctions and participates in Wnt signalling. Mutations in the human gene are implicated in the congenital heart disorder, arrhythmogenic right ventricular cardiomyopathy (ARVC), but the signalling effects of plakoglobin loss in ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in decreased heart size, reduced heartbeat, cardiac oedema, reflux of blood between heart chambers and a twisted tail. Wholemount in situ hybridisation shows reduced expression of the heart markers nkx2.5 at 24 hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve markers notch1b and bmp4 at 48 hpf. Wnt target gene expression was examined by semi-quantitative RT-PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin morphant. β-catenin protein expression is increased in morphant embryos as is its colocalisation with E-cadherin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant hearts have an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers of desmosomes and adherens junctions in the intercalated discs. These results establish the zebrafish as a model for ARVC caused by loss of plakoglobin function and indicate that there are signalling as well as structural consequences of this loss.     

氯丙嗪在斑马鱼胚胎和早期幼鱼发育过程中的神经毒性作用

目的 采用模式动物斑马鱼作为研究对象,观察氯丙嗪（chlorpromazine,CPZ）暴露对胚胎和幼鱼早期神经发育的影响.方法 在一般毒性评价的基础上,通过整体胚胎细胞凋亡检测和脑组织病理学检查,了解CPZ对神经发育的器质性改变;采用神经行为学方法,包括幼鱼触动逃避反应、自发运动以及惊恐逃避反射等,研究氯丙嗪暴露所致的神经发育功能性障碍.结果斑马鱼胚胎受精后6 h（6 hpf）～72 hpf暴露于CPZ（≥5 mg/L）可引起胚胎和幼鱼死亡、致畸和幼鱼孵化延迟,并呈浓度和时间依赖性;采用吖啶橙染色检测36 hpf整体胚胎凋亡细胞,发现凋亡细胞主要集中在胚胎中脑、后脑、丘脑以及中后脑连接区、脊索和尾部等处;脑组织病理学检测发现,7dpf幼鱼颅腔增大、脑体积减小、脑细胞缩小且细胞间隙增宽.6～72 hpf CPZ（≥0.0625 mg/L）暴露后,幼鱼神经行为学研究发现,CPZ（≥0.125 mg/L）可引起3dpf幼鱼触觉运动能力下降;CPZ（≥0 5 mg/L）可浓度依赖性地抑制幼鱼自发运动,并出现僵直不动、震颤或快速刻板式转圈运动等行为改变;光惊恐实验中,暗环境下各暴露组幼鱼对突发强光刺激均表现为惊跳逃避,并且暗-光交替期运动加速度变化与对照组无显著差异;在撤除光源后,1mg/L和2 mg/L暴露组幼鱼暗适应时程缩短,而0.125 mg/L和0.25 mg/L暴露组暗适应时程延长,提示CPZ对外界刺激引发的幼鱼活跃游动有抑制和促进双重毒性作用.结论 CPZ暴露对斑马鱼胚胎和幼鱼具有明显的神经发育毒性作用.模式动物斑马鱼作为一种高通量筛选模型在外源性化合物神经发育毒性评价中具有较好的应用前景.     

叶酸缺乏对斑马鱼背主动脉发育的影响

目的观察叶酸缺乏斑马鱼胚胎的背主动脉（DA）发育情况,初步探讨叶酸缺乏后胚胎DA发育异常的机理。方法采用将二氢叶酸还原酶（DHFR）功能阻断的方法构建叶酸缺乏斑马鱼模型,分别应用DHFR抑制剂甲氨蝶呤（MTX）以及DHFR基因knock-down技术处理斑马鱼胚胎。在胚胎发育至48 hpf时在显微镜下观察胚胎的整体发育情况,在60 hpf时应用荧光显微造影的方法观察胚胎的背主动脉发育状况。利用胚胎整体原位杂交和real-time PCR的方法检测影响DA发育的关键因子ephrinB2、Ang-1和Radar的表达情况,利用TUNEL法检测胚胎底索的凋亡情况。结果MTX处理组胚胎以及DHFR knock-down组胚胎有相似的胚胎发育异常表型。荧光显微造影显示叶酸缺乏组胚胎的DA发育异常。叶酸缺乏组胚胎的ephrinB2、Ang-1和Radar表达减弱,底索凋亡增加。结论叶酸缺乏可导致斑马鱼胚胎背主动脉发育异常,其机理与ephrinB2、Ang-1和Radar的表达减弱以及底索凋亡增加有关。     

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.     

Temporal and spatial expression of the four Igf ligands and two Igf type 1 receptors in zebrafish during early embryonic development

The insulin-like growth factor (Igf) family is an evolutionarily conserved system essential for normal growth and development in vertebrates. Unlike mammals, four distinct Igf ligands (Igf1, Igf2a, Igf2b and Igf3) and two Igf type 1 receptors (Igf1ra and Igf1rb) are present in zebrafish. However, the localization of these multiple ligands and receptors especially the recently discovered igf3 during early development of zebrafish is poorly understood. In this study, detailed expression patterns of these components of the Igf system during embryogenesis of zebrafish were analyzed. It was found that igf1 is specifically expressed in the trigeminal ganglia region from 18 hpf to 72 hpf, while igf2a is restricted to the caudal regions of the notochord from 14 hpf to 18 hpf as well as in the midbrain, dorsal hind brain and otic vesicle at 24 hpf. On the other hand, igf2a is highly expressed in the midbrain and pharyngeal arch region at 48 hpf, followed by its appearance in the liver and brain at 72 hpf, while igf2b is restricted to the floor plate and hypochord from 12 hpf to 18 hpf, and strong expression is also detected in the midbrain and dorsal hind brain at 24 hpf. The teleost specific igf3 is highly expressed in the pharyngeal arch region before 24 hpf, but is then restricted to the sternohyoideus after 48 hpf. The receptor subtype igf1ra is ubiquitously expressed before 24 hpf but is confined to the brain at 72 hpf. However, igf1rb is widely expressed before 10 hpf, but is more confined to the brain region at 24 hpf and 72 hpf. This dynamic temporal-spatial expression during embryogenesis of zebrafish, together with the unique and overlapping expression patterns of the Igf ligands and receptors suggest the coordination of the divergent functions of the Igf system during early development in zebrafish.     

Functional differentiation of bmp2a and bmp2b genes in zebrafish

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b^−/− embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a^−/− zebrafish. The results showed that the expression of runx2b in bmp2a^−/− was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.     

微小染色体维系蛋白3基因在斑马鱼早期胚胎发育过程中的表达

目的:制备地高辛标记的微小染色体维系蛋白3(MCM3)基因的RNA探针,研究MCM3在斑马鱼早期发育中的时空表达。方法:收集并固定受精后24 h时期的野生型斑马鱼胚胎,提取总RNA,制备DIG标记的MCM3 RNA反义探针,整胚原位杂交,研究MCM3在斑马鱼胚胎早期发育过程的表达。结果:斑马鱼的MCM3氨基酸序列与小鼠、人具有高度同源性,通过不同时期胚胎的原位杂交,发现MCM3在早期发育过程中普遍性表达,胚胎受精后0~2 hMCM3在增殖性区域泛表达,受精后14~22 h在中枢神经系统、发育未成熟的眼部、体节及增殖性区域表达,受精后24 h在血液、中枢神经、翼板中脑、视觉盖及增殖性区域表达,受精后48 h在头部及肛门增殖性区域表达。结论:明确了MCM3在斑马鱼胚胎发育过程中的表达模式,证明其与早期斑马鱼发育细胞增殖密切相关,为研究该基因功能提供了一定的理论基础。