Gal80 intersectional regulation of cell-type specific expression in vertebrates

Characterization and functional manipulation of specific groups of neurons in the vertebrate central nervous system (CNS) remains a major hurdle for understanding complex circuitry and functions. In zebrafish, the Gal4/UAS system has permitted expression of transgenes and enhancer trap screens, but is often limited by broad expression domains. We have developed a method for cell-type specific expression using Gal80 inhibition of Gal4-dependent expression. We show that native Gal4 is able to drive strong expression, that Gal80 can inhibit this expression, and that overlapping Gal4 and Gal80 expression can achieve "intersectional" expression in spatially and genetically defined subsets of neurons. We also optimize Gal80 for expression in vertebrates, track Gal80 expression with a co-expressed fluorescent marker, and use a temperature-sensitive allele of Gal80 to temporally regulate its function. These data demonstrate that Gal80 is a powerful addition to the genetic techniques available to map and manipulate neural circuits in zebrafish.     

Single‐cell analysis of somatotopic map formation in the zebrafish lateral line system

The zebrafish lateral line is a simple sensory system comprising a small number of neurons in addition to their sensory organs, the neuromasts. We have adopted this system as a model for single‐cell level analyses of topographic map formation and examined when and how the lateral line topographic map is established. Single‐neuron labeling demonstrated that somatotopic organization of the ganglion emerges by 54 hr postfertilization, but also that this initial map is not as accurate as that observed at 6 days postfertilization. During this initial stage, individual neurons exhibit extensively diverse behavior and morphologies. We identified leader neurons, the axons of which are the first to reach the tail, and later‐appearing axons that contribute to the initial map. Our data suggest that lateral line neurons are heterogeneous from the beginning of lateral line development, and that some of them are intrinsically fate determined to contribute to the somatotopic map. Developmental Dynamics 239:2058–2065, 2010. © 2010 Wiley‐Liss, Inc.     

长春碱增加班马鱼肿瘤耐药基因abcb4的表达

目的通过研究长春碱(VBL)对斑马鱼早期发育过程中abcb4肿瘤耐药基因表达的影响,为进一步建立抗肿瘤药物多药耐药筛选的斑马鱼模型奠定重要的实验基础。方法将长春碱稀释成不同浓度梯度的药液,在受精后0.5~1.5 h的斑马鱼胚胎上完成药物暴露实验,计算出长春碱IC_(50)的数值。设置长春碱为实验组,胚胎培养液为空白对照组,分别作用于受精后0.5~1.5 h的斑马鱼胚胎,观察受精后24~120 h的斑马鱼胚胎发育情况,记录死亡、孵化及畸形的胚胎数目;收集不同组别的斑马鱼胚胎,通过实时荧光定量PCR和胚胎整体原位杂交的方法,检测斑马鱼胚胎中abcb4基因的表达量。结果计算出长春碱在斑马鱼胚胎中的IC_(50)为3.08μmol/L;与对照组比较,实验组的斑马鱼胚胎abcb4基因的mRNA水平明显升高(P0.05);通过胚胎整体原位杂交的方法,在受精后120 h斑马鱼胚胎的小肠部位发现有abcb4基因阳性杂交信号,且实验组斑马鱼胚胎在脑和心脏部位发现abcb4基因阳性杂交信号。结论长春碱能明显增加斑马鱼早期胚胎中abcb4肿瘤耐药基因的表达水平,提示使用斑马鱼可以复制肿瘤耐药模型。     

CTLA‐4‐expression on VZV‐specific T cells in CSF and blood is specifically increased in patients with VZV related central nervous system infections

VZV‐reactivation may lead to symptomatic central nervous system (CNS) diseases, but identification of VZV as causative pathogen of CNS‐diseases is challenging. This study was performed to characterize VZV‐specific T cells from cerebrospinal fluid (CSF) and blood of patients with active CNS‐disease and to determine whether this may improve differential diagnosis. 27 patients with pleocytosis in the CSF were recruited and classified into three groups (10 VZV‐related, 10 non‐VZV‐related, 7 unclear). VZV‐specific CD4⁺ T cells were quantified in CSF and blood after simultaneous stimulation with a VZV‐antigen lysate and detection of cytokines (IFN‐γ, IL‐2, TNF‐α) and CTLA‐4. Polyclonal stimulation served as positive control. VZV‐specific CD4⁺ T‐cell frequencies were highest in both CSF (p = 0.0001) and blood (p = 0.011) of patients with VZV‐infection, and were enriched at the site of infection (p = 0.002). While cytokine‐expression profiles only showed minor differences between the groups, CTLA‐4‐expression levels on VZV‐specific T cells from CSF and blood were significantly increased in VZV‐related CNS‐infections (p = 0.0002 and p<0.0001) and clearly identified VZV‐related CNS‐diseases (100% sensitivity and 100% specificity). Polyclonally stimulated T cells did not show any quantitative and phenotypical differences between the groups. Increased frequency and CTLA‐4‐expression of VZV‐specific T cells from CSF or blood are specifically found in patients with VZV‐related CNS‐infection.     

High‐resolution MRI of early‐stage mouse embryos

Both the availability of methods to manipulate genes and the completion of the mouse genome sequence have led to the generation of thousands of genetically modified mouse lines that provide a new platform for the study of mammalian development and developmental diseases. Phenotyping of mouse embryos has traditionally been performed on fixed embryos by the use of ex vivo histological, optical and high‐resolution MRI techniques. Although potentially powerful, longitudinal imaging of individual animals is difficult or impossible with conventional optical methods because of the inaccessibility of mouse embryos inside the maternal uterus. To address this problem, we present a method of imaging the mouse embryo from stages as early as embryonic day (E)10.5, close to the onset of organogenesis in most physiological systems. This method uses a self‐gated MRI protocol, combined with image registration, to obtain whole‐embryo high‐resolution (100 µm isotropic) three‐dimensional images. Using this approach, we demonstrate high contrast in the cerebral vasculature, limbs, spine and central nervous system without the use of contrast agents. These results indicate the potential of MRI for the longitudinal imaging of developing mouse embryos in utero and for future applications in analyzing mutant mouse phenotypes. Copyright © 2012 John Wiley & Sons, Ltd.     

Differential expression patterns and developmental roles of duplicated scinderin‐like genes in zebrafish

Scinderin, the closest homologue of the actin‐severing protein, gelsolin, has two similar paralogs (Scinla and Scinlb) in zebrafish. Scinla is abundant in the adult cornea; Scinlb comprises considerably less corneal protein. Here, we show that scinla is expressed in the nose, lens, brain, cornea and annular ligament of the iridocorneal angle; by contrast, scinlb is expressed in the hatching gland, floor plate, notochord, otic vesicle, brain, pharynx, cartilage, swim bladder and cornea. Activity of scinla and scinlb promoter fragments driving the EGFP reporter gene in transgenic zebrafish resembled scinla or scinlb expression. Previously, we showed that reduction of scinla by injection of antisense morpholino oligonucleotides ventralized embryos; here, specific reduction of scinlb expression led to subtle brain abnormalities associated with increased cell death, decreased shhb expression in the floor plate, and slightly reduced eye distance. Thus, scinla and scinlb have different expression patterns and developmental roles during zebrafish development. Developmental Dynamics 238:2633–2640, 2009. Published 2009 Wiley‐Liss, Inc.     

中枢神经系统生殖细胞肿瘤中c-kit蛋白表达的临床病理学意义

目的 探讨c-kit(CD117)在原发性中枢神经系统(CNS)生殖细胞肿瘤(GCTs)中的表达及其临床病理学意义.方法 应用免疫组化EnVision法检测21例原发性CNS GCTs c-kit和PLAP的表达.结果 生殖细胞瘤100%表达c-kit,87.5%表达PLAP,c-kit和PLAP在畸胎瘤中均不表达.c-kit和PLAP在松果体母细胞瘤、垂体腺瘤、中枢神经细胞瘤、淋巴瘤、低级别星形细胞瘤、朗格汉斯组织细胞增生症和髓母细胞瘤中不表达.结论 c-kit和PLAP是原始生殖细胞较特异性标记之一,c-kit和PLAP高表达支持CNS GCTs起源于原始生殖细胞.c-kit在生殖细胞瘤中表达较PLAP更加敏感,是优于PLAP的诊断标记,可用于生殖细胞肿瘤诊断和鉴别诊断.同时也为药物靶向治疗生殖细胞瘤提供理论依据.     

Screening for ALK abnormalities in central nervous system metastases of non‐small‐cell lung cancer

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%–7% of primary non‐small‐cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non‐smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ² = 16.783) in former‐smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.     

The B7–CD28/CTLA-4 costimulatory pathways in autoimmune disease of the central nervous system

The past year has seen significant advances in our understanding of the role of the B7–CD28/CTLA-4 pathway in regulating the responses of self-reactive T cells, giving impetus to manipulation of this pathway for treating human autoimmune diseases. Recent studies have demonstrated that B7–CD28 costimulation has critical roles in stimulating both the initiation and effector phases of autoimmunity and that CD28 regulates the threshold for activation of self-reactive T cells. Recent work has also revealed critical roles for CTLA-4 in limiting the extent of Th1/Th2 cell differentiation and in downregulating the responses of self-reactive T cells during both the initiation and progression of autoimmune disease.     

Six cadm/SynCAM genes are expressed in the nervous system of developing zebrafish.

The Cadm (cell adhesion molecule) family of cell adhesion molecules (also known as IGSF4, SynCAM, Necl and TSLC) has been implicated in a multitude of physiological and pathological processes, such as spermatogenesis, synapse formation and lung cancer. The precise mechanisms by which these adhesion molecules mediate these diverse functions remain unknown. To investigate mechanisms of action of these molecules during development, we have identified zebrafish orthologs of Cadm family members and have examined their expression patterns during development and in the adult. Zebrafish possess six cadm genes. Sequence comparisons and phylogenetic analysis suggest that four of the zebrafish cadm genes represent duplicates of two tetrapod Cadm genes, whereas the other two cadm genes are single orthologs of tetrapod Cadm genes. All six zebrafish cadms are expressed throughout the nervous system both during development and in the adult. The spatial and temporal patterns of expression suggest multiple roles for Cadms during nervous system development.     

Calretinin in the peripheral nervous system of the adult zebrafish

Calretinin is a calcium-binding protein found widely distributed in the central nervous system and chemosensory cells of the teleosts, but its presence in the peripheral nervous system of fishes is unknown. In this study we used Western blot analysis and immunohistochemistry to investigate the occurrence and distribution of calretinin in the cranial nerve ganglia, dorsal root ganglia, sympathetic ganglia, and enteric nervous system of the adult zebrafish. By Western blotting a unique and specific protein band with an estimated molecular weight of around 30 kDa was detected, and it was identified as calretinin. Immunohistochemistry revealed that calretinin is selectively present in the cytoplasm of the neurons and never in the satellite glial cells. In both sensory and sympathetic ganglia the density of neurons that were immunolabelled, their size and morphology, as well as the intensity of immunostaining developed within the cytoplasm, were heterogeneous. In the enteric nervous system calretinin immunoreactivity was detected in a subset of enteric neurons as well as in a nerve fibre plexus localized inside the muscular layers. The present results demonstrate that in addition to the central nervous system, calretinin is also present in the peripheral nervous system of zebrafish, and contribute to completing the map of the distribution of this protein in the nervous system of teleosts.     

Dynamic miRNA expression patterns during retinal regeneration in zebrafish: Reduced dicer or miRNA expression suppresses proliferation of Müller Glia‐derived neuronal progenitor cells

Background: Adult zebrafish spontaneously regenerate their retinas after damage. Although a number of genes and signaling pathways involved in regeneration have been identified, the exact mechanisms regulating various aspects of regeneration are unclear. microRNAs (miRNAs) were examined for their potential roles in regulating zebrafish retinal regeneration. Results: To investigate the requirement of miRNAs during zebrafish retinal regeneration, we knocked down the expression of Dicer in retinas prior to light‐induced damage. Reduced Dicer expression significantly decreased the number of proliferating Müller glia‐derived neuronal progenitor cells during regeneration. To identify individual miRNAs with roles in neuronal progenitor cell proliferation, we collected retinas at different stages of light damage and performed small RNA high‐throughput sequencing. We identified subsets of miRNAs that were differentially expressed during active regeneration but returned to basal levels once regeneration was completed. We then knocked down five different miRNAs that increased in expression and assessed the effects on retinal regeneration. Reduction of miR‐142b and miR‐146a expression significantly reduced INL proliferation at 51h of light treatment, while knockdown of miR‐7a, miR‐27c, and miR‐31 expression significantly reduced INL proliferation at 72h of constant light. Conclusions: miRNAs exhibit dynamic expression profiles during retinal regeneration and are necessary for neuronal progenitor cell proliferation. Developmental Dynamics 243:1591–1605, 2014. © 2014 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists     

MYC protein expression is associated with poor prognosis in primary diffuse large B‐cell lymphoma of the central nervous system

MYC and BCL2 gene translocations and protein expression have recently demonstrated to be of prognostic significance in systemic diffuse large B‐cell lymphoma (DLBCL). However, their role in primary central nervous system DLBCL (CNS‐DLBCL) prognosis has been scarcely analyzed. We studied the immunophenotype, the status of the MYC, BCL2, and BCL6 genes and the clinical features of a series of 42 CNS‐DLBCL and evaluated their prognostic significance. We found high MYC protein expression in 43% of cases, and this was associated with lower overall survival (OS). Cases with concurrent expression of MYC and BCL2 showed a lower OS, although the difference did not reach statistical significance. Translocations involving the MYC or BCL2 genes were not detected. The BCL6 gene was frequently translocated, but was unrelated to survival. We conclude that MYC protein expression detected by immunohistochemistry identifies a CNS‐DLBCL subset with worse prognosis and may contribute to a more accurate risk stratification of CNS‐DLBCL patients.     

Four twist genes in zebrafish, four expression patterns.

Twist genes code for regulatory bHLH proteins essential for embryonic development and conserved across the metazoa. There are four genes that constitute the zebrafish twist family: twist1a, twist1b, twist2--orthologs of the mammalian twist1 and twist2 genes; and twist3--a gene from a new clade that does not exist in mammals. Presented here are their embryonic mRNA expression profiles. The study extends the known conservation of twist developmental patterns in tetrapods to the fish, e.g., expression in cephalic neural crest, sclerotome and lateral plate mesoderm. Some other expression domains are unique, like hypochord and dorsal aorta; some, like the notochord, may be ancestral patterns retained from protochordates; and the expression in invaginating/migrating cells may have been retained from the jellyfish. Perhaps this is one of the more ancient functions of twist--conserved from diploblasts to humans--to facilitate cell movement.     

Automated image‐based phenotypic analysis in zebrafish embryos

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high‐throughput chemical screens, and optical transparency makes them potentially suited for image‐based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to using the zebrafish as a high‐throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi‐well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high‐content reader and analyzed using an artificial intelligence‐based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)^y1) arrayed in 96‐well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image‐based high‐content screening methodology to measure complex whole organism phenotypes. Developmental Dynamics 238:656–663, 2009. © 2009 Wiley‐Liss, Inc.     

Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease‐activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3‐day‐old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.