Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.     

PhotoMorphs™: A novel light‐activated reagent for controlling gene expression in zebrafish

Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly used antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light‐ activatable knockdown reagent called PhotoMorph?. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein, No tail, and E‐cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E‐cadherin knockdown. A splice‐blocking PhotoMorph directed to the rheb gene showed light‐dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish. genesis 47:736–743, 2009. © 2009 Wiley‐Liss, Inc.     

A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord

The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

Efficient Transfection Strategy for the Spatiotemporal Control of Gene Expression in Zebrafish

Functional analyses of gene function by knockdown and expression approaches strongly enhance the genetic study of development. In vivo application of the introduction of inhibitors of gene expression, mRNA, and expression constructs in the target region make it possible to perform region- and stage-specific regulation of gene function in a simple manner. As a basic tool for the conditional regulation of gene expression in target tissue, we present methods for the efficient introduction of antisense morpholino oligonucleotide (MO), mRNA, and expression plasmid constructs into early and later stage zebrafish embryo and larva. Lipofection of a neuron-specific expression construct plasmid encoding green fluorescent protein (GFP) into optic vesicle resulted in clear GFP expression in the retinotectal pathway in hatched larva. Co-lipofection of MO and GFP mRNA to the presumptive head region resulted in brain-specific knockdown of the gene in mid-stage embryos.     

VANGL1 rare variants associated with neural tube defects affect convergent extension in zebrafish

In humans, rare non-synonymous variants in the planar cell polarity gene VANGL1 are associated with neural tube defects (NTDs). These variants were hypothesized to be pathogenic based mainly on genetic studies in a large cohort of NTD patients. In this study, we validate the potential pathogenic effect of these mutations in vivo by investigating their effect on convergent extension in zebrafish. Knocking down the expression of tri, the ortholog of Vangl2, using an antisense morpholino (MO), as shown previously, led to a defective convergent extension (CE) manifested by a shortened body axis and widened somites. Co-injection of the human VANGL1 with the tri-MO was able to partially rescue the tri-MO induced phenotype in zebrafish. In contrast, co-injection of two human VANGL1 variants, p.Val239Ile and p.Met328Thr, failed to rescue this phenotype. We next carried out overexpression studies where we measured the ability of the human VANGL1 alleles to induce a CE phenotype when injected at high doses in zebrafish embryos. While overexpressing the wild-type allele led to a severely defective CE, overexpression of either p.Val239Ile or p.Met328Thr variant failed to do so. Results from both tri-MO knockdown/rescue results and overexpression assays suggest that these two variants most likely represent “loss-of-function” alleles that affect protein function during embryonic development. Our study demonstrates a high degree of functional conservation of VANGL genes across evolution and provides a model system for studying potential variants identified in human NTDs.     

Proper Activity of Histone H3 Lysine 4 (H3K4) Methyltransferase Is Required for Morphogenesis during Zebrafish Cardiogenesis

While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.     

Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish

The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish.     

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.     

Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.     

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.