Journal of Genetics and Genomics

The Tiny Zebrafish Keep Swimming Fast in the Developmental Biology Pond

Jinrong Peng, Jian Zhang, Anming Meng

2012, 39(9): 419-420. doi: 10.1016/j.jgg.2012.08.001

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Reverse Genetic Approaches in Zebrafish

Peng Huang, Zuoyan Zhu, Shuo Lin, Bo Zhang

2012, 39(9): 421-433. doi: 10.1016/j.jgg.2012.07.004

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Zebrafish (Danio rerio) is a well-established vertebrate animal model. A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism. Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally. For a long time, targeted genome modification has been heavily relied on large-scale traditional forward genetic screens, such as ENU (N-ethyl-N-nitrosourea) mutagenesis derived TILLING (Targeting Induced Local Lesions IN Genomes) strategy and pseudo-typed retrovirus mediated insertional mutagenesis. Recently, engineered endonucleases, including ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes. Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish, including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting. Future directions and expectations will also be discussed.

Myelopoiesis during Zebrafish Early Development

Jin Xu, Linsen Du, Zilong Wen

2012, 39(9): 435-442. doi: 10.1016/j.jgg.2012.06.005

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Myelopoiesis is the process of producing all types of myeloid cells including monocytes/macrophages and granulocytes. Myeloid cells are known to manifest a wide spectrum of activities such as immune surveillance and tissue remodeling. Irregularities in myeloid cell development and their function are known to associate with the onset and the progression of a variety of human disorders such as leukemia. In the past decades, extensive studies have been carried out in various model organisms to elucidate the molecular mechanisms underlying myelopoiesis with the hope that these efforts will yield knowledge translatable into therapies for related diseases. Zebrafish has recently emerged as a prominent animal model for studying myelopoiesis, especially during early embryogenesis, largely owing to its unique properties such as transparent embryonic body and external development. This review introduces the methodologies used in zebrafish research and focuses on the recent research progresses of zebrafish myelopoiesis.

Overlapping Cardiac Programs in Heart Development and Regeneration

Yi-Song Zhen, Qing Wu, Cheng-Lu Xiao, Nan-Nan Chang, Xu Wang, Lei Lei, Xiaojun Zhu, Jing-Wei Xiong

2012, 39(9): 443-449. doi: 10.1016/j.jgg.2012.07.005

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Gaining cellular and molecular insights into heart development and regeneration will likely provide new therapeutic targets and opportunities for cardiac regenerative medicine, one of the most urgent clinical needs for heart failure. Here we present a review on zebrafish heart development and regeneration, with a particular focus on early cardiac progenitor development and their contribution to building embryonic heart, as well as cellular and molecular programs in adult zebrafish heart regeneration. We attempt to emphasize that the signaling pathways shaping cardiac progenitors in heart development may also be redeployed during the progress of adult heart regeneration. A brief perspective highlights several important and promising research areas in this exciting field.

Ribosome Biogenesis Factor Bms1-like Is Essential for Liver Development in Zebrafish

Yong Wang, Yue Luo, Yunhan Hong, Jinrong Peng, Lijan Lo

2012, 39(9): 451-462. doi: 10.1016/j.jgg.2012.07.007

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Ribosome biogenesis in the nucleolus requires numerous nucleolar proteins and small non-coding RNAs. Among them is ribosome biogenesis factor Bms1, which is highly conserved from yeast to human. In yeast, Bms1 initiates ribosome biogenesis through recruiting Rcl1 to pre-ribosomes. However, little is known about the biological function of Bms1 in vertebrates. Here we report that Bms1 plays an essential role in zebrafish liver development. We identified a zebrafishbms1l mutant which carries a T to A mutation in the gene bms1-like (bms1l). This mutation results in L¹⁵² to Q¹⁵² substitution in a GTPase motif in Bms1l. Surprisingly, bms1l mutation confers hypoplasia specifically in the liver, exocrine pancreas and intestine after 3 days post-fertilization (dpf). Consistent with the bms1l mutant phenotypes, whole-mount in situ hybridization (WISH) on wild type embryos showed that bms1l transcripts are abundant in the entire digestive tract and its accessory organs. Immunostaining for phospho-Histone 3 (P-H3) and TUNEL assay revealed that impairment of hepatoblast proliferation rather than cell apoptosis is one of the consequences of bms1l giving rise to an under-developed liver. Therefore, our findings demonstrate that Bms1l is necessary for zebrafish liver development.

Kzp Regulates the Transcription of gata2 and pu.1 during Primitive Hematopoiesis in Zebrafish Embryos

Fang Liu, Shaohua Yao, Ting Zhang, Chun Xiao, Yanna Shang, Jin Liu, Xianming Mo

2012, 39(9): 463-471. doi: 10.1016/j.jgg.2012.08.003

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Kaiso zinc finger-containing protein (Kzp), a maternally-derived transcription factor, controls dorsoventral patterning during zebrafish gastrulation. Here, we uncovered a new function for Kzp in zebrafish embryonic primitive hematopoiesis. The depletion of kzp led to defects in primitive hematopoiesis including the development of the erythroid and myeloid lineages. On the other hand, overexpression ofkzp caused the ectopic expression of gata1, gata2, and pu.1. Chromosome immunoprecipitation assays revealed that Kzp protein directly binds to gata1, gata2, and pu.1 promoters. Interestingly, the ectopic expression of gata2 was able to rescue the erythroid, but not the myeloid lineage in kzp-depleted zebrafish embryos. gata1 expression controlled by Kzp was dependent on gata2 during primitive erythropoiesis. Our results indicate that Kzp is a critical transcriptional factor for the expression of gata2 and pu.1 to modulate primitive hematopoiesis.

Large-Scale Forward Genetic Screening Analysis of Development of Hematopoiesis in Zebrafish

Kun Wang, Zhibin Huang, Lingfeng Zhao, Wei Liu, Xiaohui Chen, Ping Meng, Qing Lin, Yali Chi, Mengchang Xu, Ning Ma, Yiyue Zhang, Wenqing Zhang

2012, 39(9): 473-480. doi: 10.1016/j.jgg.2012.07.008

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Zebrafish is a powerful model for the investigation of hematopoiesis. In order to isolate novel mutants with hematopoietic defects, large-scale mutagenesis screening of zebrafish was performed. By scoring specific hematopoietic markers, 52 mutants were identified and then classified into four types based on specific phenotypic traits. Each mutant represented a putative mutation of a gene regulating the relevant aspect of hematopoiesis, including early macrophage development, early granulopoiesis, embryonic myelopoiesis, and definitive erythropoiesis/lymphopoiesis. Our method should be applicable for other types of genetic screening in zebrafish. In addition, further study of the mutants we identified may help to unveil the molecular basis of hematopoiesis.

A Surgery Protocol for Adult Zebrafish Spinal Cord Injury

Ping Fang, Jin-Fei Lin, Hong-Chao Pan, Yan-Qin Shen, Melitta Schachner

2012, 39(9): 481-487. doi: 10.1016/j.jgg.2012.07.010

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Adult zebrafish has a remarkable capability to recover from spinal cord injury, providing an excellent model for studying neuroregeneration. Here we list equipment and reagents, and give a detailed protocol for complete transection of the adult zebrafish spinal cord. In this protocol, potential problems and their solutions are described so that the zebrafish spinal cord injury model can be more easily and reproducibly performed. In addition, two assessments are introduced to monitor the success of the surgery and functional recovery: one test to assess free swimming capability and the other test to assess extent of neuroregeneration by in vivo anterograde axonal tracing. In the swimming behavior test, successful complete spinal cord transection is monitored by the inability of zebrafish to swim freely for 1 week after spinal cord injury, followed by the gradual reacquisition of full locomotor ability within 6 weeks after injury. As a morphometric correlate, anterograde axonal tracing allows the investigator to monitor the ability of regenerated axons to cross the lesion site and increasingly extend into the gray and white matter with time after injury, confirming functional recovery. This zebrafish model provides a paradigm for recovery from spinal cord injury, enabling the identification of pathways and components of neuroregeneration.

Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

Jingang Liu, Lu Gong, Changqing Chang, Cong Liu, Jinrong Peng, Jun Chen

2012, 39(9): 489-502. doi: 10.1016/j.jgg.2012.07.009

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The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

Guili Song, Qing Li, Yong Long, Perry B. Hackett, Zongbin Cui

2012, 39(9): 503-520. doi: 10.1016/j.jgg.2012.05.010

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Expression-independent gene or polyadenylation [poly(A)] trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed. Although a number of poly(A)-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome, further efforts are needed to avoid the 3′-terminal insertion bias and the splice donor (SD) read-through, and to improve the mutagenicity. Here, we present aSleeping Beauty (SB) transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding, gene-breaking and large-scale mutagenesis, respectively. The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element (ARE), which greatly reduced the SD read-through events, except that the internal ribosomal entry site (IRES) element was introduced in front of the SD signal to overcome the phenomenon of 3′-bias gene trapping. The breaking cassette consisting of an enhanced splicing acceptor (SA), a poly(A) signal coupled with a transcriptional terminator (TT) effectively disrupted the transcription of trapped genes. Moreover, theHsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11, which allows the conditional remobilization of a trap insert from a non-coding region. The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells. In addition, the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus, this poly(A)-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.

Establishment of Multi-Site Infection Model in Zebrafish Larvae for Studying Staphylococcus aureus Infectious Disease

Ya-juan Li, Bing Hu

2012, 39(9): 521-534. doi: 10.1016/j.jgg.2012.07.006

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Zebrafish (Danio rerio) is an ideal model for studying the mechanism of infectious disease and the interaction between host and pathogen. As a teleost, zebrafish has developed a complete immune system which is similar to mammals. Moreover, the easy acquirement of large amounts of transparent embryos makes it a good candidate for gene manipulation and drug screening. In a zebrafish infection model, all of the site, timing, and dose of the bacteria microinjection into the embryo are important factors that determine the bacterial infection of host. Here, we established a multi-site infection model in zebrafish larvae of 36 hours post-fertilization (hpf) by microinjecting wild-type or GFP-expressing Staphylococcus aereus (S. aureus) with gradient burdens into different embryo sites including the pericardial cavity (PC), eye, the fourth hindbrain ventricle (4V), yolk circulation valley (YCV), caudal vein (CV), yolk body (YB), and Duct of Cuvier (DC) to resemble human infectious disease. With the combination of GFP-expressing S. aureus and transgenic zebrafish Tg (coro1a: eGFP; lyz: Dsred) and Tg (lyz: Dsred) lines whose macrophages or neutrophils are fluorescent labeled, we observed the dynamic process of bacterial infection by in vivo multicolored confocal fluorescence imaging. Analyses of zebrafish embryo survival, bacterial proliferation and myeloid cells phagocytosis show that the site- and dose-dependent differences exist in infection of different bacterial entry routes. This work provides a consideration for the future study of pathogenesis and host resistance through selection of multi-site infection model. More interaction mechanisms between pathogenic bacteria virulence factors and the immune responses of zebrafish could be determined through zebrafish multi-site infection model.

2012 Vol. 39, No. 9