Haigen Huang

Stimulation of erythropoiesis by inhibiting a new hematopoietic death receptor in transgenic zebrafish

Stimulation of erythropoiesis by inhibiting a new hematopoietic death receptor in transgenic zebrafish

Cloned zebrafish by nuclear transfer from long-term-cultured cells

Although mammals have been cloned from genetically manipulated cultured cells, a comparable achievement has not been realized in lower vertebrates. Here we report that fertile transgenic zebrafish can be obtained by nuclear transfer using embryonic fibroblast cells from long-term cultures. The donor nuclei, modified by retroviral insertions expressing green fluorescent protein (GFP), were transplanted into manually enucleated eggs. Overall, a 2% success rate was achieved, resulting in 11 adult transgenic zebrafish expressing GFP. These nuclear transplants produced fertile, diploid offspring, and their F1/F2 progeny continued to express GFP in a pattern identical to that of the founder fish. This finding demonstrates that slowly dividing nuclei from cultured cells can be reprogrammed to support rapid embryonic development and sets up a foundation for targeted genetic manipulation in zebrafish.

Analysis of pancreatic development in living transgenic zebrafish embryos.

Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, germline transgenic zebrafish expressing the green fluorescent protein (GFP) reporter gene in the pancreas were generated. For both constructs, the GFP expression patterns in transgenic embryos were consistent with the mRNA expression patterns detected by RNA in situ hybridization. A deletion promoter analysis revealed that positive and negative cis-acting elements were involved in regulation of insulin gene expression. Three-dimensional reconstructions imaged from living embryos using two-photon laser-scanning microscopy (TPLSM) demonstrated that the zebrafish pancreas is formed from a single dorsal pancreatic cell mass. This is in contrast to mammals where the pancreas derives from both dorsal and ventral anlage. These transgenic fish should be useful for in vivo studies of factors involved in specifying and regulating pancreatic development and function.

A Large-Scale Zebrafish Gene Knockout Resource for the Genome-Wide Study of Gene Function

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable “base editing” system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5′-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etv2, a master regulator of endothelial cell fate, can induce fast skeletal muscle cells to transdifferentiate into endothelial cells in the zebrafish embryo.

NXT2 is required for embryonic heart development in zebrafish

BackgroundNXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes.ResultsA zebrafish mutant exhibiting specific heart defects during embryogenesis was generated by animal cloning-mediated retroviral insertions. Molecular analysis indicated that the mutant phenotype was caused by a disruption of NXT2. Whole-mount RNA in situ hybridization showed that NXT2 transcripts were clearly detectable in embryonic heart as well as other tissues. Further analysis revealed that expression level of one form of alternative splicing NXT2 mRNA transcripts was significantly reduced, resulting in deficient myocardial cell differentiation and the malformation of cardiac valve at the atrioventricular boundary. The defects could be reproduced by morpholino anti-sense oligo knockdown of NXT2.ConclusionNXT2 has a critical role in maintaining morphogenetic integrity of embryonic heart in vertebrate species.

The Zebrafish Insertion Collection (ZInC): a web based, searchable collection of zebrafish mutations generated by DNA insertion

ZInC (Zebrafish Insertional Collection, http://research.nhgri.nih.gov/ZInC/) is a web-searchable interface of insertional mutants in zebrafish. Over the last two decades, the zebrafish has become a popular model organism for studying vertebrate development as well as for modeling human diseases. To facilitate such studies, we are generating a genome-wide knockout resource that targets every zebrafish protein-coding gene. All mutant fish are freely available to the scientific community through the Zebrafish International Resource Center (ZIRC). To assist researchers in finding mutant and insertion information, we developed a comprehensive database with a web front-end, the ZInC. It can be queried using multiple types of input such as ZFIN (Zebrafish Information Network) IDs, UniGene accession numbers and gene symbols from zebrafish, human and mouse. In the future, ZInC may include data from other insertional mutation projects as well. ZInC cross-references all integration data with the ZFIN (http://zfin.org/).

High-Throughput Screening for Bioactive Molecules Using Primary Cell Culture of Transgenic Zebrafish Embryos

Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

Vertebrate MAX-1 is required for vascular patterning in zebrafish

During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tissues. However, key molecules involved in this process still remain largely elusive. Here, we report molecular cloning, expression, and functional studies of zebrafish max-1, a homolog of Caenorhabditis elegans max-1 that has been implicated in motor neuron axon guidance. During early embryonic development, zebrafish max-1 is specifically expressed in subsets of neuronal tissues, epithelial cells, and developing somites through which vascular endothelial cells migrate from large ventral axial vessels to form stereotypic intersegmental blood vessels (ISV). Blocking zebrafish max-1 mRNA splicing by morpholino injection led to aberrant ISV patterning, which could be rescued by injection of either C. elegans or zebrafish max-1 mRNA. Analysis of motor neurons in the same region showed normal neuronal axon pathfinding. Further studies suggested that the ISV defect caused by max-1 knockdown could be partially rescued by overexpression of ephrinb3 and that max-1 was involved in mediating membrane localization of ephrin proteins, which have been shown to provide guidance cues for endothelial cell migration. Our findings therefore suggest that max-1, acting upstream of the ephrin pathway, is critically required in vascular patterning in vertebrate species.