Qingshun Zhao

Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo

Retinoic acid (RA) signaling in vertebrate embryos occurs in a distinct physical and temporal pattern. Regulating this spatial distribution is crucial to the development of the embryo, as RA in excess or in inappropriate tissues is teratogenic. In order to understand how RA availability is determined in zebrafish we have investigated the expression of cyp26a1, an enzyme that inactivates RA, and its relationship to raldh2, one of the enzymes that produce RA from retinal. cyp26a1 expression follows three phases: in presumptive anterior neurectoderm and in a circumblastoporal ring during gastrulation, in the tailbud throughout somitogenesis, and in multiple specific tissue types beginning at mid-somitogenesis and continuing through 48 h postfertilization (hpf). This expression was either adjacent or opposite to those tissues expressing raldh2. We then investigated how RA production might regulate these relationships. Endogenous RA produced by raldhs did not play a role in setting cyp26a1 expression in most tissues. However, exogenous RA regulates expression of both enzymes. cyp26a1 is up regulated in the embryo in a time, concentration, and tissue-dependent manner. Conversely, raldh2 expression is reduced with RA treatment. Tests of the raldh2 promoter in cell transfections proved that RA directly represses its activity. These data demonstrate that the feedback mechanisms regulating production and degradation of RA must be considered in any experiments altering levels of RA in the developing vertebrate embryo.

Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.

Retinoid Regulation of the Zebrafish cyp26a1 Promoter

Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio‐inactive metabolites. Previous research revealed that the mouse Cyp26A1 promoter has two canonical RA response elements (RAREs) that underlie the regulation of the gene by RA. Analyzing the 2,533‐base pairs (2.5 k) genomic sequence upstream of zebrafish cyp26a1 start codon, we report that the two RAREs are conserved in zebrafish cyp26a1 promoter. Mutagenesis demonstrated that the two RAREs work synergistically in RA inducibility of cyp26a1. Fusing the 2.5 k (kilobase pairs) fragment to the enhanced yellow fluorescent protein (eYFP) reporter gene, we have generated two transgenic lines of zebrafish [Tg(cyp26a1:eYFP)]. The transgenic zebrafish display expression patterns similar to that of cyp26a1 gene in vivo. Consistent with the in vitro results, the reporter activity is RA inducible in embryos. Taken together, our results demonstrate that the 2.5 k fragment underlies the regulation of the zebrafish cyp26a1 gene by RA. Developmental Dynamics 237:3798–3808, 2008.

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.

Molecular cloning and expression pattern of myostatin gene in yellow catfish (Pelteobagrus fulvidraco)

Myostatin (Mstn), a member of transforming growth factor β (TGF-β) superfamily, plays crucial roles in negative regulation of muscle growth. Yellow catfish, Pelteobagrus fulvidraco Richardson, is one of the most important freshwater aquaculture species in China, but little is known about its genes relate to growth. Here we report molecular cloning and expression pattern of Mstn gene in yellow catfish. Our results reveal that yellow catfish Mstn comprises three exons encoding a protein of 393 amino acid residues. Protein sequence alignments show that the Mstn exhibits 94% amino acid identity with other catfish Mstn and 59.3% identity with cattle Mstn, respectively. Moreover, the predicted bioactive form of yellow catfish Mstn shares 100% identity with other catfish and 87.1% identity with cattle Mstn respectively. Employing reverse transcription polymerase chain reaction (RT-PCR) analysis, we demonstrated that the yellow catfish Mstn gene is expressed in a variety of tissues with varied levels.

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay.

N-CoR is required for patterning the anterior–posterior axis of zebrafish hindbrain by actively repressing retinoid signaling

Active repression of gene expression mediated by unliganded nuclear receptors plays crucial roles in early development of vertebrates. N-CoR (nuclear receptor co-repressor) is the first identified co-repressor that can repress retinoic acid (RA) inducible gene transcription in the absence of RA. Previously, N-CoR was reported to be required for late-stage organogenesis in mouse but whether N-CoR can affect RA-responsive early embryonic patterning is unknown. In this study, we report molecular cloning of zebrafish orthologue of N-CoR and its wide distribution pattern during zebrafish early development. Knocking down n-cor elevates endogenous RA signaling in zebrafish embryos and posteriorizes the neural ectoderm. Overexpressing or knocking down n-cor in zebrafish embryos alters the length of hindbrain in a manner similar to decreasing or increasing RA signaling in embryos, respectively. Taken together, our results demonstrate that N-CoR is essential for early hindbrain patterning by actively repressing retinoid signaling.

Retinoic Acid Signaling Plays a Restrictive Role in Zebrafish Primitive Myelopoiesis

Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10–11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10–11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.

Identification and characterization of a novel retinoic acid response element in zebrafish cyp26a1 promoter.

Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio‐inactive metabolites. Previously, we demonstrated that zebrafish cyp26a1 promoter possesses two conserved RA response elements (RAREs; proximal R1 and distal R2) in response to RA. Here, we report that it contains a novel RARE (R3) lying between R1 and R2. Mutagenesis analysis reveals that R3 works together with R1 and R2 to ensure the maximum RA inducibility of cyp26a1 promoter. Performing electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we show that RA receptor alpha can bind the novel RARE. Creating and analyzing transgenic zebrafish of Tg(cyp26a1‐R3mut:eYFP)nju3/+ that harbor enhanced yellow fluorescent protein reporter gene (eYFP) driven by cyp26a1 promoter with mutated R3, we demonstrate that the reporter is mainly expressed in tissues of endogenous RA independent but not regions of RA dependent. Like Tg(cyp26a1:eYFP)nju1/+, which harbor eYFP driven by wild‐type cyp26a1 promoter, the reporter in Tg(cyp26a1‐R3mut:eYFP)nju3/+ responds to excessive RA dose dependently. However, it is expressed in a significantly lower level than the reporter in Tg(cyp26a1:eYFP)nju1/+ in response to exogenous RA. Taken together, our results demonstrate that zebrafish cyp26a1 promoter contains a novel RARE that plays crucial roles in regulating cyp26a1 expression during early development of zebrafish. Anat Rec, 2012.

An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease.

Engineered endonucleases are a powerful tool for editing DNA. However, sequence preferences may limit their application. We engineer a structure-guided endonuclease (SGN) composed of flap endonuclease-1 (FEN-1), which recognizes the 3′ flap structure, and the cleavage domain of Fok I (Fn1), which cleaves DNA strands. The SGN recognizes the target DNA on the basis of the 3′ flap structure formed between the target and the guide DNA (gDNA) and cut the target through its Fn1 dimerization. Our results show that the SGN, guided by a pair of gDNAs, cleaves transgenic reporter gene and endogenous genes in zebrafish embryonic genome.