Critical roles of the ddx5 gene in zebrafish sex differentiation and oocyte maturation.

DEAD-box helicase 5 (Ddx5) functions as an ATP-dependent RNA helicase and as a transcriptional coactivator for several transcription factors; however, the developmental function of the ddx5 gene in vertebrates is not fully understood. We found that the zebrafish ddx5 gene was expressed in developing gonads. Using the genome editing technology transcription activator-like effector nuclease, we established a ddx5-disrupted zebrafish and examined the morphological phenotypes of the mutant. We found that the majority of ddx5-deficient mutants developed as fertile males with normal testes and a small number of ddx5-deficient mutants developed as infertile females with small ovaries. Apoptotic cell death at 31 days post fertilization was increased in thick immature gonads (presumptive developing ovaries) of the ddx5-deficient mutant compared to those of heterozygous wild-type fish, while the number of apoptotic cells in thin immature gonads (presumptive developing testes) was comparable between the mutant and wild-type animals. Histological analysis revealed that ovaries of adult ddx5-deficient females had fewer vitellogenic oocytes and a larger number of stage I and II oocytes. The amount of cyclic adenosine monophosphate in the ddx5-deficient ovaries was high compared to that of wild-type ovaries, presumably leading to the mitotic arrest of oocyte maturation. Therefore, the ddx5 gene is dispensable for testis development, but it is essential for female sex differentiation and oocyte maturation in zebrafish.

Spatiotemporal expression of the cocaine- and amphetamine-regulated transcript-like (cart-like) gene during zebrafish embryogenesis.

The cocaine- and amphetamine-regulated transcript (CART) genes are involved in the neural regulation of energy homeostasis; however, their developmental expressions and functions are not fully understood in vertebrates. We have identified a novel zebrafish cart-like gene that encodes a protein of 105 amino acids possessing sequence similarity to zebrafish and mammalian CART proteins. RT-PCR analysis revealed that the cart-like transcripts were maternally supplied and gradually decreased during the cleavage, blastula and gastrula stages; then, transcripts subsequently reaccumulated at the segmentation, pharyngula and hatching stages. Based on a whole-mount in situ hybridization analysis using an antisense cart-like RNA probe, we found that the cart-like transcript was predominantly expressed in both the Rohon-Beard neurons and trigeminal ganglia, suggesting the involvement of the cart-like gene in zebrafish neural development.

Developmental expression of the slurp-like1/ly2.3/ly97.3 and slurp-like2/ly2.2/ly97.2 genes during zebrafish early embryogenesis.

Mammalian SLURP1 and SLURP2 belong to the Ly-6/uPAR superfamily and are involved in maintaining the physiological integrity of keratinocytes. However, the developmental expression and functions of other Ly-6/uPAR family genes in vertebrates are still obscure. We have isolated novel Ly-6/uPAR family genes slurp-like1 (ly2.3/ly97.3) and slurp-like2 (ly2.2/ly97.2) in zebrafish. Both the Slurp-like1 and Slurp-like2 proteins contain the typical signal sequence and carboxy-terminal CCXXXXCN (X: an arbitrary amino acid) consensus sequence of the Ly-6/uPAR family but lack a transmembrane domain and a GPI-anchoring signal sequence, suggesting that both proteins may function as secretory proteins. Whole-mount in situ hybridization analysis revealed that slurp-like1 was predominantly expressed in the floor plate of the neural tube and in the hypochord of the notochord at 24 h post-fertilization (hpf) and detected in the liver and intestinal bulb at 72 hpf, while slurp-like2 was expressed in the midbrain and hindbrain at 24 hpf and detected in the liver and pancreas at 72 hpf. Differential expression profiles of the slurp-like1 and slurp-like2 genes suggest the distinct physiological involvement of these genes in zebrafish early embryogenesis.

Exogenous gene integration mediated by genome editing technologies in zebrafish.

Genome editing technologies, such as transcription activator-like effector nuclease (TALEN) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, can induce DNA double-strand breaks (DSBs) at the targeted genomic locus, leading to frameshift-mediated gene disruption in the process of DSB repair. Recently, the technology-induced DSBs followed by DSB repairs are applied to integrate exogenous genes into the targeted genomic locus in various model organisms. In addition to a conventional knock-in technology mediated by homology-directed repair (HDR), novel knock-in technologies using refined donor vectors have also been developed with the genome editing technologies based on other DSB repair mechanisms, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). Therefore, the improved knock-in technologies would contribute to freely modify the genome of model organisms.

Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.

The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.

Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.

The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.

Comprehensive analysis of sphingosine-1-phosphate receptor mutants during zebrafish embryogenesis.

The lipid mediator sphingosine-1-phosphate (S1P) regulates various physiological and pathological phenomena such as angiogenesis and oncogenesis. Secreted S1P associates with the G-protein-coupled S1P receptors (S1PRs), leading to the activation of downstream signaling molecules. In mammals, five S1prs have been identified and the genetic disruption of a single S1pr1 gene causes vascular defects. In zebrafish, seven s1prs have been isolated. We found that individual s1prs showed unique expression patterns with some overlapping expression domains during early embryogenesis. We generated all s1pr single-mutant zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases and analyzed their phenotypes during early embryogenesis. Zygotic s1pr1, s1pr3a, s1pr3b, s1pr4, s1pr5a and s1pr5b mutants showed no developmental defects and grew into adults, whereas zygotic s1pr2 mutant showed embryonic lethality with a cardiac defect, showing quite distinct embryonic phenotypes for individual S1pr mutants between zebrafish and mouse. We further generated maternal-zygotic s1pr1, s1pr3a, s1pr3b, s1pr4, s1pr5a and s1pr5b mutants and found that these maternal-zygotic mutants also showed no obvious developmental defects, presumably suggesting the redundant functions of the S1P receptor-mediated signaling in zebrafish.

Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish.

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish.

Maternal and Zygotic Sphingosine Kinase 2 Are Indispensable for Cardiac Development in Zebrafish.

Sphingosine 1-phosphate (S1P) is synthesized from sphingosine by sphingosine kinases (SPHK1 and SPHK2) in invertebrates and vertebrates, whereas specific receptors for S1P (S1PRs) selectively appear in vertebrates, suggesting that S1P acquires novel functions in vertebrates. Because the developmental functions of SPHK1 and SPHK2 remain obscure in vertebrates, we generated sphk1 or sphk2 gene-disrupted zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases. Both zygotic sphk1 and sphk2 zebrafish mutants exhibited no obvious developmental defects and grew to adults. The maternal-zygotic sphk2 mutant (MZsphk2), but not the maternal-zygotic sphk1 mutant and maternal sphk2 mutant, had a defect in the cardiac progenitor migration and a concomitant decrease in S1P level, leading to a two-heart phenotype (cardia bifida). Cardia bifida in MZsphk2, which was rescued by injecting sphk2 mRNA, was a phenotype identical to that of zygotic mutants of the S1P transporter spns2 and S1P receptor s1pr2, indicating that the Sphk2-Spns2-S1pr2 axis regulates the cardiac progenitor migration in zebrafish. The contribution of maternally supplied lipid mediators during vertebrate organogenesis presents as a requirement for maternal-zygotic Sphk2.

Proliferation following tetraploidization regulates the size and number of erythrocytes in the blood flow during medaka development, as revealed by the abnormal karyotype of erythrocytes in the medaka TFDP1 mutant.

BACKGROUND: For the delivery of oxygen, the correct size/number of erythrocytes is required for proper blood flow. RESULTS: By combined analyses of wild-type (WT) medaka and the kyoho (kyo) mutant, we found proliferation-mediated adaptation for size/number of erythrocytes in the blood flow during medaka development. Before the start of heart beating in the WT medaka, the karyotype of erythrocytes was 2N-4N. After the start of blood flow, the karyotype changed to 4N-8N with tetraploidization, and the cell size became larger. After the start of intersegmental and pharyngeal blood flow, the erythrocytes became smaller. The medaka mutant kyo showed erythrocytes of large size, and positional cloning of kyo demonstrated the candidate gene TFDP1, indicating higher polyploidization due to arrest in S-phase in erythrocytes of the kyo mutant. CONCLUSIONS: From our findings, we uncovered a previously unrecognized system for the regulation of the size/number in the blood flow:proliferation of erythrocytes following tetraploidization during embryonic development.