The interleukin-2 receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta, and IL-2R gamma chains, the last of which is also used in the receptors for IL-4, IL-7, and IL-9. Stimulation with IL-2 induces the tyrosine phosphorylation and activation of the Janus kinases Jak1 and Jak3. Jak1 and Jak3 were found to be selectively associated with the "serine-rich" region of IL-2R beta and the carboxyl-terminal region of IL-2R gamma, respectively. Both regions were necessary for IL-2 signaling. Furthermore, Jak3-negative fibroblasts expressing reconstituted IL-2R became responsive to IL-2 after the additional expression of Jak3 complementary DNA. Thus, activation of Jak1 and Jak3 may be a key event in IL-2 signaling.

Sphingosine-1-phosphate (S1P) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S1P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko157, that displays cardia bifida (two hearts) resembling that in the S1P receptor-2 mutant. A migration defect of myocardial precursors in the ko157 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S1P injection. We show that the export of S1P from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the ko157 mutant. Thus, Spns2 in the YSL functions as a S1P transporter in S1P secretion, thereby regulating myocardial precursor migration.

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.