Graphical Abstract

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Stress-activated protein kinase/c-Jun NH 2 -terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated The SAPK/JNK 1 is a member of the family of mitogen-activated protein kinase (MAPK). This MAPK is activated not only by many types of cellular stresses, including changes in osmolarity, heat shock, and UV irradiation, but also by serum, lysophosphatidic acid, and inflammatory cytokines (interleukin-1␤ and tumor necrosis factor-␣). The activated SAPK/JNK phosphorylates transcription factors c-Jun, Jun D, and activating transcription factor-2 to regulate gene expression for the stress response. Activation of SAPK/JNK requires the phosphorylation of Tyr and Thr residues located in a Thr-Pro-Tyr motif in the activation loop between VII and VIII of the kinase domain. The phosphorylation is catalyzed by the dual specificity kinases SEK1 (also known as MKK4) and MKK7 (SEK2), which are capable of catalyzing the phosphorylation of both Thr and Tyr residues in vitro (1, 2).Targeted gene-disruption experiments in mice demonstrate that both SEK1 and MKK7 are required for embryonic development. Sek1 Ϫ/Ϫ embryos die between embryonic day 10.5 (E10.5) and E12.5 with impaired liver formation (3-5). Furthermore, we have recently reported that SEK1 is crucial for hepatocyte growth factor-induced activation of SAPK/JNK in developing hepatoblasts of mouse embryos. On the other hand, mkk7 Ϫ/Ϫ embryos die between E11.5 and E12.5 with similar impairment of liver formation and SAPK/JNK activation (6). These results clearly show that both SEK1 and MKK7 play indispensable roles in hepatoblast proliferation during mouse embryogenesis. Distinct biochemical properties between SEK1 and MKK7 may be critical for the indispensable roles of the two activators of SAPK/JNK in vivo.In this regard, several in vitro experiments have shown that SAPK/JNK is activated synergistically by SEK1 and MKK7 (7-9). The synergistic activation may be related to the enzymatic properties of the two MAPKKs: SEK1 prefers the Tyr residue and MKK7 prefers the Thr residue of the MAPK. We have also reported that the synergistic activation of SAPK/JNK in response to stress signals is attenuated with a decreased level of its Tyr phosphorylation in sek1 Ϫ/Ϫ mouse ES cells that retain MKK7 at the same level as the wild-type cells (10).

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.