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The precise government of the left-right (LR) specification of an organ is an essential aspect of its morphogenesis. Multiple signaling cascades have been implicated in the establishment of vertebrate LR asymmetry. Recently, mTOR signaling was found to critically regulate the development of LR asymmetry in zebrafish. However, the upstream factor(s) that activate mTOR signaling in the context of LR specification are as yet unknown. In this study, we identify the SLC7 amino acid transporters Slc7a7 and Slc7a8 as novel regulators of LR asymmetry development in the small fish medaka. Knockdown of Slc7a7 and/or Slc7a8 in medaka embryos disrupted LR organ asymmetries. Depletion of Slc7a7 hindered left-sided expression of the southpaw (spaw) gene, which is responsible for LR axis determination. Work at the cellular level revealed that Slc7a7 coordinates ciliogenesis in the epithelium of Kupffer's vesicle and thereby the generation of the nodal fluid flow required for LR asymmetry. Interestingly, knockdown of Slc7a7 depressed mTOR signaling activity in medaka embryos. Treatment with rapamycin, an inhibitor of mTOR signaling, together with Slc7a7 knockdown synergistically perturbed spaw expression, indicating an interaction between Slc7a7 and mTOR signaling affecting gene expression required for LR specification. Taken together, our results demonstrate that Slc7a7 governs the regulation of LR asymmetry development via the activation of mTOR signaling.

Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Dysfunction of apoptosis is involved in many fatal diseases such as cancer. Visualization of apoptosis in living animals is necessary to understand the mechanism of apoptosis-related diseases. Here, we describe a genetically encoded fluorescent probe for imaging apoptosis in living multicellular organisms, based on spontaneous complementation of two fragments of a green fluorescent protein (GFP) variant (GFP OPT). The probe is designed for detection of mitochondria-mediated apoptosis during which a mitochondrial protein of Smac is released into cytosol. The Smac is connected with a carboxy-terminal fragment of GFP OPT (GFP11), whereas the remainder of GFP OPT (GFP(1-10)) is located in the cytosol. Under an apoptotic condition, the Smac is released from mitochondria into cytosol, allowing complementation of the GFP-OPT fragments and the emission of fluorescence. Live-cell imaging demonstrates that the probe enables detection of apoptosis in living cells with a high signal-to-background ratio. We applied the probe to living zebrafish, in which apoptotic cells were visualized with fluorescence. The technique provides a useful tool for the study of apoptosis in living animals, facilitating elucidation of the mechanisms of apoptosis-related diseases.

Stress-induced Sapk/Jnk signaling is involved in cell survival and apoptosis. Recent studies have increased our understanding of the physiological roles of Jnk signaling in embryonic development. However, still unclear is the precise function of Jnk signaling during gastrulation, a critical step in the establishment of the vertebrate body plan. Here we use morpholino-mediated knockdown of the zebrafish orthologs of the Jnk activators Mkk4 and Mkk7 to examine the effect of Jnk signaling abrogation on early vertebrate embryogenesis. Depletion of zebrafish Mkk4b led to abnormal convergent extension (CE) during gastrulation, whereas Mkk7 morphants exhibited defective somitogenesis. Surprisingly, Mkk4b morphants displayed marked upregulation of wnt11, which is the triggering ligand of CE and stimulates Jnk activation via the non-canonical Wnt pathway. Conversely, ectopic activation of Jnk signaling by overexpression of an active form of Mkk4b led to wnt11 downregulation. Mosaic lineage tracing studies revealed that Mkk4b-Jnk signaling suppressed wnt11 expression in a non-cell-autonomous manner. These findings provide the first evidence that wnt11 itself is a downstream target of the Jnk cascade in the non-canonical Wnt pathway. Our work demonstrates that Jnk activation is indispensable for multiple steps during vertebrate body plan formation. Furthermore, non-canonical Wnt signaling may coordinate vertebrate CE movements by triggering Jnk activation that represses the expression of the CE-triggering ligand wnt11.

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer's vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.

We have previously described a rat autosomal recessive mutation, creeping (cre), causing severe ataxia and disarrangement of neuronal cells in the central nervous system. The mutant strain has recently been successfully inbred, named Komeda Zucker creeping (KZC) rat. In the present study, we have performed a genetic analysis of the creeping mutation, and mapped it to rat Chromosome (Chr) 4. Comparative mapping, together with the similarity of the phenotype, suggested that the creeping mutation is homologous to the mouse reeler mutation. In fact, reelin expression was markedly reduced in the homozygous mutant (cre/cre) animals compared with the normal littermates. Thus, the KZC rat should become a useful biological model with a novel mutation in the reelin gene.