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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.

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.

The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap's TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1-mediated transactivation of photoreceptor cell genes during zebrafish retinogenesis.

During vertebrate embryogenesis, the liver develops at a precise location along the endodermal primitive gut tube because of signaling delivered by adjacent mesodermal tissues. Although several signaling molecules have been associated with liver formation, the molecular mechanism that regulates liver specification is still unclear. We previously performed a screen in medaka to isolate mutants with impaired liver development. The medaka hio mutants exhibit a profound (but transient) defect in liver specification that resembles the liver formation defect found in zebrafish prometheus (prt) mutants, whose mutation occurs in the wnt2bb gene. In addition to their liver abnormality, hio mutants lack pectoral fins and die after hatching. Positional cloning indicated that the hio mutation affects the raldh2 gene encoding retinaldehyde dehydrogenase type2 (RALDH2), the enzyme principally responsible for retinoic acid (RA) biosynthesis. Mutations of raldh2 in zebrafish preclude the development of pectoral fins. Interestingly, in hio mutants, expression of wnt2bb in the lateral plate mesoderm (LPM) directly adjacent to the liver-forming endoderm was completely lost. Conclusion: Our data reveal the unexpected finding that RA signaling positively regulates the wnt2bb gene expression required for liver specification in medaka. These results suggest that a common molecular mechanism may underlie liver and pectoral fin specification during piscine embryogenesis. (HEPATOLOGY 2009.)