YAP is essential for tissue tension to ensure vertebrate 3D body shape.

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

WDR55 is a nucleolar modulator of ribosomal RNA synthesis, cell cycle progression, and teleost organ development.

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.

Generation of transgenic medaka expressing claudin7-EGFP for imaging of tight junctions in living medaka embryos.

The tight junction (TJ) is a specialized cell-cell adhesion structure in epithelial and endothelial sheets unique to the chordates and functions as a barrier of fluidal diffusion across the cell sheets. In order to study the dynamics of TJ formation in vivo during embryogenesis, we have generated a transgenic medaka line that expresses claudin-7 protein fused to enhanced green fluorescent protein under the regulation of the red seabream beta-actin promoter in transparent medaka embryos. Claudins contain four transmembrane domains and have been identified as the key molecules that dictate the function of TJs. This transgenic medaka line will thus be useful for imaging of TJs in living embryos and hence in screening for mutations affecting cell-cell adhesion.

Conserved function of caspase-8 in apoptosis during bony fish evolution.

Caspase-8, a member of the caspase family, plays an important role in apoptotic signal transduction in mammals. Here we report the identification and characterization of the caspase-8 (casp8) gene in the zebrafish Danio rerio. The zebrafish casp8 gene has a genomic organization similar to mammalian casp8 genes, consisting of 10 exons. By chromosome mapping, we found that casp8 maps on linkage group 6 (LG6), a zebrafish chromosome segment orthologous to the long arm of human Chr. 2, which carries CASP8. In contrast, the zebrafish casp10-like gene and the cflar gene separately localize on LG9 and LG11, respectively, and these genes form a cluster with CASP8 on the human chromosome. This chromosomal segregation is unique to fish but not other vertebrates. Furthermore, we examined the function of zebrafish Casp8 protein in mammalian cells, and showed that it has pro-apoptotic activity when overexpressed. In addition, this molecule was capable of transmitting apoptotic signals mediated through not only Fas but also the TNF receptor in mouse Casp8-deficient cells. Expression analysis showed that casp8 is maternally expressed, and transcripts continue to be present throughout embryogenesis and into larval stages. These results show that zebrafish casp8 has a structure and function similar to mammalian CASP8 orthologs, and our study suggests that the role of caspase-8 in the apoptotic signal pathway has been conserved over at least 450 million years of vertebrate evolution.

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8.

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.

The Tomita collection of medaka pigmentation mutants as a resource for understanding neural crest cell development.

All body pigment cells in vertebrates are derived from the neural crest. In fish the neural crest can generate up to six different types of pigment cells, as well as various non-pigmented derivatives. In mouse and zebrafish, extensive collections of pigmentation mutants have enabled dissection of many aspects of pigment cell development, including fate specification, survival, proliferation and differentiation. A collection of spontaneous mutations collected from wild medaka (Oryzias latipes) populations and maintained at Nagoya University includes more than 40 pigmentation mutations. The descriptions of their adult phenotypes have been previously published by Tomita and colleagues (summarised in Medaka (Killifish) Biology and Strains, 1975), but the embryonic phenotypes have not been systematically described. Here we examine these embryonic phenotypes, paying particular attention to the likely defect in pigment cell development in each, and comparing the spectrum of defects to those in the zebrafish and mouse collections. Many phenotypes parallel those of identified zebrafish mutants, although pigment cell death phenotypes are largely absent, presumably due to the different selective pressures under which the mutants were isolated. We have identified mutant phenotypes that may represent the Mitf/Kit pathway of melanophore specification and survival. We use in situ hybridisation with available markers to confirm a key prediction of this hypothesis. We also highlight a set of novel phenotypes not seen in the zebrafish collection. These mutants will be a valuable resource for pigment cell and neural crest studies and will strongly complement the mutant collections in other vertebrates.

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

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.

An expression pattern screen for genes involved in the induction of the posterior nervous system of zebrafish.

The posterior nervous system, including the hindbrain and the spinal cord, has been shown to be formed by the transformation of neural plate of anterior character by signals derived from non-axial mesoderm. Although secreted factors, such as fibroblast growth factors (FGFs), Wnts, retinoic acid (RA) and Nodal, have been proposed to be the posteriorizing factors, the mechanism how neural tissue of posterior character is induced and subsequently specified along the anteroposterior axis remains elusive. To identify intercellular signaling molecules responsible for posteriorization of the neural plate as well as to find molecules induced intracellularly by the posteriorizing signal in the caudal neural plate, we screened by in situ hybridization for genes specifically expressed in posterior tissues, including the posterior neural plate and non-axial mesoderm when posteriorization of the neural plate takes place. From a subtracted library differentiating anterior versus posterior neural plate, 420 cDNA clones were tested, out of which 76 cDNA fragments showed expression restricted to the posterior tissue. These clones turned out to represent 32 different genes, including one novel secreted factor and one transmembrane protein. Seven genes were induced by non-axial mesodermal implants and bFGF beads, suggesting that these are among the early-response genes of the posteriorizing signal. Thus, our approach employing cDNA subtraction and subsequent expression pattern screening allows us to clone candidate genes involved in a novel signaling pathway contributing to the formation of the posterior nervous system.