The expression and role of Loto genes was previously investigated in arthropods and it was concluded that the morphogenetic functions of Loto genes were conserved in the last common ancestors of arthropods. In this paper, possible orthologs of Loto clade genes are analysed and it was found that they possibly play a conserved role in convergent extension of cells from the blastoderm stage to germ band extension stage. The differences and similarities in the number of and expression of Loto clade genes in arthropods and onychophorans are compared and analysed. Unlike in arthropods, the expression of Euperipatoides Loto is strongest in the head including part of the somewhat enigmatic frontal appendages (the onychophoran antennae). I also find a stripped segmental pattern of expression that is similar to that classic expression in transverse segmental stripes of Loto class genes as seen in arthropods. However, these stripes are only week and appear to be mesodermal rather than ectodermal, as in arthropods. These facts are discussed in regard to current hypothesis about the origin of segmentation in panarthropods.Insects, crustaceans and chelicerates each possess a set of several Loto class genes which are also involved in convergent extension. But the myriapod Strigamia only appears to possess one such gene.  I therefore investigated Loto genes in another distantly related myriapod, Glomeris. It appears to only have one Loto gene and this appears to be involved in convergent extension as well. Two Toll genes  of Glomeris (Gm-567 and Gm-596) were incomplete and could according to phylogenetic analysis represent Loto genes. I therefore checked their expression and found that they are not expressed like Loto genes. A third potential Glomeris Loto gene (Gm-399) could not be isolated via PCR.My data suggest a somewhat different role for onychophoran Loto than convergent extension, and that the ancestral set of panarthropod Loto genes may be one, rather than a set. / Loto-genernas roll och uttryck var tidigare undersökt inom leddjur och slutsatsen var att morfogenetiska funktioner av Loto-gener var konserverade hos närmsta förfäderna till leddjuren. I denna artikel/rapport studerades ortologer till Loto-generna, vilket kunde konstateras att dessa förmodligen har en viktig roll i konvergent förlängning (convergent extension) av celler från blastoderma stadiet till germ band stadiet. Skillnader och likheter i uttryck av Loto-gener inom leddjur och Onychophora (klomaskar) jämfördes och analyserades. Olikt från leddjur så var uttrycket av Euperipatoides Loto som starkast i antennen vid huvudet på Onychophora. Jag kunde också se ett segmenterat mönster av uttrycket som är liknande de uttryck i form av segmentell band som återfinns i klassen Loto-gener hos leddjuren. Dessa är dock svaga och verkar bara vara mesodermala snarare än ektodermala som hos leddjur. Dessa fakta diskuteras kring hypotesen kring ursprunget om segmentering hos Panarthropoda.Insekter, kräftdjur och palpkäkar har alla flera klasser av Loto gener som även de är inblandade konvergent förlängning. Men, myriapod Strigamia har bara en Loto-gen. Därför valde jag att utforska de Loto-gener i en annan långt relaterad myriapod kallad Glomeris. Det verkar som Glomeris har en Loto-gen och den är involverad i konvergent förlängning också. Två Toll-gener från Glomeris (Gm-567 och Gm-596) var inte kompletta och kunde representera Loto-gener enlig analys från phylogenetiskt träd. Därför kollade jag uttrycket på dessa gener och kunde se att de inte var uttryckta i samma grad som Loto-gener. En tredje potentiell Glomeris Loto-gen (Gm-399) kunde inte bli isolerad med hjälp av PCR.Min data tyder på att klomaskars Loto-gener har en annan roll än konvergent förlängning och attpanarthropods kanske bara hade en Loto-gene istället för ett set av gener.

No description available.

Coordinated inductive and morphogenetic processes of gastrulation establish the zebrafish body plan. Gastrulation includes massive cell rearrangements to generate the three germ layers and shape the embryonic body. Three modes of cell migration must occur during vertebrate gastrulation and include: epiboly, internalization of the presumptive mesendoderm and convergent extension (C&E). C&E movements narrow the germ layers mediolaterally (convergence) and elongate them anteroposteriorly (extension) to define the embryonic axis. The molecular mechanisms regulating coordinated cell migrations remain poorly understand and studying these has become of great interest to researchers. Understanding cell migration during development is highly relevant to a number of human physiological processes. Abnormal cell migration during early development can lead to congenital defects, with improper cell migration during adult life potentially leading to the invasion and metastasis of cancer. By studying cell migration events, in vivo, new insights are to be found to both the function and malfunction of key embryonic and postembryonic migratory events. The non-canonical Wnt pathway has been identified as an evolutionarily conserved signaling pathway, regulating C&E cell movements during vertebrate gastrulation. With the absence of the non-canonical Wnts (ncWnts), Wnt5 and Wnt11, during zebrafish development leading to a shorter and broader body axis with defects in elongation during segmentation resulting in undulation of the notochord. While it is clear ncWnts are necessary for C&E, many of the downstream effectors regulating these cell movements have not been defined. Previous research has shown that activation of ncWnt signaling through Wnt5 or Wnt11 results in an increase in intracellular Ca2+ during zebrafish gastrulation. To determine if the Ca2+/Calmodulin-dependent protein kinase, CaMK-II, is a potential downstream target of the Ca2+ increases during ncWnt activation, CaMK-II’s role in C&E was assessed. This study identifies camk2b1 and camk2g1 as being necessary for C&E movements, and outlines the phenotype of the overall embryo as well as individual cells of camk2b1 and camk2g1 morphants. The defects of CaMK-II morphants are specifically linked to alterations in C&E cell movements, while cell fate and proliferation are unaffected. An increase in CaMK-II activation during gastrulation produces similar C&E defects, demonstrating the specificity of CaMK-II’s activation in facilitating these highly coordinated cellular movements. We show that CaMK-II is working downstream Wnt 11 and in parallel to JNK signaling during gastrulation C&E. Overall, these data identify CaMK-II as a required component of C&E movements during zebrafish development, downstream ncWnt signaling, and altering cell migration through changes in cell shape

Final publication is available at http://www.plosgenetics.org/article/related/info%3Adoi%2F10.1371%2Fjournal.pgen.1005802 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19627号 / 医博第4134号 / 新制||医||1016(附属図書館) / 32663 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 松田 秀一, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Vertebrate development is regulated by cellular communication by mechanisms of cell fate and cell behavior. These crucial mechanisms are regulated by cellular signaling and in the case of cell fate, cellular signaling results in transcription of developmentally important genes. Communication between cells can also result in regulation of cell behavior by acting on cytoskeletal elements rather than nuclear factors. One of the cellular signals that regulate both cell fate and cell behavior is the family of Wnt signaling molecules. Wnt5a is one of 19 Wnt molecules and has been previously demonstrated to play critical roles in many important processes in embryonic development as well tumor suppression. Despite many studies that lend credence to a pathway that regulates cell behavior for Wnt5a rather than cell fate, the identity of the pathway(s) Wnt5a impinges upon remains unclear. Despite the possibility of Wnt5a signaling through multiple pathways, here, focus is given to the non-canonical Wnt signaling pathway, a pathway that regulates cell behavior, also known as the Wnt/Planar Cell Polarity (PCP) pathway. The involvement of Wnt5a in the Wnt/PCP pathway was demonstrated with a genetic approach: crossing Wnt5a heterozygous mice with mice heterozygous for a component of the Wnt/PCP pathway to uncover genetic interactions in vivo. Hence, Wnt5a X Looptail (Lp) (Wnt/PCP) heterozygous crosses have been performed. Double heterozygotes for this intercross did not exhibit a decrease in viable progeny as compared to the decreased numbers of Lp heterozygotes. These observations demonstrated a lack of genetic interaction between Wnt5a and the PCP pathway. Wnt5a mutants possess phenotypes associated with deficits in the Wnt/PCP pathway, namely convergent extension (CE) defects and neural tube closure defects. However, upon further investigation of the increased penetrance of craniorachischisis in Wnt5a-/-;Lp+/-, Wnt5a mutants do not display the characteristic broadening of the neural floor plate commonly associated with Lp-/-. This supports that Wnt5a and PCP signaling are parallel pathways that have converged to regulate different aspects of CE and neural tube closure. Despite the complexity of Wnt5a and its potential involvement in multiple pathways, dissection of this will explain the broad range of phenotypes observed.

No description available.

The Wnts, a family of secreted glycoprotein ligands, act through the frizzled (Fz) receptor, a family of seven-transmembrane (7TM) receptor proteins, to mediate intracellular signaling pathways that regulate cell fate determination, cell migration, or both. Whereas many molecular components of the Wnt signal transduction cascade have been identified, it remains unclear how the signal is transduced from the Fz receptors to the cytoplasm. To address this important question, a membrane-based yeast two-hybrid (MbY2H) screen was performed to identify potential Fz-interacting proteins. For this screen, the Frizzled7 (Fz7) protein was used as the bait and a mouse brain library was used the prey. This screen identified resistance to inhibitors of cholinesterase 8 homolog A (Ric8A), a 542–amino acid cytoplasmic protein, along with other proteins as putative Fz7-binding proteins. Ric8A had been studied previously in C. elegans and D. melanogaster for its function in regulating asymmetric cell division as a receptor-independent guanine nucleotide exchange factor (GEF) for Gα proteins. Additional studies in M. musculus and X. laevis further uncovered a role for this protein during gastrulation and neurulation; however, the mechanisms by which Ric8A regulated these processes remained unclear. In this thesis, I show Ric8A to be a bona fide binding partner for both Fz7; that Ric8A can also bind to the phosphoprotein Dishevelled (Dvl); and that both its interaction with Fz7 and Dvl is Wnt-regulated. The spatial and temporal mRNA expression pattern of the Xenopus homologue of Ric8A suggests a potential role in regulating Wnt signaling. The Xenopus homologue of Ric8A was cloned and gain-of-function and loss-of-function approaches in Xenopus uncovered a role for Ric8A in gastrulation and neural tube closure. Additionally, we found inhibition of Ric8A function mechanistically prevents activation of Rac1 which is required downstream of Wnt/Fz signaling during gastrulation. Overall, this study uncovers a novel regulator of Wnt signaling during early development / Biology

One of the best-known structures in Drosophila is the sex comb, an arrangement of modified bristles on the tarsal forelegs of males. This complex, sexually-dimorphic trait shows striking variation among closely related species, although most other aspects of the tarsal bristle pattern have been conserved. I studied the development of the sex comb in the model organism Drosophila melanogaster and six related species. I confirmed that the D. melanogaster sex comb, although longitudinal in the adult, originates in a transverse orientation and rotates during development, and showed that this process occurs through male-specific convergent extension. However, in the species that I examined that have longitudinally-oriented sex combs that extend the full length of the tarsus, including D. ficusphila and two species of the montium subgroup, the sex comb does not rotate, and instead forms from two longitudinal rows that converge during development. Another species of the montium subgroup, D. nikananu, has a sex comb that is convergently similar to D. melanogaster, but forms in a manner typical of its subgroup, showing that very similar combs can be formed through different processes. In all species, there is a strong correlation between the position of the sex comb and the transverse bristle row on the foreleg tarsus just proximal to it. To test whether it is possible to violate this apparent constraint on development, I perturbed the expression of the leg patterning gene dachshund to generate ectopic sex combs in D. melanogaster. I found that while most patterns showed the same correlation, a few circumvent the constraint. I also demonstrated that the ectopic combs were formed non-autonomously and that overexpression of dachshund can transform certain aspects of the sex comb phenotype to resemble the transverse bristles to which they are homologous.

One of the best-known structures in Drosophila is the sex comb, an arrangement of modified bristles on the tarsal forelegs of males. This complex, sexually-dimorphic trait shows striking variation among closely related species, although most other aspects of the tarsal bristle pattern have been conserved. I studied the development of the sex comb in the model organism Drosophila melanogaster and six related species. I confirmed that the D. melanogaster sex comb, although longitudinal in the adult, originates in a transverse orientation and rotates during development, and showed that this process occurs through male-specific convergent extension. However, in the species that I examined that have longitudinally-oriented sex combs that extend the full length of the tarsus, including D. ficusphila and two species of the montium subgroup, the sex comb does not rotate, and instead forms from two longitudinal rows that converge during development. Another species of the montium subgroup, D. nikananu, has a sex comb that is convergently similar to D. melanogaster, but forms in a manner typical of its subgroup, showing that very similar combs can be formed through different processes. In all species, there is a strong correlation between the position of the sex comb and the transverse bristle row on the foreleg tarsus just proximal to it. To test whether it is possible to violate this apparent constraint on development, I perturbed the expression of the leg patterning gene dachshund to generate ectopic sex combs in D. melanogaster. I found that while most patterns showed the same correlation, a few circumvent the constraint. I also demonstrated that the ectopic combs were formed non-autonomously and that overexpression of dachshund can transform certain aspects of the sex comb phenotype to resemble the transverse bristles to which they are homologous.

La voie de la polarité planaire cellulaire (PCP), aussi connue sous le nom de la voie non-canonique du Frizzled/Dishevelled, contrôle le processus morphogénétique de l'extension convergente (CE) qui est essentiel pour la gastrulation et la formation du tube neural pendant l'embryogenèse. La signalisation du PCP a été récemment associée avec des anomalies du tube neural (ATN) dans des modèles animaux et chez l'humain. Prickle1 est une protéine centrale de la voie PCP, exprimée dans la ligne primitive et le mésoderme pendant l'embryogenèse de la souris. La perte ou le gain de fonction de Prickle1 mène à des mouvements de CE fautifs chez le poisson zèbre et la grenouille. PRICKLE1 interagit directement avec deux autres membres de la voie PCP, Dishevelled et Strabismus/Vang. Dans notre étude, nous avons investigué le rôle de PRICKLE1 dans l'étiologie des ATN dans une cohorte de 810 patients par le re-séquençage de son cadre de lecture et des jonctions exon-intron. Le potentiel pathogénique des mutations ainsi identifiées a été évalué par des méthodes bioinformatiques, suivi par une validation fonctionnelle in vivo dans un système poisson zèbre. Nous avons identifié dans notre cohorte un total de 9 nouvelles mutations dont sept: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys et p.Ser739Phe, p.Val550Met et p.Asp771Asn qui affectent des acides aminés conservés. Ces mutations ont été prédites in silico d’affecter la fonction de la protéine et sont absentes dans une large cohorte de contrôles de même origine ethnique. La co-injection de ces variantes avec le gène prickle1a de type sauvage chez l’embryon de poisson zèbre a démontré qu’une mutation, p.Arg682Cys, modifie dans un sens négatif le phénotype du défaut de la CE produit par pk1 de type sauvage. Notre étude démontre que PK1 peut agir comme facteur prédisposant pour les ATN chez l’humain et élargit encore plus nos connaissances sur le rôle des gènes de la PCP dans la pathogenèse de ces malformations. / The planar cell polarity pathway (PCP) or the non-canonical Frizzled/Dishevelled pathway controls the morphogenetic process of convergent extension (CE) that is essential during embryogenesis for gastrulation and neural tube formation. Recently, PCP signalling was associated with neural tube defects (NTD) in humans and animal models. The core PCP protein, Prickle1, is expressed in the primitive streak and mesoderm during mouse embryogenesis. Both gain and loss of function of Prickle1 cause faulty CE movements in zebrafish and the frog. PRICKLE1 physically interacts with two other core PCP members, Dishevelled and Strabismus/Vang. In the present study we investigated the role of PRICKLE1 in the aetiology of NTDs in a large cohort of 810 patients through resequencing of its open reading frame and exon-intron junctions. The pathogenicity of the identified mutations was assessed through bioinformatics methods followed by a functional validation in a zebrafish system, in vivo. We identified in our cohort a total of nine novel mutations, of which seven affected conserved amino acids: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys, p.Ser739Phe, p.Val550Met and p.Asp771As. These mutations were predicted to affect the function of the protein in silico and were absent in a large cohort of ethnically-matched controls. Co-injection of these variants with the wild type pk1 in zebrafish oocytes revealed that one mutation, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a in a dominant fashion. Our study demonstrates that PRICKLE1 can represent a predisposing factor for human NTDs and further expands our knowledge on the role that PCP genes in the pathogenesis of these malformations.