Hiroki Oda

Structural and functional diversity of cadherin at the adherens junction

Adhesion between cells is essential to the evolution of multicellularity. Indeed, morphogenesis in animals requires firm but flexible intercellular adhesions that are mediated by subcellular structures like the adherens junction (AJ). A key component of AJs is classical cadherins, a group of transmembrane proteins that maintain dynamic cell–cell associations in many animal species. An evolutionary reconstruction of cadherin structure and function provides a comprehensive framework with which to appreciate the diversity of morphogenetic mechanisms in animals.

Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion protein

Directional migration of border cells in the Drosophila egg chambers is a developmentally regulated event that requires dynamic cellular functions. In this study, the electron microscopic observation of migrating border cells revealed loose actin bundles in forepart lamellipodia and numerous microvilli extending from nurse cells and providing multiple adhesive contacts with border cells. To analyze the dynamics of actin in migrating border cells in vivo, we constructed a green fluorescent protein‐actin fusion protein and induced its expression in Drosophila using the GAL4/UAS system. The green fluorescent protein‐actin was incorporated into the actin bundles and it enabled visualization of the rapid cytoskeletal changes in border cell lamellipodia. During the growth of the lamellipodia, the actin bundles that increased in number and size radiated from the bundle‐organizing center. Quantification of the fluorescence intensity showed that an accumulation of bundle‐associated and spotted green fluorescent protein‐actin signals took place during their centripetal movement. Our results favored a treadmilling model for actin behavior in border cell lamellipodia.

Early patterning of the spider embryo: a cluster of mesenchymal cells at the cumulus produces Dpp signals received by germ disc epithelial cells

In early embryogenesis of spiders, the cumulus is characteristically observed as a cellular thickening that arises from the center of the germ disc and moves centrifugally. This cumulus movement breaks the radial symmetry of the germ disc morphology, correlating with the development of the dorsal region of the embryo. Classical experiments on spider embryos have shown that a cumulus has the capacity to induce a secondary axis when transplanted ectopically. In this study, we have examined the house spider, Achaearanea tepidariorum, on the basis of knowledge from Drosophila to characterize the cumulus at the cellular and molecular level. In the cumulus, a cluster of about 10 mesenchymal cells, designated the cumulus mesenchymal (CM) cells, is situated beneath the epithelium, where the CM cells migrate to the rim of the germ disc. Germ disc epithelial cells near the migrating CM cells extend cytoneme-like projections from their basal side onto the surface of the CM cells. Molecular cloning and whole-mount in situ hybridization showed that the CM cells expressed a spider homolog of Drosophila decapentaplegic (dpp), which encodes a secreted protein that functions as a dorsal morphogen in the Drosophila embryo. Furthermore, the spider Dpp signal appeared to induce graded levels of the phosphorylated Mothers against dpp (Mad) protein in the nuclei of germ disc epithelial cells. Adding data from spider homologs of fork head, orthodenticle and caudal, we suggest that, in contrast to the Drosophila embryo, the progressive mesenchymal-epithelial cell interactions involving the Dpp-Mad signaling cascade generate dorsoventral polarity in accordance with the anteroposterior axis formation in the spider embryo. Our findings support the idea that the cumulus plays a central role in the axial pattern formation of the spider embryo.

Axis specification in the spider embryo : dpp is required for radial-to-axial symmetry transformation and sog for ventral patterning

The mechanism by which Decapentaplegic (Dpp) and its antagonist Short gastrulation (Sog) specify the dorsoventral pattern in Drosophila embryos has been proposed to have a common origin with the mechanism that organizes the body axis in the vertebrate embryo. However, Drosophila Sog makes only minor contributions to the development of ventral structures that hypothetically correspond to the vertebrate dorsum where the axial notochord forms. In this study, we isolated a homologue of the Drosophila sog gene in the spider Achaearanea tepidariorum, and characterized its expression and function. Expression of sog mRNA initially appeared in a radially symmetrical pattern and later became confined to the ventral midline area, which runs axially through the germ band. RNA interference-mediated depletion of the spider sog gene led to a nearly complete loss of ventral structures, including the axial ventral midline and the central nervous system. This defect appeared to be the consequence of dorsalization of the ventral region of the germ band. By contrast, the extra-embryonic area formed normally. Furthermore, we showed that embryos depleted for a spider homologue of dpp failed to break the radial symmetry, displaying evenly high levels of sog expression except in the posterior terminal area. These results suggest that dpp is required for radial-to-axial symmetry transformation of the spider embryo and sog is required for ventral patterning. We propose that the mechanism of spider ventral specification largely differs from that of the fly. Interestingly, ventral specification in the spider is similar to the process in vertebrates in which the antagonism of Dpp/BMP signaling plays a central role in dorsal specification.

Phenotypic analysis of null mutants for DE-cadherin and Armadillo in Drosophila ovaries reveals distinct aspects of their functions in cell adhesion and cytoskeletal organization

Background : DE‐cadherin is an epithelial cadherin in Drosophila, and forms adherens junctions by associating with Armadillo (β‐catenin). To investigate its role in oogenesis, we generated germ‐line clones homozygous for a null mutation in shotgun (shg) encoding this molecule, and examined their phenotypes, comparing with those of armadillo (arm) mutants.

Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein.

Abstract Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, we constructed Dα-catenin fused with a green fluorescent protein (Dα-catenin-GFP), and found that it targeted apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, we examined the dynamic performance of AJs containing Dα-catenin-GFP in epithelial morphogenetic movements. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dα-catenin-GFP occurred concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behaviour of AJs was concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension could be followed. Finally, we utilized Dα-catenin-GFP to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dα-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution.

Progressive activation of Delta-Notch signaling from around the blastopore is required to set up a functional caudal lobe in the spider Achaearanea tepidariorum

In the development of most arthropods, the caudal region of the elongating germ band (the growth zone) sequentially produces new segments. Previous work with the spider Cupiennius salei suggested involvement of Delta-Notch signaling in segmentation. Here, we report that, in the spider Achaearanea tepidariorum, the same signaling pathway exerts a different function in the presumptive caudal region before initiation of segmentation. In the developing spider embryo, the growth zone becomes morphologically apparent as a caudal lobe around the closed blastopore. We found that, preceding caudal lobe formation, transcripts of a Delta homolog, At-Delta, are expressed in evenly spaced cells in a small area covering the closing blastopore and then in a progressively wider area of the germ disc epithelium. Cells with high At-Delta expression are likely to be prospective mesoderm cells, which later express a twist homolog, At-twist, and individually internalize. Cells remaining at the surface begin to express a caudal homolog, At-caudal, to differentiate as caudal ectoderm. Knockdown of At-Delta by parental RNA interference results in overproduction of At-twist-expressing mesoderm cells at the expense of At-caudal-expressing ectoderm cells. This condition gives rise to a disorganized caudal region that fails to pattern the opisthosoma. In addition, knockdown of Notch and Suppressor of Hairless homologs produces similar phenotypes. We suggest that, in the spider, progressive activation of Delta-Notch signaling from around the blastopore leads to stochastic cell fate decisions between mesoderm and caudal ectoderm through a process of lateral inhibition to set up a functional caudal lobe.

Accumulation of Armadillo Induced by Wingless, Dishevelled, and Dominant-negative Zeste-white 3 Leads to Elevated DE-cadherin inDrosophila Clone 8 Wing Disc Cells

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (ZW-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate zw-3, which in turn down-regulatesarm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which bind to Arm, we overexpressed Dsh, ZW-3, and Arm, in theDrosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression led to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wild-type and dominant-negative forms of ZW-3 decreased and increased Arm levels, respectively, indicating that modulation in zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevated DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative ZW-3-, and truncated Arm-induced accumulation of DE-cadherin protein was accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevated Arm levels by stabilizing Arm at cell-cell junctions.

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny

SUMMARY Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic‐binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell–cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister‐groups vertebrates and urochordates.

Expression patterns of a twist-related gene in embryos of the spider Achaearanea tepidariorum reveal divergent aspects of mesoderm development in the fly and spider.

Abstract We cloned an Achaearanea tepidariorum (Chelicerata, Arachnida) gene related to Drosophila twist (twi), which encodes a basic helix-loop-helix transcription factor required to specify mesoderm fate in the Drosophila embryo. The cloned spider gene was designated At.twist (At.twi). We examined its expression by whole-mount in situ hybridization. At.twi transcripts were first detected in cells located at the polar and equatorial areas of the spherical embryo when the cumulus reached the equator. As the extra-embryonic area expanded, more cells expressed At.twi transcripts. The At.twi-expressing cells became distributed nearly uniformly in the embryonic area. At these stages, some At.twi-expressing cells were found in the surface epithelial cell layer, but other At.twi-expressing cells were at slightly deeper positions from the surface. When the embryo was transformed into a germ band, all At.twi-expressing cells were situated just beneath the surface ectoderm, where they became metamerically arranged. Although little expression was observed in the caudal lobe of the elongating germ band, new stripes of At.twi expression appeared beneath the ectoderm in accordance with the posterior growth. These observations suggested that the cells expressing At.twi were most likely mesoderm. We propose that At.twi can be used as a molecular marker for analyzing mesoderm development in the spider embryo. Moreover, comparison of the expression patterns of twi and At.twi revealed divergent aspects of mesoderm development in the fly and spider. In addition, we cloned an Achaearanea gene related to snail, which is another mesoderm-determining gene in Drosophila, and showed that its expression was restricted to the ectoderm with no indication for a role in mesoderm development.