The unique nature of body handedness, which is distinct from the anteroposterior and dorsoventral polarities, has been attracting growing interest in diverse biological disciplines. Recent research progress on the left-right asymmetry of animal development has focused new attention on the mechanisms underlying the development and evolution of invertebrate handedness. This exploratory review of currently available information illuminates the prospective value of Drosophila and pulmonate snails for innovative new research aimed at elucidating these mechanisms. For example, findings in Drosophila and snails suggest that an actin filament-dependent mechanism may be evolutionarily conserved in protostomes. The polarity conservation of primary asymmetry across most metazoan phyla, which visceral handedness represents, indicates developmental constraint and purifying selection as possible but unexplored mechanisms. Comparative studies using Drosophila and snails, which have the great advantages of using genetic and evolutionary approaches, will accelerate our understanding of the mechanisms governing the conservation and diversity of animal handedness. Developmental Dynamics 237: 3497-3515, 2008.

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Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.

Bone is a connective tissue composed of many cell types, including osteoblasts. How bones acquire their unique size and shape during development remains poorly understood. Herein we investigated the molecular and cellular mechanisms of bone morphogenesis in the zebrafish scale by using transgenic lines to enable visualization of specific types of osteoblasts. We demonstrate that the zebrafish scale contains three distinct types of osteoblasts: (i) a monolayer of central osteoblasts along the inner surface of scales; (ii) marginal osteoblasts elongated along the scale edge; and (iii) submarginal osteoblasts located between the central and marginal osteoblast populations. The size of the central osteoblasts increases progressively during development, suggesting that scale growth is mediated primarily by cell growth rather than the recruitment of new osteoblasts. In addition, the total number of central osteoblasts increases in regenerated scales and is correlated with scale size, possibly allowing for the rapid growth of regenerating scales. Moreover, osteoblast proliferation is not detected during regeneration, suggesting that the osteoblasts originate from post-mitotic precursor cells. Sonic hedgehog a (shha) is expressed in the epidermal cells that make contact with the marginal osteoblasts. Pharmacological inhibition of Hedgehog (Hh) signaling during regeneration reduces the number of marginal osteoblasts and interferes with scale growth, indicating that epidermis-derived Shh regulates scale regeneration. Finally, genetic inhibition of Wnt/planar cell polarity (PCP) signaling in the epidermis results in misorientation of scales with regard to the body axis. These results reveal a novel role for the epidermis in the regulation of bone patterning, namely the regeneration of osteoblasts and directional bone growth.

Differentiation of osteoclasts (OCs) from hematopoietic cells requires cellular interaction with osteoblasts (OBs). Due to the difficulty of live-imaging in the bone, however, the cellular and molecular mechanisms underlying intercellular communication involved in OC differentiation are still elusive. Here, we develop a fracture healing model using the scale of trap: GFP; osterix:mCherry transgenic zebrafish to visualize the interaction between OCs and OBs. Transplantation assays followed by flow cytometric analysis reveal that most trap:GFP high OCs in the fractured scale are detected in the osterix:mCherry + fraction because of uptake of OB-derived extracellular vesicles (EVs). In vivo live-imaging shows that immature OCs actively interact with osterix:mCherry + OBs and engulf EVs prior to convergence at the fracture site. In vitro cell culture assays show that OB-derived EVs promote OC differentiation via Rankl signaling. Collectively, these data suggest that EV-mediated intercellular communication with OBs plays an important role in the differentiation of OCs in bone tissue.