Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control.

Apical extracellular matrix filling the lumen controls the morphology and geometry of epithelial tubes during development, yet the regulation of luminal protein composition and its role in tube morphogenesis are not well understood. Here we show that an endosomal-retrieval machinery consisting of Rab9, retromer and actin nucleator WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog) regulates selective recycling of the luminal protein Serpentine in the Drosophila trachea. Secreted Serpentine is endocytosed and sorted into the late endosome. Vps35, WASH and actin filaments differentially localize at the Rab9-enriched subdomains of the endosomal membrane, where Serpentine containing vesicles bud off. In Rab9, Vps35 and WASH mutants, Serpentine was secreted normally into the tracheal lumen, but the luminal quantities were depleted at later stages, resulting in excessively elongated tubes. In contrast, secretion of many luminal proteins was unaffected, suggesting that retrograde trafficking of a specific class of luminal proteins is a pivotal rate-limiting mechanism for continuous tube length regulation.

Conversion of plasma membrane topology during epithelial tube connection requires Arf-like 3 small GTPase in Drosophila.

The development of tubular organs often involves the hollowing of cells into a torus (doughnut shape), as observed in blood vessel formation in vertebrates and tracheal development in insects. During the fusion of Drosophila tracheal branches, fusion cells located at the tip of migrating branches contact each other and form intracellular luminal cavities on opposite sides of the cells that open to connect the tubule lumens. This process involves the intracellular fusion of plasma membranes associated with microtubule tracks. Here, we studied the function of an evolutionarily conserved small GTPase, Arf-like 3, in branch fusion. Arf-like 3 is N-terminally acetylated, and associates with both intracellular vesicles and microtubules. In Arf-like 3 mutants, the cell adhesion of fusion cells, specification of apical membrane domains, and secretion of luminal extracellular matrix proceeded normally, but the luminal cavities did not open due to the failure of intracellular fusion of the plasma membranes. We present evidence that the Arf-like 3 mutation impairs the localized assembly of the exocyst complex, suggesting that the targeting of exocytosis machinery to specific apical domains is the key step in converting the plasma membrane topology in fusion cells.

Phenotypic analysis of a novel chordin mutant in medaka.

We have isolated and characterized a ventralized mutant in medaka (the Japanese killifish; Oryzias latipes), which turned out to have a mutation in the chordin gene. The mutant exhibits ventralization of the body axis, malformation of axial bones, over-bifurcation of yolk sac blood vessels, and laterality defects in internal organs. The mutant exhibits variability of phenotypes, depending on the culture temperature, from embryos with a slightly ventralized phenotype to those without any head and trunk structures. Taking advantages of these variable and severe phenotypes, we analyzed the role of Chordin-dependent tissues such as the notochord and Kupffer's vesicle (KV) in the establishment of left-right axis in fish. The results demonstrate that, in the absence of the notochord and KV, the medaka lateral plate mesoderm autonomously and bilaterally expresses spaw gene in a default state.

Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle.

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.