Supporting evidence for the contractile nature of fish branchial pillar cells was provided by demonstrating the presence of actin fibers and a novel four-and-a-half LIM (FHL) protein in which expression is specific for contractile tissues and sensitive to the tension applied to the pillar cell. When eel gill sections were stained with rhodamine-phalloidin, a selective fluorescent probe for fibrous actin, a strong bundle-like staining was observed around collagen columns in pillar cells, suggesting the presence of abundant actin fibers. A cDNA clone encoding a novel member of the actin-binding FHL family, FHL5, was isolated from a subtracted cDNA library of eel gill. Northern analysis revealed that FHL5 mRNA is highly expressed only in gills, heart, and skeletal muscle. In gills, FHL5 was found to be confined to pillar cells by immunohistochemistry. Confocal fluorescence microscopy showed that FHL5 is present in both cytosol and nucleus; within the cytosol, a large portion of FHL5 is colocalized with the phalloidin-positive actin bundles. Furthermore, transfection of myogenic C2C12 cells with FHL5 cDNA demonstrated, in addition to its interaction with actin stress fibers, a nuclear shuttling activity of FHL5. The mRNA and protein levels were found to be elevated on 1) transfer of eels from seawater to freshwater, 2) volume expansion by infusion of isotonic dextran-saline, and 3) constriction of gill vasculature by bolus injection of endothelin-1. These results suggest contractile nature of pillar cells and a role of FHL5 in maintaining the integrity and regulating the dynamics of pillar cells. C2C12 myoblast; endothelin; immunohistochemistry; lamella; zinc finger; four-and-a-half LIM FISH GILLS CONSIST OF a large number of filaments arranged along the gill arches. The surfaces of the filaments are greatly enlarged by a series of plate-like lamellae. Each lamella is composed of two sheets of epithelia separated by a thin space through which the blood circulates to allow the exchange of respiratory gases. The separation between the epithelial sheets is maintained by pillar cells and collagen bundles. Pillar cells are spool-shaped cells connecting two epithelial sheets of the respiratory lamella in fish gills (50,51,64). They are characterized by collagen bundles contained in the infoldings of the plasma membrane and are oriented perpendicularly to the epithelial sheets, which consist of a thin layer of pavement cells and a basal lamina that is continuous with the collagen bundles traversing the pillar cells (49, 64); for the anatomy of gill lamella, see the schematic illustration in Fig. 9D. The membrane-enclosed collagen bundles help prevent ballooning of the lamella. The inner surfaces of the two epithelial sheets are covered with flanges extended from the pillar cells, forming capillary lumen called lacunae. Another feature of the pillar cells is the presence of numerous myofilament-like structures that course through the cytoplasm in an orientation parallel to the collagen bundles. These myofilaments appear to ...

LIM-domain-binding proteins (CLIM/NLI/Ldb) are nuclear cofactors for LIM homeodomain transcription factors (LIM-HDs) and LIM-only proteins (LMOs). The LIM-interaction domain (LID) of Ldb is located in the carboxy-terminal region and encoded by the last exon (exon 10) of Ldb genes. It is known that the mammalian CLIM1/Ldb2 gene has a splice isoform, named CLIM1b, lacking the LID. However, little is known about the nature of CLIM1b or the evolutionary conservation of this type of alternative splicing in amphibians and teleost fish. Here, we demonstrate that splice isoforms lacking the LID are also present in the Ldb1 genes of mammals, chick, and Xenopus, as well as in fish paralog Ldb4. All these splicing variations occur in intron 9 and exon 10. We observed that Ldb4b (splice isoform lacking LID) is localized in the nucleus when expressed in mammalian culture cells, and binds to Ldb4a (splice isoform containing LID) but not directly to LIM proteins. However, Ldb4b binds to LMO4 via Ldb4a when coexpressed in culture cells. We also found that mouse Ldb1b lacks the ability to activate protein 4.2 promoter, which is stimulated by LMO2 and Ldb1. These findings suggest that splice isoforms of Ldb lacking LID are potential regulators of Ldb function.

S U M M A R Y Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae. (J Histochem Cytochem 55:941-953, 2007)

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