Abstract
Variable region-containing chitin-binding proteins (VCBPs) are secreted, immune-type molecules that have been described in both amphioxus, a cephalochordate, and sea squirt, Ciona intestinalis, a urochordate. In adult Ciona, VCBP-A, -B and -C are expressed in hemocytes and the cells of the gastrointestinal tract. VCBP-C binds bacteria in the stomach lumen and functions as an opsonin in vitro. In the present paper the expression of VCBPs has been characterized during development using in situ hybridization, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) technologies. The expression of VCBP-A and -C is detected first in discrete areas of larva endoderm and becomes progressively localized during differentiation in the stomach and intestine, marking the development of gut tracts. In “small adults” (1–2 cm juveniles) expression of VCBP-C persists and VCBP-A gradually diminishes, ultimately replaced by expression of VCBP-B. The expression of VCBP-A and -C in stage 7–8 juveniles, at which point animals have already started feeding, is influenced significantly by challenge with either Gram-positive or -negative bacteria. A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.
Highlights
The variable region containing chitin-binding proteins (VCBPs), which possess a domain organization consisting of a leader peptide, two tandem N-terminal immunoglobulin V-type domains and a single C-terminal chitin-binding domain, belong to a multigene family encoding secreted proteins [1,2]
Expression patterns, genetic diversity and structural similarities with V-type domains of immunoglobulins and T cell receptors suggest that VCBPs represent a unique gut-associated form of innate immune proteins [1] and could reflect an important transition between non-rearranging immune molecules and the conventional rearranging antigen-binding receptors of jawed vertebrates [3]
It has been shown that the gut is the major site of expression of VCBPs, it was unclear when these genes are expressed in the developing animal
Summary
The variable region containing chitin-binding proteins (VCBPs), which possess a domain organization consisting of a leader peptide, two tandem N-terminal immunoglobulin V-type domains and a single C-terminal chitin-binding domain, belong to a multigene family encoding secreted proteins [1,2]. In fully differentiated adult intestine, whole mount in situ hybridization (WISH) reveals expression of VCBP-C in a ringshaped structure at the junction between the stomach and the intestine, and in the hind-gut; as in stage 4 of 1st ascidian juvenile, the mid-gut is clear (Fig. 4). VCBP-C expression retraces the same pattern of hybridization observed in the 7 and 8 juvenile stages, as seen in adult stomach, in the crypts of the stomach ridges (Fig. 6F). VCBP-A could be detected in the lamina propria, both as a secreted molecule and in the granular amoebocytes populating this compartment (Fig. 9C) Quite often this type of hemocyte is associated with the basement membrane of the stomach cells and apposed to their plasma membrane (Fig. 9C and D). The opposite result occurred in response to the E. coli challenge in which a minor decrease in the expression of VCBP-A mRNA and a two-fold increase in the expression of VCBP-C mRNA (Fig. 10B) were observed
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