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Expression of Ciona intestinalis variable region-containing chitin-binding proteins during development of the gastrointestinal tract and their role in host-microbe interactions.

Liberti A, Melillo D, Zucchetti I, Natale L, Dishaw LJ, Litman GW, De Santis R, Pinto MR - PLoS ONE (2014)

Bottom Line: 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.A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Physiology and Evolution, Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy.

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.

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WISH of VCBP-A and -C at stages 4, 5 and 7 of 1st ascidian juvenile.VCBP-A is localized in the stomach structure in all the stages examined (A–C). In particular, semi-thin sections (D) of WISH samples at the stage 7 indicate that VCBP-A is expressed in scattered cells of the stomach epithelium. VCBP-C is localized in part of the developing intestine and in a ring-shaped structure at the junction between the stomach and the intestine (E and I) at stage 4, whereas at stage 5 and 7, it is expressed in both the stomach (F and G) and in the intestine (J and K). Semi-thin sections of WISH samples confirm that at stage 7 VCBP-C is confined to groups of cells localized in the ridges of the stomach (H) and distributed evenly in the intestine (L). s, stomach; arrow, ring-shaped structure; arrowhead, intestine. (Scale bars: A, B, E, F, I and J, 50 µm; C, G and K, 100 µm; D, H and L, 15 µm).
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pone-0094984-g003: WISH of VCBP-A and -C at stages 4, 5 and 7 of 1st ascidian juvenile.VCBP-A is localized in the stomach structure in all the stages examined (A–C). In particular, semi-thin sections (D) of WISH samples at the stage 7 indicate that VCBP-A is expressed in scattered cells of the stomach epithelium. VCBP-C is localized in part of the developing intestine and in a ring-shaped structure at the junction between the stomach and the intestine (E and I) at stage 4, whereas at stage 5 and 7, it is expressed in both the stomach (F and G) and in the intestine (J and K). Semi-thin sections of WISH samples confirm that at stage 7 VCBP-C is confined to groups of cells localized in the ridges of the stomach (H) and distributed evenly in the intestine (L). s, stomach; arrow, ring-shaped structure; arrowhead, intestine. (Scale bars: A, B, E, F, I and J, 50 µm; C, G and K, 100 µm; D, H and L, 15 µm).

Mentions: Gut differentiation proceeds, although feeding has not commenced, at stage 4 in the early development of juvenile ascidians [12]; the oesophagus, stomach and intestine can be distinguished. VCBP-A mRNA is localized in the stomach and restricted only to some cells in the region closer to the oesophagus (Fig. 3A). By contrast, VCBP-C is expressed in a small ring of cells at the border between the stomach and the intestine, as well as in a more extended region of the intestine that corresponds to the hind-gut (Fig. 3E and I). It has not been possible to detect expression of VCBP-C in the mid-gut. In fully differentiated adult intestine, whole mount in situ hybridization (WISH) reveals expression of VCBP-C in a ring-shaped 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). The corresponding controls carried out with sense probes are shown in Figure S2 (G and H).


Expression of Ciona intestinalis variable region-containing chitin-binding proteins during development of the gastrointestinal tract and their role in host-microbe interactions.

Liberti A, Melillo D, Zucchetti I, Natale L, Dishaw LJ, Litman GW, De Santis R, Pinto MR - PLoS ONE (2014)

WISH of VCBP-A and -C at stages 4, 5 and 7 of 1st ascidian juvenile.VCBP-A is localized in the stomach structure in all the stages examined (A–C). In particular, semi-thin sections (D) of WISH samples at the stage 7 indicate that VCBP-A is expressed in scattered cells of the stomach epithelium. VCBP-C is localized in part of the developing intestine and in a ring-shaped structure at the junction between the stomach and the intestine (E and I) at stage 4, whereas at stage 5 and 7, it is expressed in both the stomach (F and G) and in the intestine (J and K). Semi-thin sections of WISH samples confirm that at stage 7 VCBP-C is confined to groups of cells localized in the ridges of the stomach (H) and distributed evenly in the intestine (L). s, stomach; arrow, ring-shaped structure; arrowhead, intestine. (Scale bars: A, B, E, F, I and J, 50 µm; C, G and K, 100 µm; D, H and L, 15 µm).
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pone-0094984-g003: WISH of VCBP-A and -C at stages 4, 5 and 7 of 1st ascidian juvenile.VCBP-A is localized in the stomach structure in all the stages examined (A–C). In particular, semi-thin sections (D) of WISH samples at the stage 7 indicate that VCBP-A is expressed in scattered cells of the stomach epithelium. VCBP-C is localized in part of the developing intestine and in a ring-shaped structure at the junction between the stomach and the intestine (E and I) at stage 4, whereas at stage 5 and 7, it is expressed in both the stomach (F and G) and in the intestine (J and K). Semi-thin sections of WISH samples confirm that at stage 7 VCBP-C is confined to groups of cells localized in the ridges of the stomach (H) and distributed evenly in the intestine (L). s, stomach; arrow, ring-shaped structure; arrowhead, intestine. (Scale bars: A, B, E, F, I and J, 50 µm; C, G and K, 100 µm; D, H and L, 15 µm).
Mentions: Gut differentiation proceeds, although feeding has not commenced, at stage 4 in the early development of juvenile ascidians [12]; the oesophagus, stomach and intestine can be distinguished. VCBP-A mRNA is localized in the stomach and restricted only to some cells in the region closer to the oesophagus (Fig. 3A). By contrast, VCBP-C is expressed in a small ring of cells at the border between the stomach and the intestine, as well as in a more extended region of the intestine that corresponds to the hind-gut (Fig. 3E and I). It has not been possible to detect expression of VCBP-C in the mid-gut. In fully differentiated adult intestine, whole mount in situ hybridization (WISH) reveals expression of VCBP-C in a ring-shaped 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). The corresponding controls carried out with sense probes are shown in Figure S2 (G and H).

Bottom Line: 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.A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Physiology and Evolution, Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy.

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.

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