Figure 5: Chitin is expressed endogenously and its colocalization with VCBP-C is independent of microbial exposure.

(a) Signals for VCBP-C (Alexa Fluor 594, red) and chitin (Fc-CBD-C DyLight 488, green) are colocalized (yellow, arrow) in the intestinal region of the gut primordium of late rotation stage juveniles maintained under germ-free conditions and persists throughout development. Gut development is complete by stage 5/7, with the immediate onset of feeding¹³. (b) Intestinal mucus is chitin-rich (in green, arrow). (c) Magnified view of gut (from b) in which VCBP-C (in red, arrow) is distributed primarily at the edges of the gut tissue surfaces. Chitin-rich pellets are seen in the distal gut and are purged into environment before feeding. (d) Magnified view of the stomach demonstrates chitin-rich mucus and VCBP-C in the epithelium, red. (e) Chitin (green) is prominent and restricted to the stomach and midgut epithelium 2–3 weeks post fertilization; chitin-rich mucus cannot be detected in the oesophagus or branchial basket. Intense signal is evident at the outer edges and are more prominent on the ventral side (arrows) of the stomach and midgut; a chitin signal also is prominent in fecal pellets (not visible in e) but cannot be detected in the hindgut epithelium. (f) Chitin is prominent throughout the gut to the anus in the whole-mount staining of young adults (second ascidian stage and onwards); images of both sides of the stomach are included to emphasize enhanced signal in ridges of the epithelial grooves (inset). Bright field overlays are shown in a,b,e. Scale bars (a,c), 50 μm; (b,e), 100 μm; (d) 25 μm; and (f) 200 μm. BB, branchial basket; E, epithelium; es, oesophagus; hg, hindgut; L, stomach lumen; mg, midgut; st, stomach.