Fig. 4

Characterization of VAChT antibodies and VAChT-ir (VAChT, magenta) in oyster, Crassostrea gigas, tissues. a Western blot of total protein lysates from adult oyster tissue probes stained with goat polyclonal antibodies against rat VAChT. The specific band is detected in all tested oyster tissues as well as in cell lysate from the mouse spinal cord. b Double-staining for VAChT/tubulin (VAChT/TUB) of frozen sections of adult oyster tissues. A strong positive signal is detected in the anterior adductor muscle (muscle), mantle, and gills at the structures corresponding to the nerve bundles. c-e Confocal images of the larvae stained with VAChT/tubulin, right side view; the anterior is always up. c D-hinge veliger. The apical organ (AO) contains two to three cells and their basal fibers. Paired solitary neurons located posteriorly on the right and left sides of the larval body (pn). Each posterior cell sends an anteriorly directed fiber along the ventral edge of the larval body (arrows) (an asterisk marks the neuron nuclei). c1 Bodies of two AO neurons; asterisks mark the nuclei. c2 Posterior neuron, an asterisk marks the nucleus. d In the veliger stage (92 hpf), a strong VAChT-ir signal is detected within the AO/CG complex, and single fibers appear to innervate the velum (arrowheads). Immunopositive fibers run in two parallel, ventral cords (vnc), and a solitary posterior cell is visible at the caudal end of each ventral cord. Immunopositive cells appear in the PG. e At the 7-dpf veliger stage, VAChT-ir fibers from AO/CG innervate the velum, and fibers from PG innervate the foot (f) and regions around the mouth. g Summary diagram of the ontogeny of the VAChT-ir-containing structures in C. gigas. Only one lateral side is shown. Additional abbreviations: a – anus, m – mouth. Scale bars =100 μm in b and 20 μm in c