Fig. 2

FMRFamide-immunoreactivity (FMRFa-ir) in the trochophore and veliger larvae of C. gigas. Green—FMRFa-ir; magenta—cilia, acetylated tubulin immunoreactivity; blue—nuclei, DAPI. The apical pole is always upward; the ventral side is on the right. a Trochophore at 20 hpf. Dorsal (arrowheads) and ventral (asterisks) groups of early neurons located posttrochally. a1 Higher magnification of right dorsal neuron (dn); arrowheads indicate a ciliated dendritic knob (ck) and basal neurites (n). a2 and a3 Right and left ventral neurons (vn, asterisks), respectively. b Trochophore at 24 hpf. Faint dotted immunoreactivity appears at the apical region (arrows). A dorsal cell sends an axon towards the ventral side (arrowheads). Three ventral cells indicated by one arrow (flask-shaped cells) and asterisks (round cells). b1 and b2 Higher magnification of the right and left dorsal neurons (dn), arrowheads indicate the ciliated dendritic knob (ck) of each cell. b3 and b4 Higher magnification of right and left ventral groups (vn); each ventral group contains two flask-shaped cells (arrows) and two round cells (asterisks). c Trochophore at 28 hpf. Two flask-shaped cells appear in the apical organ (AO). Growth cone (arrows, gc) of the right dorsal cell (dn), where a growing axon reaches the ipsilateral ventral cell (vn); only the right sides of the larvae are shown. c1-c4 Right and left dorsal and ventral groups of neurons with DAPI. c5 and c6 Two flask-shaped neurons of the AO at two different magnifications (asterisks). c7 Higher magnification of a growing axon of the right dorsal cell; arrowhead indicates a ciliated dendritic knob, and arrows indicate a growth cone of the dorsal neuron. d D-hinge veliger stage at 36 hpf. The cells of the AO are located at the top (asterisks); no connections exist between the AO and other early cells. d1 Two flask-shaped apical neurons (asterisks) and their basal neurites. d2 and d3 Long axons of both the right and left dorsal cells reach the ipsilateral groups of the ventral cells. e Ventral view of the veliger at 36 hpf. Arrowheads indicate projections from dorsal and apical cells towards the ventral neurons. insets Higher magnification of the left and right ventral groups. e1 Higher magnification of the apical neurons demonstrating long cilia at the end of their dendrite (arrows). f The veliger stage at 52 hpf and 60 hpf (insert). Dorsal (dn) and ventral (vn) cells stain in a punctate pattern and their axons form an anlagen of the ventral nerve cord. Small immunopositive neurons appear posteriorly (posterior neurons, arrow, pn). Insert: Four flask-shaped apical neurons (asterisks) of AO.f1 Higher magnification of the AO (asterisk). f2 Long axon of a dorsal neuron (dn) to the ventral neuron (vn). f3 Neurites extending from the AO (arrow, an) follow the path established by pioneer axons of the dorsal neurons. g The veliger stage at 96 hpf. AO/CG consist of six flask-shaped cells (asterisks) and basal processes of AO/CG neurons organize into a compact central neuropil (arrow, np). A single neurite (n, arrowhead) extends from the neuropil of the AO/CG toward the velum. Paired ventral cords (vnc) with the interconnecting commissures (short arrows) are clearly visible. The anlagen of the pedal ganglia (PG) appear along each ventral cord in the region of the developing foot. g1 Flask-shaped neurons (asterisks) with neuropil forming (Z12). g2 Flask-shaped neurons (asterisks) with neuropil forming (Z14). g3 and g4 Neurons within the PG under high magnification (asterisks). g5 Posterior neurons under high magnification (asterisk). h The late veliger stage at 9 dpf. Neurites extend from the AO/CG to the dorsal side and dorsal edge of the velum (arrowheads). The anlagen of the PG are located along the ventral cord (vnc). Paired ventral cords are connected by two commissures (short arrows). h1 Compact neuropil in the center of the AO/CG. h2 and h3 Right and left PG anlagen; arrows point to the neuronal nuclei. h4 and h5 Higher magnification of the right and left posterior neurons; the asterisk marks the cell nucleus. i 3D reconstruction of the AO/CG complex, paired ventral cords and PG anlagen of 9-dpf late-veliger larvae. j Summary diagram of the ontogeny of the FMRFa-ir-containing structures in C. gigas. Only one lateral side is shown. Arrowhead indicates an axon of a dorsal cell in the late trochophore. Additional abbreviations: a – anus, f – foot, m – mouth, pmo – presumptive mouth opening, pt – prototroch, st – stomach. Scale bars =20 μm