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Fig. 4 | BMC Evolutionary Biology

Fig. 4

From: Owenia fusiformis – a basally branching annelid suitable for studying ancestral features of annelid neural development

Fig. 4

Innervation of apical organ and esophageal region in late developmental stages of O. fusiformis (21 dpf - 28 dpf). FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue). Confocal z-projections and volume renderings of 5-HT-LIR (a; a, inset) (except of (h) where 5-HT-LIR is shown in grey), FMRFamide-LIR (c, d, f) and aTub-LIR (e, inset). Ventral is left and apical is up in all images, except of (b) which shows a view from dorsal and (h) where the specimen is shown in antero-dorsal view with the anterior part down. Stages are given in days post fertilization (dpf). The shape of the esophagus is indicated by white lines. a At 21 dpf 5-HT-LIR is visible in the circumesophageal connective (cc) and its roots (vr, dr), within the esophageal nerve (en, dotted line) including all branching fibers and the tissue underlying the esophagus. Position of the apical tuft (at) is indicated by sketch. The inset shows absence of 5-HT-LIR in the apical region (indicated by white arrow heads). b The 5-HT-LIR frames the entire esophagus. Position of the esophageal nerve (en) is indicated by the dotted line. Note that -LIR is absent in the region of the apical organ. c Although both roots of the circumesophageal connective (cc) are detectable via anti-tubulin staining at 21 dpf, FMRFamide-LIR is restricted to the dorsal root (dr) including prominent lateral perikarya (arrow head), the apical nerve ring (red dotted line in inset), and the distinct apical perikarya (red asterisks). The inset shows the FMRFamide-LIR in the same view without background staining. The position of the apical ring is implicated by red dots. The distinct apical perikarya are marked (red asterisks). d A lateral view reveals prominent FMRFamide-LIR in the tissue underlying the esophagus (es) (red dotted circle). The inset shows the dorsal root of circumesophageal connective (dr) and the tissue underlying the esophagus (es) (red dotted circle) in confocal z-projection (with the same scale). Note the prominent lateral (arrow head) and apical perikarya (red asterisks). e At 28 dpf, approximately 1 week after metamorphosis, FMRFamide-LIR is can be detected within the dorsal root of the circumesophageal connective (dr), in the ventral nerve cord (vn) and numerous antero-ventral perikarya. Inset shows the FMRFamide-LIR of the prominent dorsal and ventral part of the dorsal root of the circumesophageal connective (vpdr, dpdr). Note that the overlap of the FMRFamide-LIR and the aTub-LIR causes a white color in some parts of the shown image. The inset shows only the FMRFamide-LIR. f FMRFamide-LIR reveals presence of the apical perikarya in juveniles (red asterisk) and shows a prominent dorsal assemblage of perikarya (arrow head). g 5-HT-LIR is exhibited within both roots (and the dorsal and ventral part of dr) of the circumesophageal connective (vpdr, dpdr, vr) and the tissue underlying the esophagus (dotted white line). Note that the pattern of -LIR in this tissue is invaginated compared to pre-metamorphic stages. h An antero-dorsal view reveals the presence of both parts of both circumesophageal roots (vpvr, dpvr, vpdr, dpdr) and perikarya (white asterisks) with 5-HT-LIR close to the roots. The tissue underlying the esophagus is detectable (dotted white line). Note that the pattern of -LIR in this tissue is invaginated compared to pre-metamorphic stages. at, apical tuft; cc, circumesophageal connective; dpdr, dorsal part of dr; vpdr, ventral part of dr; dpvr, dorsal part of vr; dr, dorsal root of cc; en, esophageal nerve; es, esophagus; gl, gland; vpvr, ventral part of vr; vr, ventral root of cc. Scale bars = 50 μm (a–e) and 30 μm (h). The scale bars only refer to the main images, not to the insets

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