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Fig. 3 | BMC Evolutionary Biology

Fig. 3

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

Fig. 3

Innervation of apical organ and esophageal region in early developmental stages of O. fusiformis (1 dpf - 14 dpf). FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue). Confocal z-projections and volume renderings of FMRFamide-LIR (D, E, H (inset)). Ventral is left and apical is up in all images, except (d) which shows a ventral view. Stages are given in days post fertilization (dpf). The shape of the esophagus is indicated by white lines. a Early stages exhibit weak FMRFamide-LIR in the apical organ (ao) (white dotted circle). The inset shows the apical organ (ao) (white dotted circle) in higher magnification. Note that the staining in the chaetal sacs (cs) and the chaetae (ch) is due to autofluorescence. The yellow signal along the ciliary band (cb) is caused by unspecific staining and differed between the investigated specimens. The inset shows the FMRFamide-LIR underlying the apical organ (white dotted circle) in higher magnification. b, c At 2 dpf the mitraria shows strong 5-HT-LIR in the circumesophageal connective (cc) and in the tissue underlying the esophagus (es). No 5-HT-LIR is visible underlying the apical tuft (at). Note that the purple dots surrounding the apical tuft (at) are labeling artifacts. Position of the apical tuft (at) is indicated by sketch. Note that the 5-HT-LIR in the posterior larva is restricted to the midgut and the chaetal sacs. d At 7 dpf the apical circumesophageal connective (cc) shows distinct FMRFamide-LIR. Contour of the apical nerve ring (ar) is denoted (red dotted line), prominent perikarya are marked (red asterisk). e Lateral view of previous stage. Contour of the apical ring (ar) and the circumesophageal connective (cc) is denoted (dotted red line), prominent perikaryon is marked (red asterisk). f 5-HT-LIR at 7 dpf is present in the circumesophageal connective (cc), an esophageal nerve (en) with branching nerve fibers (eb) and the tissue underlying the esophagus (es). Note distinct 5-HT-LIR in at least 1 perikaryon (arrow). The connection of the latter perikarya with the esophageal nerve (en) is indicated by white dots. The inset shows the 5-HT-LIR (now grey) within the tissue underlying the esophagus in higher resolution. Notably, the signal underlying the esophageal region may also contain some unspecific staining due to the possible presence of gland cells in the esophagus. g At 14 dpf the 5-HT-LIR (and aTub-LIR) increases, but the pattern is comparable to the previous stage. No 5-HT-LIR is detectable apically (lack of signal indicated by red dotted circle). The inset shows a higher magnification of the tissue underlying the apical tuft. The purple dots surrounding the region of interest are labeling artifacts. Note that the overlap of the 5-HT-LIR and the aTub-LIR causes a white color in some parts of the shown image. The esophageal branching nerves are not shown in the image. h FMRFamide-LIR at 14 dpf is restricted to the apical ventral (vr) and dorsal root (dr) of the circumesophageal connective, the apical nerve ring and apical perikarya, and detectable in the tissue underlying the esophagus (white dotted circle). Inset shows volume rendering of FMRFamide-LIR. Position of the apical tuft (at) is indicated by sketch. Position of perikarya (red asterisks), apical nerve ring (ar, red dotted line) and roots of the circumesophageal connective (vr, dr, red dotted line) is indicated. Both roots of the ventral circumesophageal connective (vpvr, dpvr) are detectable by tubulin staining (dotted red line). 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 the same volume rendered FMRFamide-LIR. The shape of neural structures is implemented by red dotted lines. ao, apical organ; ar, apical nerve ring; at, apical tuft; cb, ciliary band; cc, circumesophageal connective; ch, chaetae; cs, chaetal sac; dr, dorsal root of cc; dpvr, dorsal part of vr; eb, branches of the esophageal nerve; en, esophageal nerve; es, esophagus; gl, gland; mg, midgut; vr, ventral root of cc; vpvr, ventral part of vr. Scale bars = 30 μm (a), 100 μm (b), 15 μm (d, e), 50 μm (f-h). The scale bars only refer to the main images, not to the insets. c represents a more detailed picture of (b) and therefore has no scale bar

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