Kaul-Strehlow S , Urata M , Minokawa T , Stach T , Wanninger A .

M 1485 Austrian Science Fund FWF

	Fig. 1. Phylogenetic relationships of major metazoan taxa compiled from recent molecular analyses (i.e., Hejnol et al. 2009; Philippe et al. 2011). Schematic representation of the adult nervous system is included for each taxon. For more information, see text
	Fig. 2. Neurogenesis in metamorphosing stages of B. misakiensis. b, c, e–k Z-projections of confocal microscopy image stacks. a, d Scanning electron micrographs. Anterior is to the top. a SEM of early metamorphosing stage (Spengel) from lateral left. Arrowhead points to the dorsolateral slit-like depression. b Overview of the serotonin-LIR nervous system (NS) in Spengel stage, view from left. Numerous serotonin-LIR neurons are part of the apical organ. Note the serotonin-LIR opisthotroch neurite ring. c Overview of the serotonin-LIR NS of a late metamorphosing stage (Agassiz), dorsal view. d Dorsal view of metamorphosing Agassiz stage. e Detail showing the developing 5-HT+ nervous plexus in the postoral region of the Spengel stage. f Close-up of the 5-HT+ apical organ of the Spengel stage in lateral view. Note the anterior and posterior cluster of bipolar neurons connected by a central neuropil. g Detail showing the elaborated 5-HT+ nervous plexus in the prospective trunk region of an Agassiz stage larva. h Detail view of 5-HT+ apical bipolar neurons. A slender distal neurite connects the soma to the apical cell surface. i Detail of the neuite bundles passing the neck region, connecting the proboscis to the collar region. k Close-up of the dorsal collar region showing the 5-HT+ bipolar neurons. 5-HT, serotonin; ac-α-tub, acetylated α-tubulin; ao, apical organ; at, apical tuft; ci, cilia; co, collar; ne, neurite; nn, nervous plexus; np, neuropil; onr, opisthotroch nerve ring; ot, opisthotroch; pf, perianal field; pr, proboscis; sn, serotonin-LIR neuron; tr, trunk
	Fig. 3. Ultrastructural details of the developing nervous system Balanoglossus misakiensis. a Sagittal section of an Agassiz stage larva of B. misakiensis. b Sagittal section of a 2-gill-slit juvenile of B. misakiensis. c Ultrastructural detail of the red-marked box in a showing a continuous layer of neurites of 4–6-μm thickness in the Agassiz stage. Inset color-coded image of the micrograph shown in c. d Ultrastructural detail of the red-marked box in b showing only individual basiepidermal neurites in the juvenile. Inset Color-coded image of the micrograph shown in d. Color code for insets: ectoderm, light blue; neurites, yellow; basal lamina (ecm), dark blue; mesoderm, red. bl, blastocoel; ecm, extra cellular matrix; epc, epidermal cell; gp, gill pore; i, intestine; ms, mesocoel; mt, metacoel; mtc, metacoelic cell; myo, myofilaments; ne, neurite; nu, nucleus; pc, protocoel; yo, yolk
	Fig. 4. Neurogenesis in early settled stages of B. misakiensis. a, c–i, l, m Z-projections of confocal microscopy image stacks. b, k Scanning electron micrographs. a Ventrolateral overview of the serotonin-LIR NS of an early settled juvenile. Anterior is to the left. b Left side view of early settled juvenile. c Detail of the anterior trunk region of early settled juvenile showing a ubiquitous basiepidermal nervous plexus. d Partial Z-projection showing the longitudinally orientated neurites within the collar region. e Detail of the 5-HT+ proboscis plexus at the base of the proboscis and the neurite bundles passing through the subepidermal collar cord. f, g Close-up of the gill pore and mesocoelic pore showing the prebranchial nerve ring and the nervous plexus entangling the mesocoelic duct in f. h SEM of a settled juvenile in lateral right view. i Close-up of the apical tip of the proboscis. k Detail of the dorsolateral collar region showing two clusters of 5-HT+ bipolar neurons that encircle the collar. l Overview of the 5-HT+ NS of a settled juvenile 24-h postsettlement, lateral right view. m Detail showing circular 5-HT+ neurites within the posterior part of the trunk whereas more anterior a net-like pattern is still present. The apical tuft and cluster of 5-HT+ somata has disappeared. Apical is to the top. 5-HT, serotonin; ac-α-tub, acetylated α-tubulin; a, anus; ao, apical organ; cgn, ciliary groove nerves; cn, circumferential neurite; cc, collar cord; co, collar; gs, gill slit; mcb, middle circular neurite bundle; msp, mesocoel pore; ne, neurite; nn, nervous plexus; onr, opisthotroch nerve ring; pnr, prebranchial nerve ring; pr, proboscis; ps, proboscis stem; sn, serotonin-LIR neuron; tr, trunk; vnb, ventral neurite bundle
	Fig. 5. Neurogenesis in the 2-gill slit juvenile of B. misakiensis. a, b Scanning electron micrographs: c–i Z-projections of confocal microscopy image stacks. Anterior is to the right. a Dorsolateral view of a 3-day-old settled juvenile. b Higher magnification of the two gill slits and the dorsal tongue bars. c Overview of the 5-HT+ NS of a 3-day-old juvenile. d The collar cord is composed of three 5-HT+ neurite bundles of which the median one projects posteriorly into the dorsal nerve cord. e Detail of the ventral 5-HT+ neurite bundle with numerous incoming circular neurites emerging from lateral bipolar neurons. f Detail of the anterior part of the dorsal 5-HT+ neurite bundle and epidermal collar region. This part is discontinuous with the posterior part of the 5-HT neurite bundle shown in g, see also double arrowheads and dashed area. g The posterior part of the dorsal 5-HT+ neurite bundle contains few serotonin-LIR neurites and is discontinuous with the anterior part of the 5-HT neurite bundle, see double arrowheads and dashed area. h Partial Z- projection of a sagittal scan of the dorsal collar region. The collar cord comprises ventral neurite bundles and a dorsal sheath of somata, which are not serotonin-LIR positive. i Close-up of a part of the proboscis region showing the 5-HT+ bipolar neurons and the basiepidermal nervous plexus. 5-HT, serotonin; a, anus; ac-α-tub, acetylated α-tubulin; cn, circumferential neurite; cc, collar cord; ci, cilia; co, collar; dnb, dorsal neurite bundle; ep, epidermis; gs, gill slit; msp, mesocoel pore; nn, nervous plexus; onr, opisthotroch nerve ring; pnr, prebranchial nerve ring; pr, proboscis; ps, proboscis stem; sn, serotonin-LIR neuron; tb, tongue bar; tm, trunk musculature; vnb, ventral neurite bundle
	Fig. 6. Neurogenesis in S. kowalevskii. a–C, f–k, m–o Z-projections of confocal microscopy image stacks. d, e, l Scanning electron micrographs (SEM). Anterior is to the right and ventral to the bottom in all images. a Overview of a dorsal kink stage. Serotonin-LIR neurons are present in the proboscis epidermis. Note the larval apical ciliary tuft. b Detail showing the developing nervous plexus in the trunk region of a dorsal kink stage. c Partial Z-projection of the anterior tip of a dorsal kink stage highlighting the absence of serotonin-LIR somata from the apical plate. d SEM of dorsal kink stage. e SEM of 1-gill-slit stage. f Partial Z-projection of the anterior tip of a 1-gill-slit stage. g Detail showing an elaborate 5-HT+ nervous plexus in the trunk region of the 1-gill-slit stage. h Overview of a 1-gill-slit stage showing the entire serotonin-LIR nervous system. i Close-up of the dorsal collar region demonstrating the 5-HT+ part in the neurulating collar cord. Double arrowheads point to dorsal connections of the anterior portion of the collar cord that is still in contact with the epidermis. k Overview of the serotonin-LIR nervous system of a 3-gill-slit juvenile. l SEM of a 3-gill-slit juvenile in lateral view. m Detail of the anterior trunk region of a 3-gill-slit juvenile showing scattered serotonin-LIR bipolar neurons projecting into the ventral neurite bundle. n Detail of two biploar neurons within the proboscis epidermis. o Detail of the collar region showing 5-HT+ neurites within the subepidermal collar cord. 5-HT, serotonin; ac-α-tub, acetylated α-tubulin; at, apical tuft; cn, circumferential neurite; cc, collar cord; co, collar; dnb, dorsal neurite bundle; ep, epidermis; gs, gill slit; ms, mesocoel; nn, nervous plexus; ot, opisthotroch; pat, postanal tail; pc, protocoel; pf, perianal field; ph, pharynx; pr, proboscis; ps, proboscis stem; sn, serotonin-LIR neuron; tr, trunk; vnb, ventral neurite bundle
	Fig. 7. Ultrastructural details of the developing nervous system in Saccoglossus kowalevskii. a Sagittal section of a 1-gill-slit hatchling of S. kowalevskii. b Ultrastructural detail of the red-marked box in a showing a continuous layer of neurites of 2–4-μm thickness. c Color-coded image of the micrograph shown in b. d Sagittal section of a 2-gill-slit juvenile of S. kowalevskii. e Ultrastructural detail of the red-marked box in c showing only single and scattered, small neurite bundles. f Color-coded image of the micrograph shown in e. Color code for c and f: ectoderm, light blue; neurites, yellow; basal lamina (ecm), dark blue; endoderm, green; mesoderm, pale red. bl, blastocoel; ecm, extra cellular matrix; epc, epidermal cell; gp, gill pore; i, intestine; ms, mesocoel; mt, metacoel; mtc, metacoelic cell; myo, myofilaments; ne, neurite; nu, nucleus; pc, protocoel; yo, yolk

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