Balanoglossus misakiensis
Creators: name: Wanninger, Sabrina Kaul-Strehlow Makoto Urata Takuya Minokawa Thomas Stach Andreas
Publisher: name: Zenodo
Year: 2015
Subjects: Biodiversity
Taxonomy
Animalia
Hemichordata
Enteropneusta
Ptychoderidae
Balanoglossus
Balanoglossus misakiensis

description: Balanoglossus misakiensis: Spengel stage   
After about 13 days postfertilization (pf), the tornaria larvae start transforming into the Spengel stage accompanied by a series of morphological changes. The larvae are approximately 950 μm in length and are subdivided by a deep circular constriction into a slightly conical preoral region forming the future proboscis and a voluminous spherical posterior region bearing the distinct opisthotroch (Fig. 2a, b). The oral field is almost completely obliterated as the ciliary bands of the neotroch are fused together. The position of the ciliary bands is still indicated by grooves (Fig. 2a). A pair of dorsolateral slit-like depressions on the anterior part of the postoral body indicates the position of the prospective first gill pores (Fig. 2a, arrowhead).  
The entire larva is covered by short cilia. An apical tuft composed of longer cilia is present at the apical tip (Fig. 2b). Within the apical epidermis, numerous serotonin-LIR neurons are grouped together to form an anterior and a posterior cluster of cells which are interconnected by a median neuropil (Fig. 2f). The serotonin-LIR somata are slender, sometimes flask-shaped, cells with a nucleus that is positioned centrally or basally within the cytoplasm. Each soma is connected to the apical cell surface by a narrow elongation, the distal neurite (Fig. 2f). The serotonin-LIR cells are bipolar neurons that send a neurite into the median neuropil of the apical organ. Aside from the cells of the apical organ, numerous serotonin-LIR somata are scattered throughout the proboscis’ epidermis. Each bipolar neuron exhibits a distal neurite connecting to the apical surface and a proximal neurite. The proximal neurites of those bipolar neurons form a basiepidermal plexus-like arrangement throughout the proboscis epidermis. Within the voluminous postoral region, serotonin-LIR somata are only occasionally present. A conspicuous number of neurites are present at the level of the opisthotroch and form a distinct neurite bundle, namely, the opisthotroch neurite ring (Fig. 2b, e). Aside from that neurite bundle, the postoral body shows only individual neurites arranged in a loose, sometimes discontinuous pattern (Fig. 2e).  
 B. misakiensis: Agassiz stage   
The larvae of the Agassiz stage (14 days pf) are of elongated shape measuring approximately 1 mm in length and 650 μm in width. The anterior proboscis is of conical acorn shape (Fig. 2c, d). It is separated from the posterior body by a deep  
 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   
circular constriction (Fig. 2c, d). The grooves of the fused ciliary bands are no longer visible externally (compare Fig. 2a and d). A second circular constriction indicates the posterior margin of the collar region (Fig. 2d). The collar region is about 150 μm long. The remaining posterior part of the body constitutes the future trunk region. It measures approximately 400 μm in total length and bears the opisthotroch at the midlevel of the trunk region (Fig. 2c, d).  
Serotonin-LIR is present at the anterior tip of the proboscis region (Fig. 2c) and reveals numerous bipolar neurons with thin and long extensions to the cell surface where each neuron bears a single cilium (Fig. 2h). The overall morphology of the serotonin-LIR bipolar neurons of the apical organ is comparable to that of the Spengel stage. Further individual serotoninLIR neurons are interspersed throughout the proboscis with a higher density present near the base of the proboscis (Fig. 2c, sn). All serotonin-LIR somata project into a basiepidermal plexus that is present throughout the proboscis. The plexus of the proboscis region is connected to that of the collar region by two laterally condensed neurite bundles that pass through a neck-like transition into the collar region (Fig. 2c, i). A pair of bilateral muscle bundles that run anteriorly into the base of the proboscis accompanies the neurite bundles (Fig. 2i). Several serotonin-LIR bipolar neurons are present within the anterior  
 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   
 collar region (Fig. 2i, k), whereas only scattered neurons are present within the prospective trunk region. The number of neurites within the collar and trunk region has considerably increased compared to the Spengel stage, and the neurites show a distinct plexus-like pattern, particularly in the prospective trunk region (Fig. 2g). The opisthotroch nerve ring is still well developed and encircles the larva’ s body in the middle of the prospective trunk region (Fig. 2c). Ultrastructural analyses of the metamorphosing Agassiz stage of B. misakiensis revealed the presence of numerous neurites that form a 4– 6-μm-thick basiepidermal layer within the prospective trunk region (Fig. 3a, c, inset).   
 B. misakiensis: early settled juvenile   
 Early settled Balanoglossus (12-h postsettlement (ps)) have an elongated body of vermiform shape measuring approximately 1 mm in length (Fig. 4a, b). The proboscis is pointed at the anterior tip and about 450 μm long. The collar region measures 200 μm in length and is subdivided by a circular constriction into a broader anterior and narrower posterior part. The trunk region has significantly increased in length, now being 430 μm long. One pair of dorsolateral gill pores is developed at the anterior margin just behind the collar region. The opisthotroch is still present and encircles the trunk region (Fig. 4b).   
Numerous serotonin-LIR somata are distributed throughout the epidermis of the proboscis region, and the apical organ is still visible at the anterior tip; yet, the number of serotoninLIR cells is reduced (Fig. 4a) compared to the Agassiz stage. The serotonin-LIR nervous system of the proboscis region differs from the Agassiz stage in having a neurite-rich plexus at the dorsomedian base of the proboscis (Fig. 4a, e). This socalled proboscis stem comprises one of the most condensed areas of the adult enteropneust nervous system. Posterior of the proboscis plexus, two serotonin-LIR neurite bundles project into the collar region and pass through the collar on a subepidermal level (Fig. 4e). Both serotonin-LIR bundles are part of the neurulated collar cord that forms at this stage. Although DAPI staining reveals numerous nuclei accompanying the collar cord, no soma stains positively for serotonin within the collar cord. At the posterior end of the collar, the two neurite bundles become basiepidermal again and project laterally (Fig. 4e) into the prebranchial nerve ring (see below). Within the epidermis of the collar region, numerous serotoninLIR somata are intercalated between the epidermis cells. Each serotonin-LIR bipolar neuron sends a proximal neurite into the basiepidermal plexus. The majority of serotonin-LIR neurites within the collar region runs in longitudinal direction (Fig. 4d) and project into one of the two circular serotoninLIR neurite bundles within the posterior half of the collar region (Fig. 4d, mcb and 4f, pnr). The more anterior circular neurite bundle runs basiepidermally at the level of the internal margin of the collar coelom (mesocoel) (Fig. 4d, mcb), whereas the more posterior ring, the prebranchial nerve ring, is present at the very posterior margin of the collar region. The prebranchial nerve ring forms a distinct network of serotonin-LIR neurites around the paired mesocoelic pores (Fig. 4f, g). These mesocoelic pores connect the coeloms of the collar region, the mesocoels, to the opening of the first gill pore, and eventually to the exterior. At this stage of development, a basiepidermal plexus is detectable by serotonin-LIR throughout the entire trunk region (Fig. 4c), however, with only few serotonin-LIR somata. The opisthotroch neurite ring is less condensed and comprises only a loose arrangement of circular neurites (Fig. 4a, c).  
 B. misakiensis: 1-gill-slit juvenile   
After approximately 1-day postsettlement, the majority of juveniles have lost the opisthotroch (Fig. 4h). The proboscis shape has slightly changed into a blunt, rounded acorn, measuring between 250 and 350 μm in length. The collar region resembles that described for the previous stage and the trunk region is shorter than the previous stage (Fig. 4h, l).  
Within the proboscis and collar region, serotonin-LIR reveals only few changes of the overall architecture of the nervous system. Along the anterior tip of the proboscis, the number of the serotonin-LIR somata within the former apical organ is further reduced (Fig. 4i, l). Along the anterior-posterior axis of the proboscis region, distinct neurite bundles indicate the position of the former larval ciliary bands (Fig. 4i, cgn, l). Within the anterior two thirds of the collar region, two rings of numerous serotonin-LIR neurons are present (Fig. 4k). These cells are long, slender bipolar neurons with the somata placed basally, centrally, or even apically within the epidermis (Fig. 4k). At the posterior collar margin, a pair of prebranchial nerves runs circumferentially, passes the mesocoelic pores, and eventually connects to the ventral part of the nervous plexus (Fig. 4l, pnr). While the arrangement and orientation of the basiepidermal neurites within the proboscis and collar region have not changed much, the situation in the trunk region is considerably different. A plexus-like arrangement of serotonin-LIR neurites is only detectable in the anterior half of the trunk (Fig. 4l, m), whereas in the posterior part of the trunk, individual circumferential neurites are present. These circular neurites project into a longitudinal neurite bundle that is present along the ventral midline (Fig. 4l, vnb).  
 B. misakiensis: 2-gill-slit juvenile   
After about 3-day postsettlement, most of the juveniles exhibit two pairs of gill slits (Fig. 5a, b). Both gill slits are U-shaped and heavily ciliated on the inside (Fig. 5b). No synapticles are developed. The juveniles measure up to 1.5 mm in total length (Fig. 5a–c). The proboscis shows the typical acorn shape, while the mesosome has developed into a 200-μm-long threelobed collar region (Fig. 5a). Scanning electron micrographs and acetylated-α- tubulin-LIR reveal that the proboscis and collar region are evenly covered with cilia, whereas on the trunk region, only scattered tufts of cilia are present (Fig. 5a, c).  
Throughout the epidermis of the proboscis, numerous serotonin-LIR bipolar neurons are intercalated between the other epidermal cells (Fig. 5c, i). Each soma projects a single proximal neurite into the basiepidermal plexus of the proboscis region (Fig. 5i). The most condensed area of serotonin-LIR neurites is present dorsally at the base of the proboscis, comprising a part of the proboscis plexus (Fig. 5c, d). This dense network of neurites measures approximately 200 μm in width and 100 μm in length. From here, three distinct serotonin-LIR neurite bundles leave the proboscis plexus posteriorly to pass through the subepidermal collar cord within the collar region (Fig. 5d). These serotonin-LIR neurite bundles are part of the ventral layer of neurites of the collar cord (Fig. 5h). The dorsal area of the collar cord is composed of numerous cells, presumably neurons, which do not show serotonin-LIR in any of the somata (Fig. 5h). At the posterior pole of the collar region, the three serotonin-LIR neurite bundles become basiepidermal again and connect to the nervous system within the trunk region. The median neurite bundle, composed of 7–10 serotonin-LIR neurites, continues directly into the dorsal midline and is part of the dorsal nerve cord (Fig. 5f). SerotoninLIR is detectable until the posterior pole of the animals, with a discontinuity of about 150 μm in the middle of the trunk region (Fig. 5f, g, double arrowheads). Several serotoninLIR bipolar neurons are located dorsolaterally close to the dorsal midline and project a short neurite into the dorsal neurite bundle (Fig. 5f). The lateral pair of neurite bundles that leaves the collar cord posteriorly runs circumferentially to the ventral side (Fig. 5d). At the level of the dorsolateral gill slits, the bilateral neurite bundles form a distinctive network around the mesocoelic pores as also described for younger stages (Fig. 5d). The bilateral neurite bundles are part of the prebranchial nerve ring and project into the ventral nerve cord. Numerous serotonin-LIR neurites extend along the entire midline of the trunk region thereby running within the ventral nerve cord that is about 40 μm wide (Fig. 5c, e). Several serotonin-LIR somata are interspersed throughout the trunk epidermis. The majority of these bipolar neurons project a proximal neurite running circumferential into the ventral nerve cord (Fig. 5e). The serotonin-LIR somata of the very posterior end send a proximal neurite running radially into the ventral neurite bundle (Fig. 5e). It should be noted that a serotonin-LIR basiepidermal plexus is only present in the epidermis of the proboscis and collar, but completely absent within the trunk region, where instead a condensed dorsal and ventral neurite bundle is present.  
 The analysis of serial sections for electron microscopy of the trunk epidermis in juvenile B. misakiensis shows comparatively few basiepidermal neurites in the epidermis (Fig. 3b, d and inset). Neurite bundles composed of 4–8 neurites are scattered throughout the lateral areas of the trunk epidermis (Fig. 3d). The most condensed neurite bundles run along the ventral and dorsal midline of the trunk region and comprise the nerve cords.
description: Published as part of Wanninger, Sabrina Kaul-Strehlow Makoto Urata Takuya Minokawa Thomas Stach Andreas, 2015, Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords, pp. 405-422 in Organisms Diversity & Evolution 15 (2) on pages 408-412, DOI: 10.1007/s13127-015-0201-2, http://zenodo.org/record/12764142