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Hemichordata

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DOI: 10.1007/978-3-7091-1856-6

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 Hemichordata 1
2
Sabrina Kaul-Strehlow and Eric Röttinger 2

Chapter vignette artwork by Brigitte Baldrian. © Brigitte
Baldrian and Andreas Wanninger.
S. Kaul-Strehlow (*)
Department of Integrative Zoology, University of
Vienna, Althanstr. 14, Vienna 1090, Austria
e-mail: sabrina.kaul-strehlow@univie.ac.at
E. Röttinger
institute for Research on Cancer and Aging,
Université Nice Sophia Antipolis, IRCAN, UMR
7284, Nice 06107, France
CNRS, IRCAN, UMR 7284, Nice 06107, France
INSERM, IRCAN, U1081, Nice 06107, France

A. Wanninger (ed.), Evolutionary Developmental Biology of Invertebrates 6: Deuterostomia 59

DOI 10.1007/978-3-7091-1856-6_2, © Springer-Verlag Wien 2015
 60
4 Introduction
5 Synopsis
6 Hemichordata is a group of exclusively marine
7 animals, consisting of two subgroups, the sessile
8 and small colonial pterobranchs and the soli-
9 tary, vermiform enteropneusts (acorn worms)
10 (Fig.  2.1; van der Horst 1939; Hyman 1959;
11 Benito and Pardos 1997). With about 130
12 described species, Hemichordata comprises a rela-
13 tively small taxon of benthic animals (http://www.
14 marinespecies.org/index.php; Cameron 2005).
15 They are distributed worldwide and inhabit shal-
16 low coastal areas but are also found in the deep
17 sea. For a long time, pterobranchs have been
18 known only from deep waters, whereas entero-
19 pneusts were thought to burrow mainly in shallow
20 waters. However, within the last five decades,
21 about a dozen of different enteropneusts have been
22 documented in the deep sea (Osborn et al. 2012).
23 In contrast, pterobranchs have been found in inter-
24 tidal zones of tropical waters only recently (Lester
25 1985) and might have been overlooked previously
26 due to their minute size and superficial similarities
27 in their gross morphology with other tube-­dwelling
28 animals, such as polychaetes and bryozoans.
A B
Fig. 2.1  Overview of the general morphology of hemichor-
dates. (A) Habitus of an adult pterobranch, Cephalodiscus
sp (Modified from Lester 1985), 2.5 mm. (B) Adult speci-
men of Balanoglossus clavigerus (Ptychoderidae), 25 cm.
(C) Adult specimen of Saccoglossus mereschkowskii
S. Kaul-Strehlow and E. Röttinger
Enteropneusts as well as pterobranchs exhibit 29
a tripartite body organisation divided into an 30
anterior prosoma, a mesosome and a posterior 31
metasomal region (Fig. 2.1). In enteropneusts, 32
the prosoma is called “acorn” or “proboscis”, 33
while in pterobranchs, it is termed “mouth 34
shield”. The middle body region encompasses 35
the anteroventral mouth opening and is referred 36
to as the “collar” or “mesosome”, respectively. 37
The mesosome of pterobranchs holds a tentacular 38
crown for filter feeding (van der Horst 1939; 39
Benito and Pardos 1997). 40
Both hemichordate groups have a characteris- 41
tic excretory system in the prosoma. It is com- 42
posed of a contractile pericardium that encloses 43
the heart sinus that anteriorly continues into the 44
glomerulus. The glomerulus is a highly dilated 45
blood plexus lined by podocytes that are capable 46
of mediating ultrafiltration into the protocoel. 47
The accumulated ultrafiltrate then leaves the pro- 48
tocoel via the proboscis pore (Balser and Ruppert 49
1990). Indeed, such an excretory complex is also 50
present in echinoderms (axial complex), thus rep- 51
resenting a synapomorphy for Ambulacraria, yet 52
in hemichordates, the unique stomochord is 53
incorporated into this complex (Dohle 2004). 54
The stomochord is an endodermal protrusion 55
filled with vacuolated cells surrounded by a 56
(Harrimaniidae), 4 cm (B, C modified from Goldschmid
2007). a anus, cn coenecium, co collar, go gonads, gs gill
slit, gw genital wing, li liver sacs, me mesosome, ms mouth
shield, msc mesocoelic pore, pr proboscis, st stalk, te tenta-
cles, tr trunk
 2 Hemichordata 61

57 thickened extracellular matrix that serves as a at its terminal end. The nervous system of entero- 104
58 supportive structure for the pericardium-­ pneusts is developed as a basiepidermal nerve net 105
59 glomerulus complex (Balser and Ruppert 1990; throughout the body (Silén 1950; Bullock and 106
60 Mayer and Bartolomaeus 2003; Kaul-Strehlow Horridge 1965). More condensed areas are pres- 107
61 and Stach 2011; Merker et al. 2013). A possible ent in the trunk region, where they constitute a 108
62 homology between the stomochord and the noto- dorsal and a ventral median longitudinal nerve 109
63 chord of chordates is still debated. cord, respectively (Bullock 1946; Knight-­Jones 110
1952). Furthermore, the dorsal nerve cord contin- 111
64 Enteropneusta ues anteriorly into the collar region to form the 112
65 Enteropneusta comprises less than 110 described subepidermal, hollow collar cord that is reminis- 113
66 species of worm-shaped animals (Cameron 2005) cent of the neural tube present in chordates 114
67 ranging from 0.5 mm to more than 2 m in size (Morgan 1894; Kaul and Stach 2010; Miyamoto 115
68 (Hyman 1959; Worsaae et al. 2012). Acorn and Wada 2013). The collar cord links to the 116
69 worms are usually ground dwellers that live in basiepidermal plexus at the base of the proboscis, 117
70 muddy or sandy sediments; only few are known that is, the proboscis stem. Given that entero- 118
71 to be associated with the undersides of rocks pneusts mainly consist of soft body parts, they 119
72 (Spengel 1893; Hyman 1959; Cameron 2005). are bad candidates for leaving fossilising remains, 120
73 The body of acorn worms is vermiform and can explaining their poor fossil record. Nevertheless, 121
74 be subdivided into three regions, each of which is a recent finding of a tube-dwelling enteropneust 122
75 supported internally by a corresponding coelo- dates their origin back to at least the Cambrian 123
76 mic cavity (Spengel 1893; Morgan 1894). The (Caron et al. 2013). 124
77 anterior proboscis differs in shape from long and
78 slender (Fig. 2.1C) to conical and short (Fig. 2.1B; Pterobranchia 125
79 van der Horst 1939). Posteriorly, the proboscis Pterobranchia is a small taxon consisting of 126
80 connects to the collar region (mesosome) by a roughly 25 species, of which all are colonial and 127
81 robust stalk. The highly muscular proboscis is the semi-sessile animals (Cameron 2005; Nielsen 128
82 main locomotory organ and is used for the search 2011). They are tube dwellers that secrete the so-­ 129
83 for food and burrowing (van der Horst 1939; called coenecium from the anterior mouth shield 130
84 Benito and Pardos 1997). The short and barrel-­ (Fig. 2.1A). The body size of the individual zooid 131
85 shaped collar region surrounds and overlaps the lies between 1 and 5 mm and an entire colony is 132
86 posterior part of the proboscis including the pro- built from a single larva that reproduces asexu- 133
87 boscis stalk (Fig. 2.1B, C). The mouth opening is ally by budding (Anderson 1907; van der Horst 134
88 positioned ventrofrontally between the constric- 1939). The mesosome is equipped with tentacles 135
89 tion separating proboscis and collar region that are used for filter feeding and probably also 136
90 (Bateson 1884, 1885; Spengel 1893). The middle serve respiratory function. The bulbous trunk 137
91 collar region is followed by the long trunk region region harbours most of the U-shaped digestive 138
92 that forms the largest part of the enteropneust tract including the posterior pharynx, stomach 139
93 body. The trunk is functionally differentiated into and intestine. The anus opens on the dorsal side 140
94 the anterior branchio-genital region, the middle of the anterior trunk region, just behind the meso- 141
95 hepatic region and the posterior abdominal (intes- some. The nervous system of pterobranchs con- 142
96 tine) region (Fig. 2.1B, C; Spengel 1893; Hyman stitutes a basiepidermal nerve net. Additionally, 143
97 1959). A bilateral row of numerous dorsolateral a prominent dorsal brain is present at the base of 144
98 gill slits accompanies the branchio-genital region. the mesosomal tentacles (Dilly et al. 1975; 145
99 In many large species in particular, the special- Rehkämper et al. 1987; Stach et al. 2012). The 146
100 ised hepatic region is characterised by numerous brain features an anterior neuron-rich area com- 147
101 liver sacs that are projections of the intestine and posed of serotonergic and giant neurons as well 148
102 body wall (Fig. 2.1B). The tubular abdominal as a posterior neuropil. Further concentrated 149
103 region is comparatively long and bears the anus parts of the peripheral nervous system are t­ entacle 150
 62 S. Kaul-Strehlow and E. Röttinger

151 nerves, a ventral stalk nerve and a pair of bran- nemerteans, annelids or other “worms” was sug- 195
152 chial nerves in Cephalodiscus gracilis (Stach gested, but also holothurians were considered as 196
153 et al. 2012). Pterobranchs mainly live associated relatives. Interestingly, a conspicuous larva, first 197
154 with hard substrates since their secreted tubes documented around 1850, played a more impor- 198
155 build encrusting aggregates on rocks and shells. tant role in resolving this issue than initially 199
156 The majority of described species have been col- thought. In 1849, Johannes Müller found a pecu- 200
157 lected in the deep sea and only few species are liar larva in the plankton near Marseille, France. 201
158 known from shallow waters. These include He named this larva “tornaria” and placed it in a 202
159 Rhabdopleura normani and C. gracilis that can closer connection to the bipinnaria of sea stars, 203
160 easily be accessed by snorkelling in shallow because of similarities in the arrangement of the 204
161 waters on the Bermuda Islands (Lester 1985, ciliary bands (Müller 1850). At the same time, 205
162 1988a, b) or Rhabdopleura compacta that lives a similar larva was found by August Krohn, but 206
163 off the south coasts of England (Sato et al. 2008). this one had a much more sinuously running cili- 207
164 Due to their secreted tubes, pterobranchs are ary band and he suggested this to be a different 208
165 associated with the fossil group Graptolithina species (Krohn 1854). In those days, it was not 209
166 that are known from the Cambrian through the known that these varying larvae actually display 210
167 Carboniferous. Graptolithina composes a quite successive developmental stages. Nowadays, this 211
168 diverse group of fossils of pelagic tube-dwelling particular larval stage with secondary lobes and 212
169 colonies that are supposed to be closely related to saddles of the ciliary feeding band (neotroch) is 213
170 the extant genus Rhabdopleura (Sato et al. 2008). called “Krohn stage”, referring to its original 214
171 Within the last couple of years, Sato and col- describer. In fact, all of the succeeding larval 215
172 leagues have begun to develop Rhabdopleura stages have later on been given specific names, 216
173 compacta, a species that can be found in the being in connection to the historical background 217
174 south of England, as an emerging model organ- of their discovery. This will be discussed 218
175 ism that can be used for developmental studies more precisely in the paragraph dealing with late 219
176 (Sato et al. 2008, 2009; Sato and Holland 2008). development. Nevertheless, it was Elias 220
Metschnikoff who made a promising finding in 221
1870, when he collected a larva that showed 222
177 Historical Background similarities with the worm of Balanoglossus
­ 223
(Metschnikoff 1870), yet his colleagues did not 224
178 The very first mentioning of an enteropneust can believe in a connection between echinoderm-like 225
179 be traced back to the work of Friedrich tornaria and the wormlike enteropneust. It took 226
180 Eschscholtz in 1825, who described a wormlike three more years until Alexander Agassiz suc- 227
181 animal named Ptychodera that he interpreted as a cessfully documented the metamorphosis of a 228
182 new species of holothuroid echinoderms tornaria larva into a juvenile Balanoglossus, thus 229
183 (Eschscholtz 1825). Only a few years later, and finally unravelling the larva’s unknown affiliation 230
184 without knowledge of Eschscholtz’ description, (Agassiz 1873). In the following years, a number 231
185 Stefano Delle Chaje documented a sand-dwelling of descriptive studies were added to the existing 232
186 worm that he named Balanoglossus clavigerus list. Bateson published a study of direct develop- 233
187 (Delle Chaje 1829). However, both reports were ment of Saccoglossus kowalevskii without a tor- 234
188 only short notes showing sketchy drawings, and naria larva and at the same time he was the first 235
189 it was not before 1866 until the first detailed ana- who observed early cleavage stages in entero- 236
190 tomical description of an enteropneust was pub- pneusts (Bateson 1884, 1885, 1886). He sug- 237
191 lished by Alexander Kowalevsky (1866). At that gested a close relationship of enteropneusts with 238
192 time, phylogenetic relationships of enteropneusts chordates, because of shared characters such as 239
193 were controversially discussed. Because of a ver- gill slits, stomochord and a neurulated collar 240
194 miform body, a closer relationship to either cord. 241
 2 Hemichordata 63

242 Since then, consecutive studies on various the Ambulacraria (Fig. 2.2; Bourlat et al. 2006; 286
243 aspects of enteropneusts have significantly Cannon et al. 2009; Hejnol et al. 2009; 287
244 increased our understanding of the group and Edgecombe et al. 2011). At present, Ambulacraria 288
245 were summarised in several classical text- seems to be widely accepted and is also morpho- 289
246 books and treatises such as Spengel’s mono- logically supported by a shared larval type 290
247 graph on enteropneusts (1893), van der Horst’s (dipleurula with specialised neotroch ciliary 291
248 “Enteropneusta” in Bronn’s Klassen und band) as well as similar coelomic systems and 292
249 Ordnungen des Tierreichs (1939) or Hyman’s excretory organs. Pterobranchs and enteropneusts 293
250 The Invertebrates (1959), to name but a few. are classically treated as sister groups, yet molec- 294
251 Strangely, the interest in enteropneust research ular phylogenetic analyses strongly support the 295
252 declined within the last half of the twentieth cen- position of pterobranchs as a sister group to 296
253 tury and relatively few works were published. Harrimaniidae (an enteropneust subclade), thus 297
254 Only recently, with upcoming immunocyto- rendering Enteropneusta paraphyletic (Fig. 2.2; 298
255 chemical as well as molecular techniques and Cameron et al. 2000; Cannon et al. 2009; Osborn 299
256 the new field of “EvoDevo” research, people et al. 2012). The possibility that pterobranchs 300
257 have rediscovered the potential and importance evolved a sessile and colonial lifestyle second- 301
258 of enteropneusts in unravelling evolutionary arily from a solitary, wormlike enteropneust 302
259 developmental questions (Tagawa et al. 1998a, ancestor has further been supported by the recent 303
260 b; Lowe et al. 2003, 2006; Röttinger and discovery of a tubicolous enteropneust from the 304
261 Martindale 2011; Röttinger and Lowe 2012; and Cambrian (Caron et al. 2013). 305
262 references therein). There are three main subclades to which the 306
majority of enteropneust species belong to, 307
namely, Ptychoderidae (Spengel 1893), 308
263  ystematics and Phylogenetic
S Spengelidae (Willey 1898) and Harrimaniidae 309
264 Relationships (Spengel 1901) (Fig. 2.2). A fourth taxon has 310
recently been described, the Torquaratoridae 311
265 Morphologically, hemichordates are well sup- (Holland et al. 2005), comprising enteropneusts 312
266 ported as deuterostome animals, because the from the deep sea (Osborn et al. 2012). Members 313
267 main coelomic cavities originate from the arch- of the Torquaratoridae are characterised by lack- 314
268 enteron by enterocoely and the mouth is formed ing a stomochord as adults. Moreover, the pro- 315
269 secondarily during development. However, the boscis skeleton is either reduced to a small 316
270 exact position of Hemichordata within the plate or completely absent. The Ptychoderidae 317
271 Deuterostomia and its putative sister group has includes the first enteropneust species described, 318
272 been controversially discussed within the last Ptychodera flava, and is characterised by the 319
273 decade, due to incongruent morphological as presence of gill-slit synapticles and a distinct 320
274 well as molecular data. On the one hand, shared trunk region featuring liver sacs and genital 321
275 chordate features such as gill slits, stomochord wings (Figs. 2.1B and 2.2). Members of the 322
276 and neurulated collar cord in enteropneusts lead Spengelidae do not exhibit genital wings at the 323
277 already Bateson (1885) and Barrington (1965) to trunk region and liver sacs were only described 324
278 suggest a closer relationship of Hemichordata for the genus Schizocardium (Spengel 1893; 325
279 and Chordata. Indeed, phylogenetic analyses Cameron 2005). 326
280 based on morphological characters strongly sup- Ptychoderidae and Spengelidae possess a 327
281 port this view (Young 1962; Maisey 1986; biphasic life cycle and develop indirectly via a 328
282 Schaeffer 1987; Ax 2001). However, more recent pelagic tornaria larva. Harrimaniidae includes the 329
283 molecular phylogenetic analyses consistently smallest representative of an acorn worm 330
284 reveal a sister group relationship between described so far, Meioglossus psammophilus 331
285 Hemichordata and Echinodermata, comprising (Worsaae et al. 2012), with less than 1 mm in 332
 64 S. Kaul-Strehlow and E. Röttinger

333 length. This taxon possesses the simplest anat- cies live in fingerlike branched coenecia and 351
334 omy, as their members lack liver sacs as well as have individual zooids, although in some spe- 352
335 genital wings within the dorsal trunk region cies, the zooids are linked to each other by the 353
336 (Fig. 2.1C). Also, the gill slits in harrimaniids are posterior stalk (Lester 1985). Cephalodiscus spp. 354
337 not supported by synapticles as in other entero- are characterised by a globular metasomal 355
338 pneust subclades (van der Horst 1939; Cameron region, one pair of gill slits, two gonads and 356
339 2005). Harrimaniid enteropneusts such as mesocoel pores and between five and nine pairs 357
340 Saccoglossus develop directly, not passing of tentacles on the mesosome (Hyman 1959). 358
341 through an extended pelagic stage. The hatch- A muscular stalk is present at the posterior end of 359
342 lings resemble young adults and soon settle in the the metasomal region and aids in its withdrawal 360
343 sediment to grow into a juvenile enteropneust. into the coenecium. Species of the genera 361
344 Pterobranchs are grouped into two genera, Rhabdopleura, in contrast, bear only a single 362
345 Cephalodiscus (M’Intosh 1882) and Rhabdo­ pair of tentacles on the mesosome, have only one 363
346 pleura (Allman 1869) (Fig. 2.2). A putative third gonad and lack gill pores altogether (Schepotieff 364
347 genus is monotypic with Atubaria heterolopha 1907; van der Horst 1939). The zooids of one 365
348 (Sato 1936) from deeper Japanese waters, yet colony live in tubular coenecia and stay intercon- 366
349 the validity of this genus is questionable nected by the stalk throughout lifetime (Lankester 367
350 (Mierzejewski 2004). Many Cephalodiscus spe- 1884; Hyman 1959). 368

Fig. 2.2  Deuterostome phylogeny. Cladogram compiled debated and not shown here (Hejnol et al. 2009; Philippe
from recent phylogenetic analyses (Osborn et al. 2012; et al. 2011)
Worsaae et al. 2012). The position of Xenacoelomorpha is
 2 Hemichordata 65

369 Early Development body and the larva exhibits a conspicuous ventral 383
depression (Fig. 2.3F, G). After a short pelagic 384
period, the larva tests the substrate and eventually 385
370 Development in Pterobranchs settles with the ventral side secreting a cocoon. 386
Inside the cocoon, the metamorphosing larva 387
371 Pterobranchs are less frequently encountered as develops the anlagen of the tentacles, the mouth 388
372 enteropneusts, which is why knowledge on the shield and the metasome; thus, the future tripar- 389
373 reproduction and development of these minute tite body organisation is already established 390
374 animals is still fragmentary. So far, it is known (Fig.  2.3H–K; Lester 1988b). After a few days, 391
375 from Rhabdopleura that the fertilised, yolk-rich the cocoon breaks and the zooid starts to form its 392
376 egg undergoes holoblastic, radial and equal coenecium, thereby founding a new colony 393
377 cleavage, eventually leading to a uniformly cili- (Fig. 2.3L). In Cephalodiscus, even less is known 394
378 ated larva (Fig. 2.3A–G; Stebbing 1970; Dilly about development, although a few accounts on 395
379 1973; Lester 1988a). The larva is of elongated single developmental stages are present (Harmer 396
380 shape with a tapering posterior end (Sato et al. 1905; Anderson 1907; Schepotieff 1907; Dilly 397
381 2008). Its colour is opaque and yellowish. 2013). A recent ultrastructural study shows an 398
382 A brown spotty pigmentation is present over the elongated, three-layered embryonic stage of C. 399

A B C D E

F G H

I K L

Fig. 2.3  Development and metamorphosis of
Rhabdopleura normani. (A) Zygote. (B) Two-cell stage.
(C) Four-cell stage. (D) Eight-cell stage. (E) Spherical,
ciliated embryo. (F) Swimming larva from dorsal.
(G) Swimming larva from ventral. (H) Metamorphosing
larva in secreted cocoon, approx. 12 h after settlement.
Lateral view. (I) Same as in H, approx. 33 h after settle-
ment. (K) Hatching juvenile, approx. 100 h after settle-
ment. (L) Zooid in secreted coenecium. Drawings not to
scale (Modified from Lester 1988a, b). ar arm holding
tentacles, cn coenecium, co cocoon, ms mouth shield, so
sensory organ, st stalk, te tentacle, tr trunk, vd ventral
depression
 66 S. Kaul-Strehlow and E. Röttinger

400 gracilis (Stach 2013). The prospective meso- and cells will form the middle and posterior ecto- 444
401 metacoels are already separated from the endo- derm (Colwin and Colwin 1951; Darras et al. 445
402 derm, whereas the prospective protocoel is still 2011). Only the cells of the lower vegetal tier 446
403 continuous with the endoderm. The one pair of will differentiate into endo- as well as mesoderm 447
404 gill pores seems to develop asynchronously, in (Fig.  2.4). Continuous cleavages lead to a 448
405 favour of the left side. The creeping larva lacks rounded coeloblastula. Before gastrulation, the 449
406 any planktonic specialisations and might develop blastula becomes cup-shaped by flattening of the 450
407 directly into a young zooid without passing animal-­ vegetal axis, while at the same time, 451
408 through a pelagic stage. the thickened vegetal pole invaginates circumfer- 452
entially (Colwin and Colwin 1953). After 453
­gastrulation of the prospective endomesoderm, 454
409 Development in Enteropneusts the blastopore closes and the embryo again 455
elongates along the anterior-posterior axis
­ 456
410 Fertilisation is external in all studied enteropneusts (Fig.  2.5A). At this time of development, the 457
411 and spawning is usually dependent of the species’ opisthotroch (ciliary band), composed of numer- 458
412 habitat and correlated with seasons, whereby tem- ous long compound cilia, is visible demarcating 459
413 perature and light intensity play a major role the postanal field. The embryos start to spin 460
414 (Hadfield 1975). As outlined before, two different around within the vitelline membrane as soon as 461
415 modes of development have been observed in the cilia start to beat. Only a few hours later, a 462
416 enteropneusts. Whereas members of Ptychoderidae circumferential groove starts to subdivide the 463
417 and Spengelidae develop a pelagic larval stage, the embryo in an anterior proboscis region and a 464
418 tornaria, harrimaniid enteropneusts develop posterior region constituting the future collar and 465
419 directly from yolk-rich eggs. Early development trunk (Fig. 2.5B). At this stage, the anterior part 466
420 including cleavage patterns has been described for of the invaginated archenteron separates as the 467
421 several enteropneust species (Bateson 1884; first coelomic cavity, thereby forming the future 468
422 Stiasny 1914a; Burdon-Jones 1952; Colwin and protocoel (Figs. 2.4 and 2.6A). The paired meso- 469
423 Colwin 1953; Tagawa et al. 1998a; Urata and and metacoels are present as separated evagina- 470
424 Yamaguchi 2004). In all studied enteropneusts, tions from the middle and posterior regions of 471
425 cleavage is radial, holoblastic and nearly equal. the archenteron (Figs. 2.6A), yet are still con- 472
nected to the archenteron. Around 4 days after 473
426  mbryology in Direct Developing
E fertilisation, the embryos are of elongated shape 474
427 Enteropneusts with a perianal field that is bent ventrally 475
428 In direct developing enteropneusts such as (Fig 2.5C). A second circular groove forms the 476
429 Saccoglossus kowalevskii, a gravid female border between the collar and trunk region. By 477
430 spawns between 200 and 1.000 oocytes with a this stage, the meso- and metacoels are pinched 478
431 diameter of about 300 μm. After fertilisation, a off and enclose the endoderm almost completely. 479
432 thick vitelline membrane is formed around the A sixth, yet small coelomic cavity is situated at 480
433 zygote. Subsequently, the fertilised egg under- the posterodorsal base of the proboscis and will 481
434 goes radial cleavage of which the two first cleav- later differentiate into the pericardium or heart 482
435 ages are meridional and the third is latitudinal vesicle. In contrast to the pro-, meso- and meta- 483
436 (Bateson 1884; Colwin and Colwin 1953). The coels that originate from the endoderm by entero- 484
437 fourth cleavage results in a single animal tier of coely, the pericardium develops by schizocoely 485
438 eight cells (Fig. 2.4), whereas the vegetal cells from the ectoderm in S. kowalevskii (Kaul-­ 486
439 divide latitudinally to yield an upper tier of four Strehlow and Stach 2011). Schizocoely is typical 487
440 larger cells as well as a lower tier of four smaller for formation of the mesoderm in various proto- 488
441 cells. Cell labelling in Saccoglossus showed that stomes and only rare cases in deuterostomes are 489
442 the cells of the animal tier give rise to the ante- known (Technau and Scholz 2003). The endo- 490
443 rior ectoderm, while the upper tier of vegetal derm connects to the exterior on the ventral side 491
 2 Hemichordata
Fig. 2.4  Early embryology of direct and indirect devel-
oping enteropneusts. Upper row: the direct developing
harrimaniid Saccoglossus kowalevskii. Lower row: the
a b
d e
Fig. 2.5  Scanning electron micrographs of developmen-
tal stages of Saccoglossus kowalevskii. (A–C) Fertilisation
membrane removed. (A) Late gastrula. (B) Early kink
stage. (C) Dorsal flexure stage. (D) One-gill-slit hatch-
ling. (E) Early settling juvenile. Note the ventral creeping
492 just between the proboscis and collar region,
493 forming the mouth opening, whereas the anus is
494 still closed. The developing stomochord
495 ­protrudes into the proboscis base as a short, rod-
496 like extension from the anterodorsal roof of the
497 endoderm. Closely behind the posterior margin
67
indirect developing ptychoderid Ptychodera flava. The
exact timing of separation of endo- and mesoderm remains
unclear
c
f
sole (arrowheads) and the postanal tail. (F) Three-gill-slit
juvenile with adult-like gross morphology. co collar, gp
gill pore, mo mouth opening, op opisthotroch, pa perianal
field, pat postanal tail, pr proboscis, tr trunk (© Sabrina
Kaul-Strehlow 2015. All Rights Reserved)
of the collar region, the endoderm pierces 498
through the metacoel of the trunk region and 499
establishes contact with the ectoderm, thereby 500
forming the anlagen of the first pair of gill pores 501
(Fig  2.6C). Embryogenesis is usually complete 502
at around 5 days after fertilisation in embryos 503
 68
A B
D E
Fig. 2.6  3D reconstructions of major organ systems in
different developmental stages of Saccoglossus kowa­
levskii based on complete serial semithin sections. Note,
only the centralised parts of the nervous system are recon-
structed in (D–F); the nerve net is not shown. Anterior is
(almost) to the left in all images. (A) Late gastrula with
anlagen of the prospective meso- and metacoels. The
anterior protocoel has already pinched off of the endo-
derm. (B) Early kink stage. Meso- and metacoels are
separated from endoderm. The pericardium emerges at
the dorsal base of the protocoel. (C) Dorsal flexure stage
504 cultured at 22 °C and hatch from the fertilisation
505 membrane (Colwin and Colwin 1953; Lowe
506 et al. 2004). At that time, Saccoglossus exhibits
507 an elongated body shape measuring ~500 μm in
508 length with a ventrally bent perianal field
S. Kaul-Strehlow and E. Röttinger
C
showing anlagen of the first pair of gill pores. (D) One-
gill-slit hatchling with opened pair of gill pores and neu-
rulating collar cord in the mesosome. (E) Two-gill-slit
juvenile with resembling adult morphology. (F) Close-up
showing the inner organisation of the proboscis, collar
and anterior trunk region in detail. agp anlage of gill pore,
cc collar cord, dnc dorsal nerve cord, gb gill bar, gl glom-
erulus, gp gill pore, hs heart sinus, pat postanal tail, pd
pericardium, psk proboscis skeleton, pst proboscis stem,
st stomochord, vnc ventral nerve cord
(Figs. 2.5D and 2.6D). One pair of dorsolateral 509
gill pores is present in the anterior part of the 510
trunk region. After hatching, the animals swim in 511
the water column for a couple of hours and soon 512
start to burrow and feed in the sediment. 513
 2 Hemichordata 69

514  mbryology in Indirect Developing

E posed of pharynx, stomach and intestine. In 562
515 Enteropneusts contrast to P. flava, which has a comparably 563
516 The ontogeny including metamorphosis of indi- long larval development for enteropneusts, the 564
517 rect developing enteropneusts has been studied in Japanese species B. misakiensis exhibits a 565
518 species of the genera Balanoglossus and shortened larval cycle and its larva hatches 566
519 Ptychodera (Morgan 1891; Stiasny 1914a, b; already at 24 h pf, thereby skipping the Müller 567
520 Tagawa et al. 1998a; Urata and Yamaguchi 2004; stage (Urata and Yamaguchi 2004). Early hatched 568
521 Nielsen and Hay-Schmidt 2007; Miyamoto et al. larvae of B. misakiensis have already developed 569
522 2010). Only recently, the first study of the devel- an opisthotroch that typically characterises the 570
523 opment of a member of the Spengelidae, Heider (second) stage of tornaria larvae 571
524 Glandiceps hacksi, has been investigated (Urata (Fig.  2.7A). In all enteropneusts studied so far, 572
525 et al. 2014). Although the later development of the hydropore opens only after the protocoel has 573
526 indirect developers differs considerably from separated from the endoderm (Spengel 1893; 574
527 direct developers such as Saccoglossus, cleavage Hyman 1959). However, in the recently studied 575
528 patterns and fate maps are identical and moreover spengelid Glandiceps hacksi, the hydropore 576
529 show strong resemblance to sea urchins (Henry forms prior to this event. This “precocious hydro- 577
[AU1]
530 et al. 2001; see Chap. XX). The 16-cell stage of pore formation” is so far unique for the entero- 578
531 Ptychodera flava comprises an animal tier of pneust G. hacksi, yet it is known also from 579
532 eight cells (primordial anterior ectoderm), an various holothuroid echinoderms (Urata et al. 580
533 upper vegetal tier of four cells (primordial poste- 2014 and references therein). 581
534 rior ectoderm) as well as a lower vegetal tier of
535 four cells (primordial endomesoderm) (Fig. 2.4;
536 Tagawa et al. 1998a). Subsequent cleavages give Late Development 582
537 rise to a coeloblastula. Cleavage speed varies
538 greatly between enteropneust species. Whereas Late Development in Direct 583
539 in P. flava it takes about ~18 h post fertilisation Developing Enteropneusts 584
540 (pf) until gastrulation starts (cultured at 22–24 °C)
541 (Tagawa et al. 1998a), it begins at around ~13 h When direct developing enteropneusts such as 585
542 pf in Balanoglossus clavigerus (cultured at Saccoglossus kowalevskii hatch, the first pair of 586
543 20 °C) (Stiasny 1914a), and in B. misakiensis, gill pores is open and the animals swim actively 587
544 this stage is already reached within ~9 h pf (cul- by the propelling opisthotroch. As soon as the 588
545 tured at 26 °C) (Urata and Yamaguchi 2004). At animals start burrowing in the sand, the opis- 589
546 the end of gastrulation, the blastopore is closed thotroch is remodelled and extends on the ventral 590
547 and the protocoel is pinched off from the anterior side of the trunk in order to serve as a creeping 591
548 region of the archenteron. The protocoel soon sole for the juvenile worms (Fig. 2.5E; Burdon-­ 592
549 attaches to the epidermis of the animal pole and Jones 1952; Stach and Kaul 2012). As the worms 593
550 fuses with the dorsal ectoderm to form the hydro- grow older, gill pores are added successively and 594
551 pore. After ~45 h pf, embryonic development is the body size gains considerably in length. The 595
552 completed in P. flava and the larva hatches from juveniles measure up to a few millimetres at this 596
553 the fertilisation membrane to instantly start point and resemble adult worms in many aspects, 597
554 swimming (Tagawa et al. 1998a; Nielsen and except for the still present postanal tail and the 598
555 Hay-Schmidt 2007). The early larva is of more or lower number of gill pores (Figs. 2.5F and 2.6E). 599
556 less spherical shape and soon develops into the The protocoel within the proboscis region is lined 600
557 typical tornaria larva. The first tornaria stage is by a myoepithelium that forms the body wall 601
558 called Müller stage and is characterised by a musculature, composed of an outer layer of cir- 602
559 closed mouth as well as anus, open hydropore cular muscles and an inner layer of longitudinal 603
560 and a developing neotroch (circumoral ciliary muscles. A single proboscis pore is located pos- 604
561 band). The digestive tract is tripartite and com- terodorsally on the left side and opens to the 605
 70
A B
D E
H I
Fig. 2.7  Scanning electron micrographs of developmen-
tal stages of Balanoglossus misakiensis. (A) Early hatched
Heider stage larva (30 h pf). (B) Tornaria at late Heider
stage (48 h pf) with ventral neurotroch and oral field
developed. (C) Early Metschnikoff stage (120 h pf) show-
ing the beginning of primary lobe formation of the ciliary
band of the oral field (neotroch). (D) Fully grown tornaria
of B. misakiensis at late Metschnikoff stage (10 days pf)
with deep primary lobes. (E) The Spengel stage (13 days
pf) designates the beginning of metamorphosis and is
characterised by the fusion of the neotroch, eventually
obliterating the oral field and reducing the size of the
606 exterior. The number and position of the probos-
607 cis pores in enteropneusts is species dependent.
608 Accordingly, in some species, bilateral pores are
609 present, while in others only a single one is found
S. Kaul-Strehlow and E. Röttinger
C
F
body. (F) Agassiz stage tornaria (14 days pf) with acorn-
shaped preoral region (future proboscis). This stage is
competent to undergo settlement. (G) Early settled juve-
nile (12 h post settlement) with elaborated collar and elon-
gated trunk region. The larval opisthotroch is still present.
(H) Two-gill-slit juvenile (3 days post settlement) that
already resembles a minute adult enteropneust. af aboral
field, at apical tuft, co collar, gs gill slit, mo mouth open-
ing, ne neotroch, nu neurotroch, of oral field, op opis-
thotroch, pa perianal field, pr proboscis (© Sabrina
Kaul-Strehlow 2015. All Rights Reserved)
on the right side. The pericardium is situated pos- 610
teriorly within the protocoel and is dorsally 611
attached to the stomochord. The pericardium is a 612
small coelomic cavity that is lined ventrally by 613
 2 Hemichordata 71

614 epithelial muscle cells overlying the heart sinus, Neurogenesis in Direct Developing 662
615 which is in principle an enlarged area within the Enteropneusts 663
616 extracellular matrix (ECM) filled with colourless Neurogenesis in direct developing enteropneusts 664
617 blood fluid (Balser and Ruppert 1990). By con- such as Saccoglossus has been studied thor- 665
618 traction of the pericardial epithelial muscle cells, oughly by molecular genetic analyses, yet mor- 666
619 the blood fluid is forwarded into the anterior phogenetic data are still scarce. At late gastrula 667
620 glomerulus. The glomerulus is a highly ramified stage (Fig. 2.5A), serotonergic neurons form 668
621 enlargement of the ECM that is lined with podo- throughout the future proboscis region and proj- 669
622 cytes on the protocoelic side. These podocytes ect neurites posteriorly (Cunningham and Casey 670
623 have fingerlike extensions and are involved in 2014). In stages close to hatching, a considerable 671
624 filtration of the blood fluid. Together with the basiepidermal nerve net is developed throughout 672
625 efferent proboscis pore, the heart-glomerulus the entire embryo (Kaul and Stach 2010). Before 673
626 complex represents the excretory system of hatching, the collar cord at the dorsal midline of 674
627 hemichordates. The digestive tract in these juve- the collar region neurulates gradually from ante- 675
628 nile worms is already subdivided into the typical rior to posterior to finally occupy a subepidermal 676
629 regions, the anterior buccal cavity followed by position underneath the epidermis (Figs. 2.6D–F 677
630 the pharyngeal region harbouring the dorsolat- and 2.8). Just after neurulation, the collar cord 678
631 eral gill pores, connected to the stomach by a comprises a large area of neuronal precursors that 679
632 short and thin tubular esophagus ending in a surround a central lumen (central canal) and 680
633 short hindgut region that opens into the anus. small ventral areas filled with neurites (Fig. 2.8C). 681
634 The paired meso- and metacoels are lined by a In older juvenile worms, a circumferential 682
635 single layer of epithelial cells that contain basal basiepidermal nerve net is present within the pro- 683
636 myofilaments. Within the metacoels, these myo- boscis and collar region. Within the trunk region, 684
637 filaments constitute a substantial longitudinal the majority of neurites seems to run within the 685
638 musculature, in particular on the ventral side. longitudinal nerve cords, i.e., the dorsal and the 686
639 The mesocoels send a pair of extensions anteri- ventral cord (Kaul and Stach 2010), whereas only 687
640 orly through the proboscis stalk into the base of scattered neurites are present laterally. The dorsal 688
641 the proboscis. These mesocoelic protrusions nerve cord extends posteriorly until the anus and 689
642 flank the stomochord and contain longitudinal is anteriorly continuous with the collar cord. The 690
643 muscle strands that are involved in moving the ventral nerve cord is usually broader and runs 691
644 entire proboscis. The few gill pores situated dor- along the midline of the trunk region to end in 692
645 solaterally at the anterior trunk region are kid- front of the postanal tail. Within the collar region, 693
646 ney-shaped with the depression facing dorsally the collar cord is differentiated into a dorsal 694
647 (Fig. 2.5F). Only later a dorsal tongue bar grows sheath of somata including unipolar giant neu- 695
648 ventrally to eventually give the gill pores their rons as well as smaller ependymal cells lining the 696
649 slitlike U shape. Tongue bars are supported inter- central canal. About two-thirds of the collar cord 697
650 nally by a collagenous bar that forms within the are filled with numerous neurites that form a ven- 698
651 ECM. It is the same material of which the pro- tral neuropil (Fig. 2.8D). The collar cord contin- 699
652 boscis skeleton is made of. The proboscis skele- ues anteriorly into the proboscis stem, a thickened 700
653 ton supports the stomochord ventrally and area of neurites located at the dorsal base of the 701
654 bifurcates within the collar region to flank the proboscis (Fig. 2.6E, F). 702
655 buccal cavity on either side. As the juveniles
656 grow, subsequent development primarily
657 involves increase of size, particularly trunk elon- Late Development in Indirect 703
658 gation, and addition of gill slits. The number of Developing Enteropneusts 704
659 gill slits in adult S. kowalevskii varies greatly as
660 new pairs seem to be added continuously After hatching, the larvae of the indirect devel- 705
661 throughout lifetime. oper Balanoglossus misakiensis are of slightly 706
 72 S. Kaul-Strehlow and E. Röttinger

A B C D

Fig. 2.8  Semi-schematic illustration of the neurulation trally. (C) The neural plate has formed a subepidermal
process in Saccoglossus kowalevskii as revealed by trans- tubular nerve cord. (D) The collar cord comprises a dorsal
mission electron microscopy. (A) Early stage showing sheath of soma and a ventral neuropil. (Modified from
dorsal neural plate. (B) the neural plate invaginats ven- Kaul and Stach 2010). nc neural canal, np neuropil

707 elongated shape and feature an apical hood Ruppert and Balser 1986). The meso- and meta- 742
708 below which the mouth opening will soon break coels in B. misakiensis form as a lateral pair of 743
709 through on the ventral side. A less pronounced protrusions from the intestine region (Figs. 2.4 744
710 perianal ciliary ring and the opisthotroch are and 2.9A). They elongate anteriorly to subse- 745
711 present near the posterior end of the larvae quently constrict in the middle to subdivide into 746
712 (Heider stage) (Fig. 2.7A). Approximately 1 day the anterior mesocoel and posterior metacoel. 747
713 after hatching, a simple neotroch is developed, This mode of development has also been reported 748
714 separating the aboral field from the oral field that from B. clavigerus (Bourne 1889; Spengel 1893; 749
715 is used for food collection and transport Stiasny 1914a) and Glandiceps sp. (Rao 1953). 750
716 (Fig. 2.7B). On the ventral side, another longitu- In tentaculated tornaria of, e.g., Ptychodera, 751
717 dinal yet short ciliary band is present, the neu- however, the meso- and metacoels form from 752
718 rotroch. The larvae swim actively in the water multiple clusters of mesenchymatic cells within 753
719 column and feed on phytoplankton as soon as the the blastocoel (Morgan 1894). After the 754
720 mouth and anus have opened. A few days later, Metschnikoff stage, indirect developers typically 755
721 the tornaria has increased in size and the neo- enter the so-called Krohn stage by developing 756
722 troch develops a more complex pattern of ciliary secondary lobes and saddles on the neotroch 757
723 bands on the anterior half of the larva by forming without obvious changes of their internal anat- 758
724 primary lobes (Fig. 2.7C). The beginning of pri- omy. In this stage, the tornaria exhibits a more 759
725 mary lobe formation is characteristic for the compact shape with a nearly planar perianal 760
726 early Metschnikoff stage. At the anterior tip, a field. The Krohn stage larva may differ morpho- 761
727 pair of dark eye spots is visible alongside the logically between species and exhibit species-­ 762
728 central ciliary tuft. During subsequent develop- specific characters. For instance, the Krohn 763
729 ment, the tornaria of B. misakiensis grows to a tornaria of Ptychodera flava develops a highly 764
730 remarkable size of 1.5 mm and the primary lobes sinuous neotroch eventually resulting in small 765
731 of the neotroch form deep protrusions to enlarge tentacles (Hadfield 1975; Nielsen and Hay- 766
732 the oral field considerably (Fig. 2.7D). At the Schmidt 2007). In contrast, the neotroch in B. 767
733 Metschnikoff stage, adult structures such as the clavigerus never develops tentacles on the sec- 768
734 proboscis vesicle (pericardium) and the meso- ondary lobes, and in B. misakiensis, the Krohn 769
735 and metacoels become apparent. The proboscis stage is skipped completely by proceeding 770
736 vesicle forms a small coelomic cavity close to directly into the Spengel stage (Figs. 2.7E and 771
737 the hydropore on the right side (Fig. 2.9A). Its 2.9B). An elaborated neotroch with tentacles as 772
738 origin seems to be species dependent and has found in P. flava is likely to result in a more effi- 773
739 been reported from the ectoderm (Spengel 1893; cient food uptake and correlates with the 774
740 Stiasny 1914b) or mesoderm by pinching off of extended pelagic period of up to 5 months in this 775
741 the protocoel (Dawydoff 1907; Morgan 1894; species. 776
2 Hemichordata
A B
D E
F G
Fig. 2.9  3D reconstructions of major organ systems in
different developmental stages of Balanoglossus
misakiensis revealed from complete serial semithin sec-
tions. Opisthotroch and ciliary bands are omitted. Anterior
to the top in (A–C) and to the left in (D–G). (A) Overview
of the inner anatomy of a typical tornaria at the
Metschnikoff stage. (B) Spengel stage. All coelomic cavi-
ties have enlarged considerably. The digestive tract is
pulled backwards. Anlagen of the first gill pores are visi-
ble. (C) Agassiz stage with first pair of gill pores opened.
(D) Early settled juvenile. The gill slit is U-shaped,
because of a dorsal tongue bar. The central nervous sys-
tem forms at this stage. (E) Two-gill-slit juvenile (3 days
73
C
post settlement). (F) Close-up of D showing the inner
organisation of the proboscis, collar and anterior trunk
region in detail. (G) Close-up of E showing the inner
organisation of the proboscis, collar and anterior trunk
region in detail. agp anlage of the gill pore, ams anlage of
the mesocoel, amt anlage of the metacoel, ao apical organ,
cc collar cord, dnc dorsal nerve cord, gl glomerulus, gp
gill pore, gs gill slit, hs heart sinus, i intestine, mo mouth
opening, msp mesocoelic pore, pd pericardium, ph phar-
ynx, phc perihaemal cavity, pnr peribranchial nerve ring,
pp proboscis pore, psk proboscis skeleton, pst proboscis
stem, sm stomochord, st stomach
 74 S. Kaul-Strehlow and E. Röttinger

777 All the preceding larval stages (Müller, spans the anterior tip of the protruding stomoch- 825
778 Heider, Metschnikoff, Krohn) comprise the so-­ ord and is posterodorsally adjoined by the peri- 826
779 called progressive larval development where the cardium. In B. misakiensis, one pair of 827
780 larvae increase in size. The following Spengel dorsolateral gill pores is present at this stage at 828
781 stage marks the beginning of the regressive larval the anterior margin of the trunk region (Fig. 2.9C). 829
782 development and is characterised by a decrease Time and number of formation of gill pores is 830
783 in size of the larva and remodelling of the preoral species specific, since reports from other entero- 831
784 part by fusion of the ciliary bands of the neotroch pneust species show competent Agassiz larvae 832
785 (Stiasny 1914b; Agassiz 1873; Nielsen and with several pairs of gill pore anlagen (Agassiz 833
786 ­Hay-­Schmidt 2007). As a result, the future pro- 1873; Morgan 1894). The meso- and metacoels 834
787 boscis gets a more and more smooth surface and progressively reduce the blastocoel to the dorsal 835
788 the position of the neotroch is indicated by and ventral midline by which the haemal system 836
789 grooves. At the same time, a number of internal is formed. The paired metacoel sends anterodor- 837
790 modifications occur. The protocoel enlarges sig- sal projections into the base of the proboscis, that 838
791 nificantly and begins to fill up the preoral part of is, the perihaemal cavities. Larvae at the Agassiz 839
792 the larva, thereby subsequently reducing the blas- stage usually stop swimming in the upper water 840
793 tocoel (Fig. 2.9B). The proboscis vesicle has column and instead begin to visit the bottom 841
794 increased in size and is now situated dorsally more frequently. The pair of apical eye spots 842
795 onto the developing stomochord. The mouth degenerates at this stage and the larvae are now 843
796 opening and pharynx have shifted backwards and competent for settlement. After settlement, the 844
797 the anlagen of the gill pores are visible as paired, larva grows into a young juvenile worm mainly 845
798 lateral evaginations from the posterior pharyn- by elongation of the trunk region. In settled juve- 846
799 geal region (Fig. 2.9B). The meso- and metacoels niles of B. misakiensis approximately 12 h post 847
800 have extended anteriorly as well as medially and fertilisation, the opisthotroch is still present in the 848
801 surround the digestive tract almost completely. middle of the trunk region in the majority of 849
802 The Spengel stage in Balanoglossus misakiensis specimens (Fig. 2.7G). The overall morphology 850
803 lasts only for a couple of hours and marks the shows only minor changes compared to the com- 851
804 transition from the fully grown tornaria into the petent Agassiz stage which concern the collar 852
805 competent Agassiz stage. The Agassiz stage is region, shape of the gill pores and the coelomic 853
806 the last stage before the animals settle and grow cavities. The collar region is subdivided into an 854
807 into a juvenile acorn worm. This stage is charac- anterior and a posterior part by a circular con- 855
808 terised by the complete absence of the neotroch striction. A dorsal tongue bar grows ventrally and 856
809 and potential tentacles. The larvae of B. misakien­ gives the gill slit its final U shape. Moreover, 857
810 sis are of elongated shape and the future three paired mesocoel ducts open into the first gill slit 858
811 body regions can be distinguished (Figs. 2.7F and on both sides and connect the mesocoel to the 859
812 2.9C). The anterior proboscis region is conical exterior (Fig. 2.9F). Within the posterior part of 860
813 and separated from the posterior part by a deep the protocoel, the stomochord-heart-glomerulus 861
814 constriction where the mouth opens into on the complex is almost completely developed. The 862
815 ventral side. The collar region is short and subdi- coelomic system gets more and more intricate as 863
816 vided from the posterior trunk region by a shal- the animals grow and aside from the perihaemal 864
817 low circular depression. The former planar cavities, that are extensions from the trunk coe- 865
818 perianal field is now highly convex as it has lom (metacoel), the collar coelom (mesocoel) 866
819 started to grow out posteriorly. The opisthotroch also sends bilateral projections anteriorly into the 867
820 is still well developed and continues to propel the base of the proboscis, thereby flanking the sto- 868
821 larva through the water. The protocoel has com- mochord (Fig. 2.9G). At ~3 days post settlement, 869
822 pletely extended and opens to the exterior through the juvenile worms of B. misakiensis have com- 870
823 the proboscis pore on the left side of the dorsal pletely lost the opisthotroch (Fig. 2.7H). The pro- 871
824 base of the proboscis (Fig. 2.9C). The glomerulus boscis is short and conical and the collar region 872
 2 Hemichordata 75

873 exhibits a three-lobed shape (Figs. 2.7H and However, further studies including serial sections 920
874 2.9E, G). The proboscis skeleton is present and for TEM and characterisation of the molecular 921
875 supports the fragile neck region by underlying signature of the eyes in tornaria larvae are neces- 922
876 the stomochord (Fig. 2.9G). sary in order to substantiate or reject this postula- 923
877 Approximately 1 week after settlement, for- tion. For instance, the composition of the sea 924
878 mation of a premature juvenile featuring a dis- urchin eye was also discovered only recently 925
879 tinct hepatic region and three pairs of gill slits is (Ullrich-Lüter et al. 2011) and revealed a solely 926
880 completed in Balanoglossus misakiensis (Urata rhabdomeric photoreceptor. 927
881 and Yamaguchi 2004). In older tornaria larvae, serotonin-LIR as well 928
as FMRFamide-LIR neurons of the apical organ 929
882 Neurogenesis in Indirect Developing are arranged in two clusters of cells, one situated 930
883 Enteropneusts in the preoral part of the neotroch and the other in 931
884 In particular, the broad usage of antibody stain- the postoral part of the neotroch (Fig. 2.10C, D). 932
885 ings to visualise specific parts of the nervous sys- Both clusters of neurons are interconnected by a 933
886 tem has contributed significantly to our comprehensive central neuropil (Nezlin and 934
887 knowledge of neurogenesis in indirect develop- Yushin 2004). The opisthotroch nerve ring con- 935
888 ing enteropneusts. Several papers are available tains synaptotagmin and tyrosine hydroxylase in 936
889 that describe the nervous system in single tor- early stages and later also serotonin (Nielsen and 937
890 naria stages, yet only two detailed studies docu- Hay-Schmidt 2007; Miyamoto et al. 2010). The 938
891 menting a complete developmental series have neural arrangement in the tornaria shows strong 939
892 been published so far (Nielsen and Hay-Schmidt congruence to that in echinoderm larvae, further 940
893 2007; Miyamoto et al. 2010). In particular, the supporting the assumption that both larval types 941
894 exact mode of neural remodelling from metamor- are homologous and evolved from a common 942
895 phosis through juvenile stages is still unclear. ambulacrarian ancestor (Byrne et al. 2007). At 943
896 The nervous system in tornaria larvae devel- the time of metamorphosis when tornariae reach 944
897 ops gradually from anterior to posterior. In early the Agassiz stage, the larval nervous system 945
898 hatched larvae, the nervous system comprises a degrades and the adult nervous system starts to 946
899 small apical organ of few synaptotagmin-like develop (Miyamoto et al. 2010). It could be 947
900 immunoreactive (LIR) as well as serotonin-LIR shown that the majority of nervous cells of 948
901 cells with neurites projecting posteriorly the ciliary band degrade and contribute little to 949
902 (Fig. 2.10A, B) (Nielsen and Hay-Schmidt 2007; the adult nervous system. During settlement, a 950
903 Miyamoto et al. 2010). As the neotroch develops basiepidermal nerve net within the proboscis and 951
904 neurite bundles, neurons form along the ciliary collar region becomes apparent and the nerve 952
905 band. In addition, the pan-neuronal marker syn- cords develop along the ventral and dorsal mid- 953
906 aptotagmin reveals a nerve net throughout the line of the trunk region. The collar cord within 954
907 oral field of the larvae (Miyamoto et al. 2010). In the collar region neurulates in a similar way as in 955
908 older larvae, the apical organ usually consists of Saccoglossus kowalevskii (Fig. 2.8) and eventu- 956
909 numerous serotonin- as well as FMRFamide-LIR ally becomes situated subepidermally (Morgan 957
910 neurons and a pair of eye spots situated laterally 1894; Miyamoto and Wada 2013). From approx. 958
911 within the apical organ (Fig. 2.10C, D) (Nezlin 3 days post settlement in Balanoglossus misakien­ 959
912 and Yushin 2004). The exact ultrastructure of the sis, all main parts of the centralised nervous sys- 960
913 eyes has not been investigated in detail. The tem are present (Fig. 2.9E, F), that is, anterior 961
914 scarce data available describe a mixed photore- proboscis stem, neurulated collar cord, circum- 962
915 ceptor cell with a rhabdomere as well as a modi- ferential peribranchial nerve ring and a dorsal as 963
916 fied cilium (Brandenburger et al. 1973). If true, well as a ventral longitudinal nerve cord within 964
917 tornaria larvae would feature a photoreceptor cell the trunk region. Unfortunately, almost nothing is 965
918 type that is unique in the animal kingdom, i.e., known about the formation and distribution of 966
919 a combined rhabdomeric and ciliary photoreceptor. specific neurotransmitters such as serotonin or 967
 76
a b
d e
Fig. 2.10  Antibody stainings of the developing nervous
system in Balanoglossus misakiensis. (A) Early tornaria
(Heider stage). (B) Same stage as in (A). The apical organ
consists of about 18 serotonin-LIR (5-HT) sensory cells.
(C) Fully grown tornaria (late Metschnikoff). Dorsal
view. (D) Close-up of the apical region of fully grown tor-
naria showing the lateral pair of eye spots and the course
968 FMRFamide in juvenile enteropneusts. One sole
969 immunocytochemical study has been published
970 on the nervous system of the miniaturised species
971 Meioglossus psammophilus (Worsaae et al.
972 2012). The adult nervous system of M. psam­
973 mophilus comprises several serotonergic sensory
974 neurons within the anterior and middle part of the
975 proboscis. A basiepidermal nerve net extends
976 throughout the proboscis and is most prominent
977 at the dorsal base (proboscis stem). Posterior to
978 the proboscis stem, individual serotonergic
979 ­neurites pass through the collar cord. A circum-
980 ferential ring of serotonergic neurons is present
981 in the collar region. These sensory neurons proj-
982 ect with a single neurite at first posteriorly, until
983 the end of the collar region, and then ventrally
984 into a median nerve cord. Serotonin-LIR reveals
985 a comparably short ventral nerve cord that bifur-
986 cates at the level of the midgut into a pair of ven-
987 trolateral neurite bundles that further extend
988 posteriorly until the end of the trunk region.
S. Kaul-Strehlow and E. Röttinger
c
of the ciliary bands. (E) Same region as in (D). The apical
organ consists of numerous sensory cells (5-HT), subdi-
vided into a ventral and a dorsal cluster. ao apical organ,
at apical tuft, cb ciliary band, eye eye spot, onr opis-
thotroch nerve ring, op opisthotroch, pa perianal field, pec
preoral cluster, poc postoral cluster, pn posteriorly neu-
rites, sn sensory cell
Individual serotonergic neurons are scattered 989
throughout the trunk epidermis and project into 990
one of the two ventral nerve cords. 991
Taken together, the few available data on neu- 992
rogenesis of the adult nervous system in entero- 993
pneusts make it considerably difficult to compare 994
it to other deuterostomes and accordingly render 995
testing of homology hypotheses difficult. 996
Comparative Aspects 997
of Hemichordate Development 998
Hemichordates and particularly enteropneusts 999
seem to have retained a number of ancestral deu- 1000
terostome traits, such as radial cleavage, entero- 1001
coely and bilateral symmetry. Early development 1002
including cleavage and cell fates is highly con- 1003
served among hemichordates (Colwin and 1004
Colwin 1951, 1953; Tagawa et al. 1998a). The 1005
radial, equal and holoblastic cleavage leads to a 1006
 2 Hemichordata 77

1007 coeloblastula that subsequently undergoes gastru- early from the anterior end of the endoderm. In 1055
1008 lation. Hemichordates share this cleavage pattern this aspect, the precocious hydropore formation 1056
1009 with echinoderms (Henry et al. 2001; McClay documented in some holothurians and the 1057
[AU2] 2011; Chap. XX) and, depending on the yolk con-
1010 spengelid enteropneust G. hacksi (Urata et al. 1058
1011 tent, also with chordates (Bertrand and Escriva 2014) is very interesting. The mode of develop- 1059
1012 2011; Lemaire 2011; Chap. XX). Radial cleavage ment is so strikingly similar that an independent 1060
1013 is a plesiomorphic feature for Ambulacraria and evolution can hardly be assumed. It shows that 1061
1014 Chordata and was most likely inherited from an irrespective of the derived phylogenetic position 1062
1015 early ancestor (Ax 2001; Nielsen 2011). of holothurians within Echinodermata, they nev- 1063
1016 The five main coelomic cavities (single ertheless may have retained more ancestral traits 1064
1017 ­protocoel, paired meso- and metacoels) in hemi- than previously thought. It should be repeated 1065
1018 chordates originate from the endoderm by entero- here that it was already Eschscholtz (1825) who 1066
1019 coely. While the protocoel derives from the compared and related the first described entero- 1067
1020 anterior end of the archenteron in all species pneust Ptychodera flava to holothurians. 1068
1021 studied, the formation of the meso- and meta- The sixth coelomic cavity, the pericardium, is 1069
1022 coels varies considerably. For instance, the meso- part of the heart-glomerulus complex and thus 1070
1023 and metacoels in Saccoglossus kowalevskii and indirectly involved in excretory function. 1071
1024 Glandiceps hacksi (Urata et al. 2014) develop Homology of this heart-glomerulus complex of 1072
1025 from separate evaginations of the middle and hemichordates with the axial complex of echino- 1073
1026 posterior endodermal region (Bateson 1884; derms is widely accepted, because of a number of 1074
1027 Kaul-Strehlow and Stach 2013), whereas in functional and structural similarities (Balser and 1075
1028 Balanoglossus clavigerus and B. misakiensis, Ruppert 1990; Mayer and Bartolomaeus 2003; 1076
1029 they emerge from a single pair of evaginations Kaul-Strehlow and Stach 2011; Nielsen 2011; 1077
1030 that eventually subdivide into the more anterior Merker et al. 2013). These include a contractile 1078
1031 mesocoels and posterior metacoels (Stiasny pericardium (pulsatile vesicle in echinoderms), 1079
1032 1914b; Spengel 1893; Urata and Yamaguchi filtrating podocytes on the protocoelic (axocoelic) 1080
1033 2004). A closer look at other enteropneust spe- side, an excretory hydropore and a glomerulus. 1081
1034 cies and echinoderms reveals enormous intraphy- However, despite the unquestioned homology of 1082
1035 letic variation of coelom formation (for review, the differentiated structures, the ontogenetic ori- 1083
1036 see Nielsen 2011). Although an ancestral deu- gin shows considerable variations between entero- 1084
1037 terostome pattern may be hard to reconstruct, it pneust species as well as echinoderm species. For 1085
1038 seems that at least in all cases, the mesoderm is instance, the pericardium in Saccoglossus kowa­ 1086
1039 formed from the endoderm. Moreover, in echino- levskii develops from the ectoderm by schizocoely 1087
1040 derms as well as hemichordates, the mesoderm (Kaul-Strehlow and Stach 2011), a mode of devel- 1088
1041 forms as three successive pairs of coelomic cavi- opment that is usually associated with proto- 1089
[AU3] ties (proto-, meso- and metacoels) (Chap. XX).
1042 stomes (Technau and Scholz 2003). On the other 1090
1043 During the development of the cephalochordate hand, a mesenchymatic (Morgan 1891; Rao 1953) 1091
1044 amphioxus, the larva passes a similar tricoelo- and further enterocoelic origin (Dawydoff 1907) 1092
1045 mate stage, before additional coelomic pouches of the pericardium has been reported from other 1093
1046 are added (Stach 2002) (Chap. XX). The fact that enteropneust species. The same holds true for 1094
1047 three pairs of coelomic cavities are present in echinoderms (for review, see Hyman 1955) and 1095
1048 members of all main deuterostome groups, at demonstrates that homologous structures indeed 1096
1049 least at a certain developmental stage, leads to the may have different ontogenetic origins. However, 1097
1050 conclusion that this is an ancestral condition for a general or even ancestral mode of pericardial 1098
1051 deuterostomes. As mentioned before, the devel- formation for Ambulacraria is thus hard to infer. 1099
1052 opment of the protocoel seems to be rather con- The situation is even more complicated by the fact 1100
1053 served among ambulacrarians in being always that corresponding counterparts of the nephridial 1101
1054 the first coelomic cavity that pinches off very complex of ambulacrarians may be present in 1102
 78 S. Kaul-Strehlow and E. Röttinger

[AU4]
1103 chordates (Chap. XX). In particular, homology of Gene Expression 1151
1104 the pericardium with Hatschek’s left diverticu-
1105 lum or Hatschek’s pit in cephalochordates
1106 (Goodrich 1917; Franz 1927; Nielsen 2011) or The pivotal phylogenetic position of hemichor- 1152
1107 with Hatschek’s nephridium in cephalochordates dates, the shared fate map during ambulacrarian 1153
1108 (Stach 2002) has been suggested earlier. In any embryonic development (Colwin and Colwin 1154
1109 case, if homologous structures are present in 1951; Cameron et al. 1987, 1989; Cameron and 1155
1110 chordates, then the nephridial complex of Davidson 1991; Henry et al. 2001) and the above-­ 1156
1111 Ambulacraria may represent a plesiomorphic mentioned classical and modern morphological 1157
1112 character within deuterostomes rather than con- descriptions have suggested homologies between 1158
1113 stituting a synapomorphy of Echinodermata and various hemichordate, echinoderm and chordate 1159
1114 Hemichordata. features. Hence, hemichordates are particularly 1160
1115 The nervous system of the tornaria larva con- appealing to investigate the evolution of deutero- 1161
1116 sists of an apical organ comprising different types stome developmental mechanisms, and in the 1162
1117 of neurons and nerves along the ciliary bands past decades, the growing community working 1163
1118 (Hay-Schmidt 2000; Nezlin and Yushin 2004; on hemichordates has developed a basic toolset 1164
1119 Miyamoto et al. 2010). Serotonin-LIR cells in the to gain insight into the molecular mechanisms 1165
1120 apical organ are arranged in bilateral clusters that drive embryonic development, the patterning 1166
1121 interconnected by a median neuropil (Nezlin and of the larval and adult body plan as well as the 1167
1122 Yushin 2004). During ontogeny, the nervous sys- molecular signature of particular structures, i.e., 1168
1123 tem develops gradually from anterior to poste- gill slits (Rychel and Swalla 2007; Gonzalez and 1169
1124 rior, and at metamorphosis, the larval nervous Cameron 2009; Gillis et al. 2011). 1170
1125 system degrades and the adult nervous system is As for most “non-model” organisms, classical 1171
1126 formed (Miyamoto et al. 2010). The nervous sys- degenerative PCR approaches (Tagawa et al. 1172
1127 tem of the different echinoderm larvae develops 1998b) or, more recently, the analysis of tran- 1173
1128 likewise and features in principle the same com- scriptomic data sets were used to identify genes 1174
1129 ponents. Of course, taxon-specific traits are pres- and characterise their expression in hemichor- 1175
1130 ent, but the general neural body plan of an apical dates (Lowe et al. 2003; Röttinger and Martindale 1176
1131 organ with sensory cells resting in the apical cili- 2011; Chen et al. 2014). Lately, the genomes of 1177
1132 ary band and nerves along the neotroch is present Ptychodera flava and Saccoglossus kowalevskii 1178
1133 (Hay-Schmidt 2000; Burke et al. 2006; Byrne have been sequenced and used for genome com- 1179
1134 et al. 2007). As in hemichordate tornariae, the parisons between these two species (Freeman 1180
1135 echinoderm larval nervous system contributes et al. 2012). This will provide an important 1181
1136 little if anything to the pentameral nervous sys- resource to identify the genetic toolkit and regula- 1182
1137 tem of the juveniles (Byrne and Cisternas 2002; tory elements of acorn worms. Protocols have 1183
1138 Cisternas and Byrne 2003; Nakano et al. 2006). been developed and optimised for whole mount 1184
1139 Because of numerous resemblances between the or section in situ hybridisation or immunocyto- 1185
1140 morphology of hemichordate tornariae and echi- chemistry and are now routinely applied on sev- 1186
1141 noderm larvae, they have been grouped together eral hemichordate species (Tagawa et al. 1998b; 1187
1142 under the term dipleurula-type larvae Okai et al. 2000; Lowe et al. 2003; Smith et al. 1188
1143 (Metschnikoff 1881). However, since the sister 2003; Sato et al. 2009; Miyamoto et al. 2010; 1189
1144 group of Ambulacraria, that is, Chordata, do not Miyamoto and Wada 2013). In order to determine 1190
1145 have primary larvae, it remains uncertain if a relative spatial gene expression for a set of genes, 1191
1146 dipleurula larva was already present in the last double fluorescent in situ hybridisation has been 1192
1147 common ancestor of Deuterostomia. Thus, the developed in S. kowalevskii (Pani et al. 2012). 1193
1148 dipleurula larva with its specific neotroch is During the reproductive season, controlled 1194
1149 likely to be a synapomorphy uniting Hemichordata spawning and fertilisation produce large numbers 1195
1150 and Echinodermata (Nielsen 2011). of synchronously developing embryos and larvae 1196
 2 Hemichordata
1197 that are amenable for pharmacological drug or
1198 recombinant protein treatments to analyse the
1199 effects of perturbing signalling pathways on the
1200 developmental process (Lowe et al. 2006; Darras
1201 et al. 2011; Röttinger and Martindale 2011; Pani
1202 et al. 2012; Green et al. 2013). However, specific
1203 gene knockdown experiments using siRNA and
1204 mRNA that are microinjected into fertilised
1205 oocytes have so far been reported only from
1206 Saccoglossus kowalevskii (Lowe et al. 2006;
1207 Darras et al. 2011; Pani et al. 2012; Cunningham
1208 and Casey 2014; Green et al. 2013). S. kowalevskii
1209 appears also to be the most suitable acorn worm
1210 species for classical embryological experiments
1211 (Colwin and Colwin 1950) that have recently
1212 inspired researchers to combine blastomere isola-
1213 tion and grafting experiments with molecular
1214 analysis to investigate the inductive capacities of
1215 individual blastomeres or animal-­vegetal explants
1216 (Darras et al. 2011; Green et al. 2013).
1217 Endomesoderm Formation
1218 and the Posterior Organiser
1219 In metazoans, canonical ß-catenin/Wnt (cWnt)
1220 signalling plays crucial roles during various
1221 aspects of embryonic development such as
1222 embryonic polarity, germ layer specification, pos-
1223 terior growth and anterior-posterior axis pattern-
1224 ing (Croce and McClay 2006; Lee et al. 2006;
1225 Martin and Kimelman 2009; Cho et al. 2010;
[AU5] Niehrs 2010; Chaps. XX, YY and ZZ). A recent
1226
1227 study in Saccoglossus kowalevskii has dissected
1228 the role of cWnt signalling during enteropneust
1229 development (Darras et al. 2011). Combining
1230 classical embryology, gene-specific knockdown
1231 experiments and gene expression analysis, the
1232 authors showed that ß-catenin is accumulated at
1233 the vegetal pole, the future site of gastrulation,
1234 which is required for endomesoderm specifica-
1235 tion. In addition, the endomesoderm secretes yet
1236 undefined signals that determine the posterior fate
1237 of the adjacent ectoderm, as the ectoderm will
1238 adopt default anterior fates when the endomeso-
1239 derm is removed (Darras et al. 2011). This mech-
1240 anism is very similar to the one observed in
1241 echinoderms (Angerer et al. 2011) and vertebrates
79
(Niehrs 2010), suggesting a conserved function 1242
of cWnt signalling at the base of deuterostomes in 1243
germ layer specification and the formation of a 1244
posterior organiser (Darras et al. 2011). 1245
In hemichordates, mesoderm forms by entero- 1246
coely (Bateson 1884), a process that is shared 1247
with echinoderms and basal chordates such as 1248
amphioxus and ascidians. The FGF signalling 1249
pathway plays a crucial role in mesoderm induc- 1250
tion in vertebrates and basal chordates (Slack 1251
et al. 1989; Kim et al. 2000; Imai et al. 2002; 1252
Fletcher et al. 2006; Kimelman 2006; Bertrand 1253
et al. 2011; Chap. XX). In order to investigate the [AU6]
1254
evolution of mesoderm formation, a recent study 1255
has examined the role of FGF signalling during 1256
mesoderm formation in Saccoglossus kowa­ 1257
levskii (Green et al. 2013). Expression of the FGF 1258
ligand fgf8/17/18 is restricted to ectodermal 1259
regions overlying sites of mesoderm specifica- 1260
tion within the archenteron, while the regions 1261
that will form mesoderm express the receptor 1262
fgfr-B. The resulting suggestion that mesoderm 1263
induction in the archenteron requires contact 1264
with the ectoderm to allow FGF/FGFR signalling 1265
is confirmed by embryological experiments that 1266
are combined with gene expression analysis of 1267
the downstream target snail. Gene-specific 1268
knockdown and gain-of-function experiments 1269
show that FGF8/17/18 is required and sufficient 1270
for mesoderm induction in S. kowalevskii and 1271
support the idea that FGF signalling played an 1272
ancestral role in deuterostome mesoderm forma- 1273
tion (Green et al. 2013; Chaps. XX and YY). [AU7]
1274
Dorsoventral Patterning 1275
BMP, a ligand of the TGFß family, and its antago- 1276
nist Chordin play a central role in establishing the 1277
dorsoventral axis and the specification of the 1278
­central nervous system (CNS) in bilaterian ani- 1279
mals (Arendt and Nübler-Jung 1996; De Robertis 1280
and Sasai 1996; Holley and Ferguson 1997; De 1281
Robertis et al. 2000; De Robertis and Kuroda 1282
2004; Chaps. XX, YY and ZZ). Gene expression [AU8] 1283
analysis in Saccoglossus kowalevskii and 1284
Ptychodera flava has shown expression of bmp2/4 1285
and its potential downstream target dlx in dorsal 1286
 80 S. Kaul-Strehlow and E. Röttinger

A B C D E

F G H I J

Fig. 2.11  Comparison of dorsoventral gene expression flava (F–J). All illustrations are based on published data:
patterns in hemichordates. Illustrations of ventral (purple) A, B and E, Lowe et al. (2006); C, Röttinger and Lowe
and dorsal (yellow) gene expression patterns in late gas- (2012); D, Darras et al. (2011); and F–J, Röttinger and
trulae of Saccoglossus kowalevskii (A–E) and Ptychodera Martindale (2011)

1287 territories (Fig. 2.11D, E, I, J), while chordin functional molecular studies that have been per- 1313
1288 ­transcripts (Fig.  2.11C, H) are localised in the formed in harrimaniid (Saccoglossus kowa­ 1314
1289 ventral ectoderm, suggesting that these proteins levskii) and also in ptychoderid (Ptychodera 1315
1290 are also involved in dorsoventral patterning in flava) enteropneusts aim to describe the dorso- 1316
1291 hemichordates (Harada et al. 2001, 2002; Lowe ventral patterning event in hemichordates (Lowe 1317
1292 et al. 2006; Röttinger and Martindale 2011). et al. 2006; Röttinger and Martindale 2011). This 1318
1293 Functional studies in S. kowalevskii demonstrate enables comparing the molecular mechanism 1319
1294 the implication of BMP signalling in this process, controlling dorsoventral patterning within ambu- 1320
1295 as overexpression and knockdown of BMP2/4 lacrarians (Fig. 2.12). 1321
1296 result in dorsalised or ventralised embryos, While bmp2/4 expression in enteropneusts is 1322
1297 respectively (Lowe et al. 2006). Based on func- restricted to dorsal structures (Fig. 2.11D, I; 1323
1298 tional studies in other bilaterian animals, the pre- Harada et al. 2002; Lowe et al. 2006; Röttinger 1324
1299 diction would be that overactivating BMP and Martindale 2011), bmp2/4 expression in echi- 1325
1300 signalling represses neural fates (De Robertis and noderms is localised on the opposite site in the 1326
1301 Kuroda 2004). However, this is not the case in S. ventral ectoderm (Angerer et al. 2000; Duboc 1327
1302 kowalevskii embryos treated with recombinant et al. 2004). Interestingly, functional studies in 1328
1303 BMP4 protein, suggesting that BMP signalling in Saccoglossus kowalevskii and echinoderms have 1329
1304 hemichordates is involved in dorsoventral pattern- shown that regardless the expression domain of 1330
1305 ing but not in neurogenesis (Lowe et al. 2006). the diffusible ligand bmp2/4, its activity is always 1331
1306 The molecular mechanisms underlying dorso- confined to the dorsal ectoderm (Duboc et al. 1332
1307 ventral patterning have been extensively studied 2004; Lowe et al. 2006). In S. kowalevskii and 1333
1308 in echinoderms (Angerer et al. 2000; Duboc et al. Paracentrotus lividus (echinoderm), dlx is an indi- 1334
1309 2004; Su and Davidson 2009; Saudemont et al. rect downstream target of BMP signalling (Lowe 1335
[AU9] 2010; Chap. XX) and are represented in a simpli-
1310 et al. 2006; Saudemont et al. 2010). In Ptychodera 1336
1311 fied version in Fig. 2.12A (for a more compre- flava, dlx transcripts are detected in the dorsal ecto- 1337
1312 hensive version, see Chap. XX). To date, the only derm (Harada et al. 2001; Röttinger and Martindale 1338
 2 Hemichordata
A B
Fig. 2.12  Comparison of dorsoventral patterning mecha-
nisms in ambulacrarians. In echinoderms (A), Nodal sig-
nalling is essential to determine ventral fates and induce
expression of bmp2/4 that acts on the dorsal side to spec-
ify dorsal ectoderm. In Saccoglossus kowalevskii (B),
BMP signalling is crucial for specifying the dorsal ecto-
derm as well, which might potentially also be the case for
Ptychodera flava (C). While a NiCl2-sensitive signal is
involved in specifying ventral fates in enteropneusts (B,
C), the relation to Nodal signalling remains unknown. (A)
1339 2011), suggesting that BMP signalling is active in
1340 this territory as well. However, additional experi-
1341 ments are required to confirm this hypothesis.
1342 The Nodal signalling pathway plays crucial
1343 roles in various deuterostome developmental
81
Paracentrotus lividus (echinoderm), simplified diagram
based on data from Saudemont et al. (2010). (B)
Saccoglossus kowalevskii (harrimanid enteropneust), dia-
gram based on data from Lowe et al. (2006), Saudemont
et al. (2010) and Röttinger and Martindale (2011). (C)
Ptychodera flava (ptychoderid enteropneust), diagram
based on data from Tagawa et al. (1998b), Taguchi et al.
(2000), Harada et al. (2001, 2002) and Röttinger and
Martindale (2011)
processes such as endo- and mesoderm forma- 1344
tion and axial patterning events along the 1345
anterior-­
posterior, dorsoventral and left-right 1346
axis (Whitman 2001; Hamada et al. 2002; 1347
Morokuma et al. 2002; Stainier 2002; Yu et al. 1348
 82 S. Kaul-Strehlow and E. Röttinger

[AU10] 2002;
1349 Chaps. XX, YY and ZZ). In echinoderms, developer Ptychodera flava and, more recently, 1394
1350 Nodal signalling is not only crucial for establish- in another ptychoderid hemichordate species, 1395
1351 ing left-right asymmetries in the larva (Duboc Balanoglossus simodensis. The recent sequenc- 1396
1352 et al. 2005) but plays also an essential role in the ing and comparison of the S. kowalevskii and P. 1397
1353 establishment of the dorsoventral axis during flava genomes has revealed the identical genomic 1398
[AU11] embryonic development (Duboc et al. 2004; Chap.
1354 organisation of their 12-gene Hox clusters 1399
1355 XX). In fact, nodal expression in the ventral ecto- (Freeman et al. 2012), which is reminiscent of the 1400
1356 derm induces ventral expression of bmp2/4 which Hox cluster organisation of B. simodensis (Ikuta 1401
1357 in turn diffuses to the dorsal ectoderm to induce et al. 2009). With the exception of differences at 1402
1358 expression of its downstream targets (Fig. 2.12A; the posterior end of these clusters, the hemichor- 1403
1359 Duboc et al. 2004; Lapraz et al. 2009; Su and date organisation is strikingly similar to that of 1404
1360 Davidson 2009; Saudemont et al. 2010). Among chordates, supporting the idea that the ambu- 1405
1361 the downstream targets of Nodal signalling in lacrarian ancestor possessed minimally a 12-gene 1406
1362 echinoderms are the ventrally expressed genes Hox cluster with at least nine genes organised 1407
1363 chordin, foxA and bra (Saudemont et al. 2010; and oriented the same as their chordate orthologs 1408
1364 Chap. XX). With the exception of the strictly (Freeman et al. 2012). 1409
1365 endodermal expression of foxA in Saccoglossus In vertebrates, expression of Hox genes as well 1410
1366 kowalevskii (Darras et al. 2011; Fritzenwanker as other transcription factors such as barH, 1411
1367 et al. 2014), bra and chordin transcripts are also engrailed, pax2/5/8, six3, etc., is restricted to the 1412
1368 detected in ventral domains in S. kowalevskii and central nervous system (CNS). To gain insight into 1413
1369 Ptychodera flava (Fig. 2.11; Tagawa et al. 1998b; the origin of the chordate CNS, previous studies in 1414
1370 Röttinger and Lowe 2012), suggesting that a Saccoglossus kowalevskii have analysed the 1415
1371 Nodal-dependent mechanism may be required to expression patterns of these genes and showed 1416
1372 define ventral domains in hemichordates. their circumferential epidermal expression during 1417
1373 NiCl2 treatments in echinoderms ventralise the early development that potentially reflects the 1418
1374 embryos and induce radialised expression of nodal broad and diffuse distribution of neurons in these 1419
1375 (Duboc et al. 2004). Interestingly, NiCl2 treat- stages (Lowe et al. 2003; Aronowicz and Lowe 1420
1376 ments in Saccoglossus kowalevskii and Ptychodera 2006; Lemons et al. 2010; Pani et al. 2012). In 1421
1377 flava also ventralise the embryos and radialise adult tissue, however, gene expression analysis of 1422
1378 expression of bra and foxA in P. flava (Röttinger neuronal markers such as Elav, synaptotagmin, 1423
1379 and Martindale 2011), further strengthening the VAChT, serotonin, Hb9, Drg11 and GABA has 1424
1380 idea that a NiCl2-sensitive and potentially Nodal- shown the existence of a centralised ventral as well 1425
1381 dependent mechanism is involved in dorsoventral as dorsal nerve cord that is internalised at the level 1426
1382 patterning in hemichordates. However, the poten- of the enteropneust worm’s collar into the collar 1427
1383 tial molecular link between NiCl2 and Nodal and cord (Nomaksteinsky et al. 2009). Intriguingly, the 1428
1384 the molecular connection between the ventralising observed centralisation of the enteropneust ner- 1429
1385 NiCl2-sensitive signal and dorsalising BMP effects vous system was in contrast to previous studies 1430
1386 in hemichordates remain unclear, and additional that described the presence of a diffuse nerve net in 1431
1387 work is required to understand the degree of con- hemichordates (Lowe et al. 2003). One idea to 1432
1388 servation to the mechanism of dorsoventral pat- explain these fundamental differences was that the 1433
1389 terning in echinoderms. developing ectoderm may represent a transient dif- 1434
fuse nerve net unrelated to that of the adult 1435
(Nomaksteinsky et al. 2009). In order to gain a 1436
1390 Anterior-Posterior Patterning ­better understanding of the relation between the 1437
embryonic and adult nervous systems, a recent 1438
1391 The bulk of molecular studies in hemichordates study has analysed a broad range of genes associ- 1439
1392 have been carried out in the direct developing ated with neurogenesis during early S. kowalevskii 1440
1393 species Saccoglossus kowalevskii, the indirect development (Cunningham and Casey 2014). 1441
 2 Hemichordata 83

1442 This analysis has revealed that already during resembling domains in Saccoglossus kowalevskii 1490
1443 embryonic development, expression of most of the (Lowe et al. 2006). Hence, because of the lack of 1491
1444 analysed genes transitions from a circumferential clear molecular data, its homology remains con- 1492
1445 to a dorsal and ventral midline localisation. Hence, troversial (Ruppert 2005; Nomaksteinsky et al. 1493
1446 this ­observation suggests that developmental cen- 2009; Kaul and Stach 2010; Holland et al. 2013). 1494
1447 tralisation of the nervous system in hemichordates Recent work in the ptychoderid enteropneust 1495
1448 occurs earlier than initially anticipated (prior to Balanoglossus simodensis has analysed the expres- 1496
1449 hatching) (Cunningham and Casey 2014). sion of genes known to be crucial of formation and 1497
1450 Perturbation of BMP signalling in patterning of the chordate neural tube (Miyamoto 1498
1451 Saccoglossus kowalevskii affects dorsoventral and Wada 2013). This study reports expression of 1499
1452 patterning but not the distribution of neurons in bmp2/4, dlx, pax3/7 and soxE in dorsal regions of 1500
1453 the analysed embryos (Lowe et al. 2006). This the collar cord but failed to observe expression of a 1501
1454 observation has led to the idea that nervous sys- potential ventral marker, pax6, thus suggesting a 1502
1455 tem formation in hemichordates may be insensi- partially conserved patterning mechanism between 1503
1456 tive to a BMP/Chordin gradient that is crucial for the hemichordate collar cord and the chordate neu- 1504
1457 the formation of the CNS in protostomes and ver- ral tube (Miyamoto and Wada 2013). In chordates, 1505
1458 tebrates. However, this analysis was carried out Hedgehog (hh) signalling emitted from the noto- 1506
1459 on embryos that presented a diffuse expression chord and received by the neural plate (via the 1507
1460 pattern of neuronal markers (Lowe et al. 2006). Hedgehog receptor patched (ptc)) is essential for 1508
1461 With the recent observations of a ventral and dor- patterning the neural tube along the dorsoventral 1509
1462 sal CNS in hemichordates (Nomaksteinsky et al. axis (Echelard et al. 1992). In B. simodensis, 1510
1463 2009; Cunningham and Casey 2014), it would be expression of hh appears restricted to the stomoch- 1511
1464 crucial to re-analyse the effects of perturbing ord and the anterior endoderm, which lies beneath 1512
1465 BMP signalling on the centralisation of the ner- the collar cord, during metamorphosis. In contrast, 1513
1466 vous system at adequate embryonic stages. ptc is expressed in the mesoderm surrounding hh- 1514
1467 Taken together, the studies described above do expressing endoderm as well as the midline of the 1515
1468 not contradict the classical idea that portions of neural plate (Miyamoto and Wada 2013). These 1516
1469 the hemichordate central nervous system may be results suggest that Hedgehog signalling from the 1517
1470 homologous to the chordate CNS (Knight-Jones underlying endoderm may be received by the col- 1518
1471 1952; Lowe et al. 2003; Nomaksteinsky et al. lar cord. However, gene-­specific functional assays 1519
1472 2009). However, the current data make it impos- are required to determine if Hedgehog signalling is 1520
1473 sible to unequivocally settle this issue at present required for dorsoventral patterning of the neural 1521
1474 (Holland et al. 2013). tube in hemichordates. 1522
1475 The tubular organisation of the collar cord of In summary, the molecular studies currently 1523
1476 enteropneusts has been proposed to be homolo- available show striking similarities between ver- 1524
1477 gous to the chordate neural tube (Morgan 1894; tebrates and enteropneusts in regard to the spatial 1525
1478 Bateson 1886; Ruppert 2005; Kaul and Stach deployment of transcription factors (Fig. 2.13) 1526
1479 2010; Luttrell et al. 2012). However, gene expres- and signalling centres involved in neuronal 1527
1480 sion data of bmp and chordin (see above) support anterior-­posterior and dorsoventral patterning of 1528
1481 the theory of dorsoventral inversion of body axes the neural tube. The implications of these obser- 1529
1482 at the base of chordates (Lowe et al. 2006). vations in establishing potential homologies 1530
1483 According to this, the chordate neural tube would between enteropneust and vertebrate body plans 1531
1484 be homologous to the ventral nerve cord of entero- and on the evolution of the chordate CNS are cur- 1532
1485 pneusts, yet it is the dorsal collar cord that neuru- rently highly debated (Holland et al. 2013) and 1533
1486 lates in enteropneusts. Moreover, genes such as additional work is required on hemichordates as 1534
1487 pax6, nkx 2.2 and msx that have similar domains well as invertebrate chordates (ascidians and 1535
1488 in the chordate neural tube and the protostome amphioxus) to gain more insights into this long-­ 1536
1489 nerve cord (Denes et al. 2007) do not at all have lasting question (see Chaps. XX and YY). [AU12]
1537
 84 S. Kaul-Strehlow and E. Röttinger

Fig. 2.13  Ectodermal anterior-posterior gene expression Saccoglossus kowalevskii (Represented gene expression
in hemichordates. Diagram summarising anterior-poste- patterns based on data from Lowe et al. (2003), Aronowicz
rior regionalisation of gene expression domains in the and Lowe (2006), Pani et al. (2012))
ectoderm of the harrimanid enteropneust hemichordate

1538 Open Questions • Development of the muscular system in direct 1544

and indirect developing enteropneusts 1545
1539 • Neurogenesis of the adult nervous system • Roles of canonical Wnt and FGF signalling in 1546
1540 • Function of the gill slits ptychoderids and of Nodal signalling in direct 1547
1541 • Investigations and characterisation of the light and indirect developing enteropneusts 1548
1542 sense organs in tornaria larvae and adult • All aspects of pterobranch embryogenesis and 1549
1543 enteropneusts development 1550
 2 Hemichordata 85

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