Evolution and development of the adelphophagic, intracapsular Schmidt’s larva of the nemertean Lineus ruber

Oviposition and timing of development of Lineus ruber

Adult specimens of L. ruber collected from the field displayed a dark red pigmentation, which was lighter at
the region of the head (Fig. 2A). L. ruber is a dioecious species. In mature female animals, multiple individual ovaries are
distributed along two lateral rows, with each ovary connecting to the epidermis through
a small gonoduct and a gonopore 1]. We did not directly observe fertilization, but it occurs internally after pseudocopulation
46]. Oviposition occurs spontaneously (Fig. 2B). Eggs are released through the lateral gonopores packed into multiple pyriform
egg capsules (Additional file 3: Video S2). As the animal releases the eggs, epithelial glands secrete an enclosing
jelly. The female nemertean progressively glides out of the jelly and lays the gelatinous
cocoon, where development takes place (Fig. 2C). Animals kept under lab conditions over a whole year were still able to reproduce,
which indicates that L. ruber can display iteroparous reproduction, at least in captivity.

Fig. 2. Oviposition in Lineus ruber. A–C Photographs of live specimens taken under the stereomicroscope. A Adult specimens of L. ruber show the typical elongated ribbon-like appearance of nemertean worms, with a dark red pigmentation throughout the body. The head region is of lighter red color, and contains several pairs of eyespots (white arrows) and a terminal anterior proboscis (asterisk). B Oviposition occurs through the multiple gonopores located on both lateral sides of
the female. Several fertilized oocytes are packed within individual pyriform capsules,
which are bounded together inside a jelly mass. As the female releases the eggs and
secretes the jelly, it glides out of the forming egg string. C Laid egg mass containing multiple egg capsules. The entire development occurs inside
this mass. E.g., egg capsules, hd head, jl jelly cover, wm worm

Development takes approximately 42 days at 14 °C (Fig. 3) (Additional file 4: Figure S2). Oocytes are rich in yolk content and display a stereotypical spiral
cleavage (personal observation and 33]) that results in the formation of a blastula 4 days after oviposition (Fig. 3A) (Additional file 4: Figure S2B). Many embryos display abnormal cell division patterns during cleavage
and get arrested at this early stage of embryogenesis. The number of unviable embryos
varies between capsules, as well as between egg masses. Gastrulation starts after
6 days of development and occurs by internalization of cells at one pole of the embryo,
resulting in the formation of an archenteron and a rounded blastopore (Fig. 3B?, B?) (Additional file 4: Figure S2C). After 10 days of development, the embryo adopts the first signs of
bilateral symmetry (Fig. 3C?, C?), with the future anterior end more elongated than the prospective posterior
side. The blastopore is still clearly open in a centered position, and the ectodermal
walls of the embryo appear more compacted. The intracapsular Schmidt’s larva forms
12 days after oviposition (Fig. 3D?, D?) (Additional file 4: Figure S2D). The larva exhibits a clear bilateral symmetry, with the anterior side
pointier than the posterior. The blastopore turns into the mouth of the larva, and
the blastoporal ectodermal rim cells appear more developed. In addition, four ectodermal
discs occupy the anterior left, anterior right, posterior left and posterior right
sides of the larva. A thin ciliated epidermis connects these four discs and encloses
the larval body. Internally, the archenteron forms a blind gut. When dissected out
of the egg capsules, the Schmidt’s larvae spin and swim slightly. Inside the capsules,
they feed on the unviable arrested embryos with which they often cohabitate. As a
result of the ingestion of other siblings, the larva increases in size and the blind
gut becomes filled with nutrients (Fig. 3E?–E???) (Additional file 4: Figure S2E). Approximately 18 days after deposition, the ectodermal discs of the
Schmidt’s larva are more developed and extend over the whole body, which is now more
elongated along the anteroposterior axis and shows a more worm-like shape (Fig. 3F?, F?). At this point of development, the original larval epidermis covers the body,
but starts to detach from the developing definitive epidermis in some regions of the
larva. The mouth is still open in an antero-ventral position.

Fig. 3. The embryonic development of Lineus ruber. A–H Photographs of fixed dissected embryos of L. ruber at representative stages of development. A A stereotypical quartet spiral cleavage results in the formation of a yolky coeloblastula.
B?, B? Gastrulation occurs at one pole of the embryo and results in the formation of a central
round blastopore and an internal archenteron. C?, C? The late gastrula shows the first signs of bilateral symmetry, with the future anterior
end pointier than the posterior side, which is more round (more evident in C?). D?, D? The Schmidt’s larva forms after 12 days of development. It exhibits an anterior pair
and a posterior pair of ectodermal imaginal discs (arrowheads), and the blastopore has become the mouth of the larva. A thick ectodermal tissue lines the mouth opening. E?–E??? The Schmidt’s larva of L. ruber can feed on other siblings that occupy the same egg capsule. The ingestion of this
yolk material increases the size of the larva and fills up the blind gut. At this
stage, the imaginal discs are still visible (arrowheads) and a conspicuous epidermis covers the larva (arrows). F?, F? About 18 days after oviposition, the Schmidt’s larva metamorphoses into the juvenile.
The imaginal discs (arrowheads) expand and the larval epidermis (arrows) detaches from the body. G?, G? After metamorphosis, the juvenile shows a worm-like shape, elongated along the anteroposterior axis but not along the dorsoventral axis,
mostly because of the high amounts of yolk present in the blind gut. The head region
is more developed and the rudiment of the proboscis is now evident. H 8 weeks after oviposition, the juveniles are morphologically similar to the adults,
and have absorbed part of the ingested yolk. I Schematic summary of the embryonic development of L. ruber at 14 °C. The light blue area depicts the ingested yolk. Drawings are not to scale. A, B?, C?, D?, E?, E???, F?, G?, H show lateral views. B?, C?, D?, E?, F?, G? show blastoporal/ventral views. In C?–H anterior is to the left. bc blastocoel, bg blind gut, bp blastopore, id imaginal discs, iy ingested yolk, le larval epidermis, mo mouth, pb proboscis, pr pharyngeal rudiment. Scale bars (A–D?) 50 ?m; (E?, E???, F?) 100 ?m; (G?, H) 500 ?m

The metamorphosis into the early juvenile is accomplished in most of the larvae about
20 days after oviposition (Fig. 3G?, G?) (Additional file 4: Figure S2G). The early juvenile lacks the larval epidermis. We could not directly
follow the fate of this tissue, and it is thus uncertain whether the larval epidermis
is ingested by the developing juvenile, as in the sister species L. viridis37], resorbed by the definitive epidermis, or simply discarded. The early juvenile of
L. ruber has a clear worm-shape and the larval imaginal discs are no longer obvious. The head
has an anterior terminal proboscis, but the eyes are not yet formed. The mouth occupies
an antero-ventral position and the gut in most of the juveniles is full of yolk content
and still blind. Juveniles actively move inside the capsules (Additional file 5: Video S3). As development proceeds, the definitive tissues and organs mature, the
yolk is gradually absorbed and the juvenile progressively adopts the morphology of
a small adult (Fig. 3H) (Additional file 4: Figure S2H). Juveniles can escape out of the capsule, and glide within the jelly
enclosing the egg mass. About 30 days after oviposition, the juveniles show the first
signs of eyespots (Additional file 4: Figure S2I), and after about 40 days, they also show body pigmentation (Additional
file 4: Figure S2I). Hatching of the juveniles varies between egg masses, but it often occurs
after about 40 days of development (Additional file 4: Figure S2I; Additional file 6: Video S4). At the moment of hatching, the size and morphology of the juveniles can
vary (Additional file 7: Video S5), but most of them exhibit a normal behavior, being capable of preying
on eggs of the annelid P. dumerilii (Additional file 8: Video S6). A graphical summary of the main developmental events during L. ruber embryogenesis is shown in Fig. 3I.

The formation of the intracapsular Schmidt’s larva

To better characterize the development of the Schmidt’s larva, we first analyzed the
patterns of actin labeling from gastrula to early juvenile stages by confocal laser
scanning microscopy (Figs. 4, 5, 6). As also observed under transmitted light (Fig. 3B?), the 8-day-old gastrula shows a central blastoporal opening in the future ventral
side of the animal that connects with an internal archenteron (Fig. 4A?, A?). Internally, the archenteron cavity and the cells lining it occupy most of
the blastocoel, which is also populated by isolated cells (Fig. 4A?). We could not discriminate whether these come from the endoderm or the ectoderm.

Fig. 4. Formation of the Schmidt’s larva in Lineus ruber. A?–G z projections of confocal scans of embryos and larvae labeled against F-actin (grayA?–D???) and incorporated EdU (blueE–G) counterstained with the nuclear marker Sytox Green (yellow) at 8, 10, 12 and 14 days after oviposition (dao). A?, A?? The gastrula has a central ventral blastopore and a wide open archenteron, with isolated
cells occupying the blastocoel (arrowheadsA??). B?–B?? After 10 days of development, the embryo shows 4 paired ectodermal discs (marked
by dashed linesB?), an incipient proboscis rudiment, and the ventral ectodermal ring around the blastopore
(dashed circleB?). Internally, the archenteron widens and bends backwards, forming a blind gut. There
are still numerous cells in the blastocoel (arrowheads). C After 12 days, an anterior indentation forms between the two anterior imaginal discs,
and the whole embryo becomes covered by a thin epidermis. D?–D??? The mature Schmidt’s larva shows 7 separate imaginal discs covered by a larval epidermis
(arrowheadsD??): the anterior terminal proboscis imaginal disc (arrowheadD?); the four lateral imaginal discs (cephalic pair and trunk pair); the imaginal disc
associated with the mouth; and the imaginal disc of the gut. At this stage of development,
the larva feeds on other siblings (D???). E–G After gastrulation, proliferative cells are scattered and distributed in the ectoderm
and cells in the blastocoel (E). With the formation of the Schmidt’s larva, proliferation concentrates mostly on
the regions that form the imaginal discs (arrowheadsF; imaginal discs indicated by dashed lines). However, isolated cells in the space between the imaginal discs also proliferate
(arrowheadsG). A?–D??, E–G are ventral views. D??? Is a lateral view, with the dorsal side to the top. In all panels, anterior is to the left. ac archenteron, bg blind gut, bp blastopore, id imaginal disc, iy ingested yolk, le larval epidermis, mo mouth, pb proboscis. Scale bars (A?–B??, D??–G) 50 ?m; (C, D?) 10 ?m

After 10 days of development, the embryo shows the cephalic (anteriorly) and the trunk
(posteriorly) pairs of discoidal ectodermal concentrations, clearly segregated from
the ring of ectodermal cells that surround the blastoporal opening (Fig. 4B?). In addition, the first signs of the unpaired proboscis rudiment are visible (Fig. 4B?). The archenteron cavity is expanded and bent backwards (Fig. 4B??), and isolated cells are still present inside the former blastocoel. After 12 days,
the embryo adopts the appearance of a Schmidt’s larva (Fig. 3D?, D??). At this stage, the ectodermal discs are monostratified epitheliums (Fig. 4C), and the proboscis rudiment is more evident. Importantly, a thin ciliated epidermis
now covers the entire surface of the larva (Fig. 4C). After 14 days of development, the indentation of the proboscis rudiment is more
pronounced (Fig. 4D?). The Schmidt’s larva is now composed of at least seven distinct epithelial aggregates
that will be the source of the definitive tissues of the juvenile during metamorphosis:
an unpaired anterior proboscis rudiment, two cephalic discs, the ventral mouth/pharynx
rudiment, the internal endodermal blind gut, and two trunk discs (Fig. 4D??). We could not identify a separate pair of cerebral organ discs at this stage
(Additional file 9: Figure S3A), which is present in most of the pilidium larvae 17] and was previously described in the Schmidt’s larva 35]. Likewise, we did not observe the formation of a well-defined dorsal rudiment (Additional
file 9: Figure S3B). At this stage, the Schmidt’s larva feeds on other siblings, filling
the blind gut with yolk (Fig. 4D???). Finally, the analysis of EdU incorporation during the formation of the Schmidt’s
larva shows that cell proliferation is mostly concentrated in the regions of the embryo
where the imaginal discs form (Fig. 4E–G), although isolated cells in the internal cavity also proliferate, which is similar
to what is observed in pelagic planktotrophic pilidium 47].

Metamorphosis of the Schmidt’s larva and organogenesis in the early juvenile

After 16 days of development, the Schmidt’s larva has significantly increased in size
due to the feeding event, the blind gut is expanded and the blastocoel obliterated
(Fig. 5A). The larva, however, still has a spherical morphology. At this stage, cells below
the surface epidermis start to form actin-positive projections (inset in Fig. 5A), which might be the earliest signs of muscle development. Eighteen days after oviposition,
the larva adopts a worm-like morphology, with the posterior tip of the trunk more
elongated (Fig. 5B?). The presence of actin-positive fibers below the epidermis is more evident, and
the cephalic discs are much more developed (Fig. 5B??). The larval epidermis is still present in some regions of the body (Fig. 5B???). After 20 days of development, the larva of L. ruber has fully metamorphosed into a juvenile (Fig. 5C?, C??). At this stage, the mouth has a clear antero-ventral position, with a well-developed
musculature (Fig. 5C?). The head shows two bilateral lobes, with a conspicuous longitudinal and transverse
musculature, and the cerebral organ canals become visible (Fig. 5C??). In addition, longitudinal and circular muscle fibers form the body wall musculature
of the developing juvenile (Fig. 5C????). During metamorphosis, the amount of proliferative cells increases (Fig. 5D, E). After 16 days of development, they are abundant in the cephalic discs, although
an important number of proliferative cells are also scattered throughout the trunk
region. In the early juvenile, proliferation occurs throughout the entire body, but
most intensely on the lateral sides of the head.

Fig. 5. Metamorphosis of the Schmidt’s larva of Lineus ruber. A–E z projections of confocal scans of larvae and early juvenile labeled against F-actin
(grayA–C????) and incorporated EdU (blueD, E) counterstained with the nuclear marker Sytox Green (yellow) at 16, 18 and 20 days after oviposition (dao). A Larvae at 16 days of development are rounded and show the first signs of cell differentiation
(arrowheads in the inset mark F-actin projections). B?–B??? After 18 days of development, the larvae are more elongated along the anteroposterior
axis, and circular and longitudinal fibers extend below the epidermis (arrowheadsB?). The imaginal discs are more developed (B??) and the larval epidermis is only present in some parts (arrowheadsB???). C?–C???? After 20 days of development, the imaginal discs have formed the basic anatomical
features of the juvenile. The rhynchocoel occupies the central region of the head,
and the mouth opening occupies the antero-ventral side of the animal. The musculature
is now much more developed, in particular in the head region around the developing
brain lobes (arrowheadsC???) and in the body wall (C????). D, E During metamorphosis of the Schmidt’s larva into the juvenile, proliferation is widespread,
although more concentrated in the anterior imaginal discs at early stages (D) and in the lateral sides of the head in the early juvenile (E). A is a lateral view, with the dorsal side to the top. The rest of the panels are ventral views. In all panels, anterior is to the left. In D, E the blue staining in the ingested yolk is background. coc cerebral organ canals, id imaginal disc, iy ingested yolk, le larval epidermis, mo mouth, ph pharynx, pb proboscis. Scale bars (A, B?, C?, C??, D, E) 100 ?m; (B??, B???, C???, C????) 25 ?m

After 25 days of development, the shape of the juveniles can vary considerably, mostly
depending on the amount of yolk ingested during the larval stage (Fig. 6A–C). Those with more yolk retain a more rounded morphology, although they still exhibit
a well-developed musculature, proboscis, mouth, pharynx, and head region (Fig. 6A). Some of these large specimens contain another developing embryo (Fig. 6B), which demonstrates that the Schmidt’s larva not only feeds on arrested embryos,
but can also ingest apparently viable embryos. Finally, those juveniles with less
ingested yolk exhibit a more mature anatomy (Fig. 6C–E). The elongated shape along the anteroposterior axis is more evident, and the
body is also flatter along the dorsoventral axis. The body wall musculature is conspicuous
(Fig. 6C), and the pharynx shows a strong muscular plexus (Fig. 6D?, D??). The mouth opens behind the brain, which is composed of two bilateral lobes
surrounded by the head musculature (Fig. 6D??). Some muscular fibers cross the brain lobes (Fig. 6D??). The anterior median region is occupied by the proboscis (Fig. 6D??, D???), and the dorsal ectoderm posterior to the brain exhibits well formed cerebral
organ canals on each side of the head (Fig. 6D???, E) 48]. They exhibit a heavily ciliated epidermis and terminate in a wider ampulla, in correspondence
with the putative chemotactile function 1]. Labeling against F-actin cannot resolve the presence of a cerebral organ associated
with the cephalic slit at this stage. Altogether, these results indicate that the
juvenile of L. ruber acquires the basic anatomy of the adult after 25 days of development at 14 °C.

Fig. 6. The early juvenile of Lineus ruber. A–E z projections of confocal scans of juveniles labeled against F-actin (gray) counterstained with the nuclear marker Sytox Green (yellow) 25 days after oviposition (dao). A Juveniles with a large volume of ingested yolk keep a spherical shape, although they
exhibit mature tissues. B The adelphophagy of the Schmidt’s larva not only affects unviable embryos, but also
developing siblings. C–E Juveniles with a moderate quantity of yolk soon adopt a worm-like shape. The body
wall musculature is well developed and the head region has a muscular mouth (D?), and a central rhynchocoel with the two brain lobes at each side (D??–D???). Posterior to the brain, the ectoderm on the left and right sides of the head invaginates
and forms the cerebral organ canal (D???, E), the connection of the cephalic organ with the exterior. Note in (E) the particular ciliation of the inner canal compared with the outer epidermis. A A lateral view, with the dorsal side to the top. B–E Are ventral views. In all panels, anterior is to the left. bl brain lobes, coc cerebral organ canal, iy ingested yolk, le larval epidermis, mo mouth, pb proboscis. Scale bars (A–D???) 100 ?m; (E) 25 ?m

Molecular specification during the formation of the Schmidt’s larva

To characterize in greater detail the formation of the Schmidt’s larva, we identified
and studied the expression pattern of genes involved in the specification of anterior
and cephalic tissues (foxQ2, six3/6, goosecoid [gsc], orthodenticle [otx]) 40], 45], 49]–60], endomesodermal cell fates (foxA, GATA456–a, twist–a [twi–a]) 56], 60]–70], and posterior territories (even–skipped [evx] and caudal [cdx]) 59], 60], 70]–77] during blastula and gastrula stages, and in the Schmidt’s larva.

Anterior genes are first detected at the gastrula stage (Fig. 7A–L??). At this stage, gsc is expressed in two clusters of ectodermal cells of the blastoporal rim (Fig. 7H), and otx is more broadly expressed around the blastoporal opening (Fig. 7K). All analyzed anterior genes are expressed in the Schmidt’s larva. In the intracapsular
larva, foxQ2 is expressed in the most anterior region of the cephalic discs and the proboscis
rudiment (Fig. 7C?, C??), six3/6 is expressed in the anterior region of the cephalic discs and the anterior ectoderm
of the mouth (Fig. 7F?, F??), gsc is expressed in two antero-lateral clusters of cells (Fig. 7I?, I??), and otx is expressed in the cephalic discs, anterior mouth, and the blind gut (Fig. 7L?, L??).

Fig. 7. Gene expression during Schmidt’s larva formation in Lineus ruber. A–AA?? Whole-mount in situ hybridization in blastulae, gastrulae and Schmidt’s larvae of
L. ruber. A–C??foxQ2 is expressed in the anterior region of the Schmidt’s larva. D–F??six3/6 is expressed in the anterior cephalic imaginal discs and in the anterior mouth. G–I??gsc is detected in two ventro-lateral clusters at each side of the blastopore (arrowheadsH) and mouth. J–L??otx is expressed around the blastopore opening, and in the anterior imaginal discs and
gut of Schmidt’s larva. M–O??foxA is expressed in the mouth and larval pharynx. P–R??GATA456–a is detected in the blind gut of the larva. S–U??twi–a is expressed in all imaginal discs of the larva. V–X??evx is expressed in one pole of the blastula (arrowheadV), one side of the gastrula and in the posterior end of the Schmidt’s larva. Y–AA??cdx is weakly detected in the posterior end of the larva (arrowhead in AA??). All panels of blastula embryos are lateral views, and panels of gastrulae are blastoporal views. In the Schmidt’s larva, panels on the left are ventral views and panels on the right side are lateral views. In all cases, anterior of the Schmidt’s larva is to the left. bp blastopore, mo mouth

Endomesodermal genes are only detected late in the Schmidt’s larva (Fig. 7M–U??). The fox gene foxA is expressed in the mouth and pharynx of the larva (Fig. 7O?, O??), the endodermal gene GATA456–a is detected in the blind gut (Fig. 7R?, R??) and the mesoderm-associated gene twi–a is broadly expressed in all the imaginal discs of the Schmidt’s larva (Fig. 7U?, U??).

Finally, the posterior gene evx is expressed already at one pole of the blastula (Fig. 7V), and is later restricted to the presumably posterior side of the gastrula (Fig. 7W) and the posterior end of the Schmidt’s larva (Fig. 7X?, X??). The gene cdx is however only weakly detected at the posterior tip of the Schmidt’s larva (Fig. 7Y–AA??). Altogether, the expression of anterior, endomesodermal and posterior genes
suggest that the establishment of the basic molecular regionalization of the embryo
of L. ruber occurs during the formation of the Schmidt’s larva.

Molecular specification during metamorphosis and organogenesis in the early juvenile

We next studied the expression of the anterior, endomesodermal and posterior markers
during metamorphosis and in the early juvenile of L. ruber. During metamorphosis, the anterior gene foxQ2 is expressed in the most anterior region of the cephalic discs and in the proboscis
(Fig. 8A?, A??). In the juvenile, foxQ2 is expressed in the anterior head, including the proboscis (Fig. 8B?, B??). The gene six3/6 is expressed in the cephalic discs and in the anterior mouth during metamorphosis
(Fig. 8C?, C??), and broadly in the head region and anterior mouth in the juvenile (Fig. 8D?, D??). The gene gsc is expressed in two antero-lateral domains during metamorphosis (Fig. 8E?, E??), and in two clusters of cells associated to the cerebral organ canals and
that might correspond to the cerebral organs in the juvenile (Fig. 8F?, F??) (Additional file 10: Figure S4). Finally, the anterior gene otx is expressed broadly during metamorphosis, mostly in the anterior region, the mouth
and scattered cells on the dorsal side (Fig. 8G?, G??). In the early juvenile, otx is expressed in the head region, and in subsurface isolated cells of the dorsal side
of the trunk (Fig. 8H?, H??). The cell types expressing otx on the dorsal side are unknown.

Fig. 8. Gene expression during metamorphosis in Lineus ruber. A?–R?? Whole-mount in situ hybridization during metamorphosis and in early juveniles of
L. ruber. A?–B??foxQ2 is expressed in the anterior end of the metamorphic embryo and juvenile. C?–D??six3/6 is detected in the anterior mouth and cephalic lobes of the metamorphic embryo and
in the head region and anterior mouth of the juvenile. E?–F??gsc is expressed into antero-lateral domains during metamorphosis and in the juvenile.
G?–H??otx is broadly expressed in the anterior region and dorsal side of the metamorphic larva
and juvenile. I?–J??foxA is expressed in the mouth and pharynx, as well as three clusters (arrowheadsI?, I??) of the metamorphic larva. In the juvenile, foxA is expressed in the mouth, inner head, ventral side and posterior end of the endoderm
(arrowheadsJ?, J??). (K?–L??) GATA456–a is detected in the gut during metamorphosis and in the juvenile. M?–N??twi–a is broadly expressed throughout the metamorphic larva and juvenile. O?–P??evx is detected in the posterior tip of the larva (arrowheadO??) and in postero-lateral scattered cells during metamorphosis. In the juvenile, evx is expressed in the posterior end of the animal (arrowheadP??) and in the lateral and dorsal nerve cords. Q?–R??cdx is expressed in posterior end of the larva during metamorphosis and in the posterior
end and endoderm of the juvenile. In metamorphic larvae and early juveniles, panels on the left are ventral views and panels on the right side are lateral views. In all cases, anterior is to the left. mo mouth

During metamorphosis, the endomesodermal marker foxA is expressed strongly in the mouth, as well as in three clusters of cells anterior
and lateral to the mouth and in scattered cells of the ventral region of unknown type
(Fig. 8I?, I??). In the juvenile, foxA is detected in the mouth, ventral side of the trunk and in the posterior tip (Fig. 8J?, J??). The endodermal marker GATA456–a is expressed in the blind gut during metamorphosis (Fig. 8K?, K??), and in the definitive endoderm and central part of the head in the juvenile
(Fig. 8L?, L??). The mesodermal gene twi–a is expressed throughout the entire body during metamorphosis and in the early juvenile
(Fig. 8M?–N??).

The posterior gene evx is expressed in a small cluster of cells at the posterior tip during metamorphosis,
as well as in two lateral bands of scattered cells in the lateral sides of the larva,
presumably the developing lateral nerve cords (Fig. 8O?, O??). In the early juvenile, expression of evx is detected in the lateral nerve cords, the dorsal nerve cord and in the posterior
end of the juvenile (Fig. 8P?, P??). The gene cdx is strongly expressed in the posterior end during metamorphosis (Fig. 8Q?, Q??) and in the posterior ectoderm and endoderm of the juvenile (Fig. 8R?, R??). Therefore, the expression of anterior, endomesodermal and posterior genes
during metamorphosis and in the juvenile together is congruent with the fates of the
imaginal discs assigned after the morphological analyses in the Schmidt’s larva of
the nemertean L. ruber.