Download The embryonic development of the flatworm

Dev Genes Evol (2004) 214:220–239
DOI 10.1007/s00427-004-0406-4
ORIGINAL ARTICLE
Joshua Morris · Ramachandra Nallur ·
Peter Ladurner · Bernhard Egger · Reinhard Rieger ·
Volker Hartenstein
The embryonic development of the flatworm Macrostomum sp.
Received: 20 January 2004 / Accepted: 15 March 2004 / Published online: 9 April 2004
Springer-Verlag 2004
Abstract Macrostomid flatworms represent a group of
basal bilaterians with primitive developmental and morphological characteristics. The species Macrostomum sp.,
raised under laboratory conditions, has a short generation
time of about 2–3 weeks and produces a large number of
eggs year round. Using live observation, histology, electron microscopy and immunohistochemistry we have
carried out a developmental analysis of Macrostomum sp.
Cleavage (stages 1–2) of this species follows a modified
spiral pattern and results in a solid embryonic primordium
surrounded by an external yolk layer. During stage 3, cells
at the anterior and lateral periphery of the embryo evolve
into the somatic primordium which gives rise to the body
wall and nervous system. Cells in the center form the
large yolk-rich gut primordium. During stage 4, the brain
primordium and the pharynx primordium appear as symmetric densities anterior-ventrally within the somatic primordium. Organ differentiation commences during stage
5 when the neurons of the brain primordium extend axons
that form a central neuropile, and the outer cell layer of
the somatic primordium turns into a ciliated epidermal
epithelium. Cilia also appear in the lumen of the pharynx
primordium, in the protonephridial system and, slightly
later, in the lumen of the gut. Ultrastructurally, these
differentiating cells show the hallmarks of platyhelminth
epithelia, with a pronounced apical assembly of microfilaments (terminal web) inserting at the zonula adherens,
and a wide band of septate junctions underneath the
Edited by J. Campos-Ortega
J. Morris · R. Nallur · V. Hartenstein ())
Department of Molecular, Cell and Developmental Biology,
University of California,
Los Angeles, CA, 90095, USA
e-mail: [email protected]
Tel.: +1-310-2067523
Fax: +1-310-2063987
P. Ladurner · B. Egger · R. Rieger
Institute of Zoology and Limnology,
University of Innsbruck,
Technikerstrasse 25, 6020 Innsbruck, Austria
zonula. Terminal web and zonula adherens are particularly well observed in the epidermis. During stage 6, the
somatic primordium extends around the surface dorsally
and ventrally to form a complete body wall. Muscle
precursors extend myofilaments that are organized into a
highly regular orthogonal network of circular, diagonal
and longitudinal fibers. Neurons of the brain primordium
differentiate a commissural neuropile that extends a single pair of ventro-lateral nerve trunks (the main longitudinal cords) posteriorly. The primordial pharynx lumen fuses with the ventral epidermis anteriorly and the
gut posteriorly, thereby generating a continuous digestive
tract. The embryo adopts its final shape during stages 7
and 8, characterized by the morphallactic lengthening of
the body into a U-shaped form and the condensation of
the nervous system.
Keywords Platyhelminth · Embryo · Morphogenesis ·
Organogenesis · Differentiation
Introduction
Flatworms represent a large and diverse taxon of simple
invertebrate animals. Recently they have again attracted
the attention of developmental biologists because, in regard to numerous morphological criteria, all or at least
some of their taxa may have branched off the phylogenetic tree near the root of the bilaterian stock (Rieger et
al. 1991; Ax 1996; Tyler 2001; Jondelius et al. 2002).
This makes them a highly relevant system in which to
study basic bilaterian developmental processes, such as
establishment of the body axes and organogenesis (e.g.,
Hartenstein and Ehlers 2000; Younossi-Hartenstein and
Hartenstein 2000a, b; Hartenstein and Jones 2003). Flatworms share a number of fundamental characters with
coelenterates, which evolved before the bilaterians. Most
notable among these characters is a single gut opening
that serves as both mouth and anus, and a ciliated epidermis used for locomotion (for recent comparative description of adult flatworm anatomy, see Ehlers 1985;
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Rieger et al. 1991; Ax 1996). Like higher animals, most
flatworms have a subepidermal muscular layer of circular, diagonal and longitudinal fibers (see Rieger et al.
1991, 1994; Hooge 2001), and a central nervous system
that comprises an anterior brain and a number of longitudinal nerve cords (Rieger et al. 1991; Reuter and
Halton 2001). However, the central nervous system lacks
compactness, being also penetrated by muscle and gland
cells. Nerve tracts issuing forth from the brain extend
dorsally, laterally and ventrally. These tracts are connected to up to three subepidermal nerve plexus. The
connectivity between neurons and muscles in flatworms
is different from higher animals, in that, frequently, long
muscle processes (sarco-neuronal processes) approach
the nerve fibers, instead of the other way around (see
Rieger et al. 1991, similar to the well studied situation in
nematodes). The internal structure of flatworms is usually referred to as being very simple. Flatworms do not
possess a coelom. The interior of the body is filled with
the gut tube and reproductive organs, with complex
muscle and connective tissue cells. A specialized vascular system or respiratory system is absent in free-living
platyhelminths. The only tubular structures are comprised of protonephridia (see Rohde 2001).
Embryonic development has been studied in a number
of different flatworm species, studying live embryos and
using classical techniques of histology and electron microscopy, as well as, to a very limited extent, cell typespecific markers, including markers for muscle and nerve
cells (Thomas 1986; Bagu and Boyer 1990; Reiter et
al. 1996; Boyer et al. 1996; Ladurner and Rieger 2000;
Younossi-Hartenstein et al. 2000; Younossi-Hartenstein
and Hartenstein 2000a, b, 2001; Hartenstein and Jones
2003). Molecular tools, both as markers and as agents to
disrupt function, have been developed to study regeneration and cell differentiation in planarians, members of
the flatworm clade Tricladida (Sanchez-Alvarado and
Newmark 1999; Sanchez-Alvarado et al. 2002; Pineda
et al. 2000, 2002; Salo et al. 2002; Cebria et al. 2002;
Ogawa et al. 2002). In order to initiate a molecular
analysis of embryonic development we have chosen the
microturbellarian Macrostomum sp. as a species that can
be easily raised in the laboratory, produces a multitude of
eggs year round, and has a very short generation time of
approximately 2–3 weeks. Macrostomum sp. is a new
species that was first briefly characterized in Ladurner et
al. (2000), and is now being described by Ladurner et al.
(2004). The new species belongs to the clade Macrostomida within the Macrostomorpha. Based on morphological and molecular phylogenetic analysis, the latter occupy a position near the root of the rhabditophoran flatworm tree, which includes the majority of free-living and
parasitic taxa (Ehlers 1985; Rieger 2001; Tyler 2001;
Littlewood and Olson 2001). Thus, macrostomids produce yolk-rich egg cells (archoophoran mode of development), rather than supplying yolk to the egg by means
of specialized yolk cells surrounding a small oocyte
(neoophoran mode of development in all other rhabditophoran flatworms except polyclads). Furthermore, the
simple pharynx shared by all macrostomids places this
taxon near the base of the rhabditophoran platyhelminths
(see Doe 1981 and references cited above), a classification that is supported by recent molecular data (Bagu et
al. 2001; Curini-Galletti 2001; Jondelius et al. 2001;
Littlewood et al. 2001). Macrostomid development has
been studied several times, but only very cursorily: histological and much less in vivo by Seilern-Aspang (1957)
in M. appendiculatum, in vivo by Reisinger (1923) in M.
viride, by Papi (1953) in M. appendiculatum, by Bogomolow (1949, 1960) in M. viride and M. rossicum and
finally by Ax and Borkott (1968a, b) in M. romanicum,
who provided the first film material about the embryonic
development, but still left open a number of important
questions. The formation of the body wall was addressed
in several more recent light and electron microscopic as
well as histochemical studies (Tyler 1981; Reiter et al.
1996; E. Robatscher and B. Egger, Innsbruck, unpublished results). These studies show that Macrostomum sp.
as well as other Macrostomum species of the M. hystricinum species clade (see Rieger 1977) share with other
primitive flatworm groups a spiral pattern of cleavage.
However, development starts to deviate after the first
three to four rounds of divisions from the typical spiralian
mode, in agreement with the observations by SeilernAspang (1957). Thus, blastomeres at the vegetal pole
seem to spread out at the surface of the embryo and form
an outer yolk mantle (Tyler 1981). This process was
called “inverse epiboly” by Thomas (1986). The transition from this stage to the final generation of body wall
and internal organs has not been followed in any detail
satisfactorily. In this paper, we have undertaken a developmental analysis of Macrostomum sp. on the basis of
observation of live embryos, histological staining of
sectioned and whole-mount material, electron microscopy
and immunohistochemistry. We define a series of morphological stages that are in accordance with a system
introduced for other platyhelminth taxa during recent
years (Younossi-Hartenstein et al. 2000). Our work is
aimed at providing a guide for the molecular-genetic
analysis of platyhelminth embryogenesis, in particular the
interpretation of gene expression data from in situ hybridization studies which are currently underway (V.
Hartenstein, UCLA, and P. Ladurner, Innsbruck, unpublished data).
Materials and methods
Animals
Macrostomum sp. is a new marine flatworm from the Mediterranean, which is presently being described by Ladurner et al.
(2004). It lives in laboratory cultures of the diatom Nitzschia
curvilineata. Petri dishes with the artificial growth medium “F2”
(Guillard and Ryther 1962) are inoculated with Nitzschia. By 10–
14 days the algae form a dense lawn at the bottom of the dish.
Groups of adult Macrostomum (approximately 1.5 mm in length)
are transferred onto the algal dish. They produce eggs continuously,
and the eggs, if left undisturbed, will develop into sexually mature
adults in approximately 2–3 weeks. For preparation, eggs are col-
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lected and fixed with 4% formaldehyde in PBS buffer. They are
washed in PBS-Triton (detergent) and sonicated to permeabilize the
tough eggshell. Subsequently, embryos (and juveniles or adults) are
prepared for histology and immunohistochemistry following the
same protocols that have been established for Drosophila embryos
(Ashburner 1989).
Electron microscopy and histology
Timed stages of embryos of Macrostomum sp. were punctured with
electrolytically sharpened tungsten needles and immediately fixed
according to Eisenman and Alfert (1982) using the weak osmium
cocktail method. After dehydration in standard acetone series,
specimens were embedded in Spurr’s low viscosity resin. Blocks
were sectioned with an LKB Ultratome. Alternating 1-mm semi-thin
sections and sets of 80-nm (silver) ultrathin sections were taken.
Ultrathin sections were mounted on net grids (Ted Pella) and
treated with uranyl acetate and lead citrate. Semi-thin sections (1 or
0.5 mm) were stained with a 1:1 mixture of methylene blue and
toluidine blue/borax (Ashburner 1989).
Results
Overview and staging system
Embryonic development of Macrostomum sp. takes approximately 120 h at 20C. We have subdivided this
phase into eight stages, which can be distinguished on the
basis of several morphological criteria in living material,
as well as fixed and sectioned preparations. The definition
Fuchsin labeling of whole-mounts
The whole-mount technique that has been extensively used by us
and others to label whole embryos of insects and other invertebrates
was adapted from Zalokar and Erk (1977). Briefly, following fixation in 4% PBS buffered formaldehyde, embryos, contained within
small wire mesh baskets holding 20–50 specimens, were washed in
70% ethanol (three changes of 5 min each) and distilled water
(5 min). They were placed in 2 N HCL (10 min) at 60C for DNA
denaturation. Following one wash in distilled water (5 min) and two
washes in 5% acetic acid, embryos were stained for 15 min in 2%
solution of filtered basic fuchsin (in 5% acetic acid). Embryos were
washed in 5% acetic acid until cytoplasmic fuchsin labeling was
removed, dehydrated in graded ethanol, and transferred to Epon,
and individually mounted on slides.
Immunohistochemistry
To visualize neurons and ciliated cells in whole-mount preparations
of embryos, a monoclonal antibody against acetylated tubulin
(Sigma; dilution 1:100) and tyrosinated tubulin (Sigma; dilution
1:1,000) were used. Embryos ranging in age between stage 4 and 8
(see Results and Younossi-Hartenstein et al. 2000) that had been
fixed in 4% formaldehyde (see above) were washed in PBT (PBS
plus 0.3% Triton X-100; pH 7.2; for washing, PBT solution was
changed three to five times over a 10-min period) and incubated
overnight in PBT containing the antibody at 1:1,000 dilution. After
another washing step in PBT the preparations were incubated for
4 h in PBT containing the secondary antibody (peroxidase- or
FITC-conjugated rabbit anti-mouse immunoglobulin; Jackson
Labs) at a dilution of 1:800. The preparations were washed and
incubated with diamino-benzidine (DAB, Sigma) at 0.1% in 0.1 M
phosphate buffer (pH 7.3) containing 0.006% hydrogen peroxide.
The reaction was stopped after 5–10 min by diluting the substrate
with 0.1 M phosphate buffer. Preparations were dehydrated in
graded ethanol (70%, 90%, 95%, 5 min each; 100%, 15 min) and
acetone (5 min) and left overnight in a mixture of Epon and acetone
(1:1). They were then mounted in a drop of fresh Epon and coverslipped. Representative specimens were embedded in Epon and
sectioned (1–3 mm) on an LKB ultramicrotome. Sections were
counterstained with methylene blue/toluidine blue/borax (Ashburner 1989). Preparations were analyzed and photographed with a
Zeiss Axiophot photomicroscope and a Biorad MRC1024ES microscope using Laser sharp version 3.2 software. Figures were assembled and lettered with Adobe Photoshop 6.02 (Adobe).
Fig. 1 Overview of stages of Macrostomum sp. development.
Panels show highly schematic drawings of embryos of increasing
age, shown in lateral view, illustrating the major events of Macrostomum sp. embryogenesis. Anterior is to the left and dorsal to
the top. The number at the top left of each drawing indicates the
embryonic stage as defined in the text. Tissues are shaded in different colors as shown at the bottom left of the panel. For details see
text (br brain primordium, cx cortex, emp embryonic primordium,
ep epidermal primordium, eye precursors of the eye, eym external
yolk mantle, ge gut epithelium, gp gut primordium, hc hull cell, mln
main longitudinal nerve cord, np neuropile, ph pharynx primordium, phe pharynx epithelium, phm pharynx muscle, sm body wall
muscles, sop somatic primordium, tp tail plate primordium)
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Fig. 2a–d Early stages of Macrostomum sp. development: cleavage (stage 1). a Photograph of living embryo at four-cell stage. b, c
Photographs of embryo at eight-cell stage, view from animal pole;
focal plane through micromere quartet 1a–d (b) and macromere
quartet (c). Note larger size of 1D macromere and relatively large
size of micromeres. d Parasagittal section showing yolk-rich animal
blastomeres (bl) and incipient hull cells (hc; pb polar bodies; scale
bar 20 mm)
of these embryonic stages is largely based on a similar
system that was developed for the rhabdocoel flatworm
Mesostoma lingua (Younossi-Hartenstein et al. 2000), and
has since then been adapted for other archoophoran and
neoophoran flatworm species (Younossi-Hartenstein and
Hartenstein 2000a, b, 2001; Hartenstein and Ehlers 2000;
Ramachandra et al. 2002). In the following, we will
briefly introduce and define the characteristic features of
the eight stages (Fig. 1).
Stage 1 (0–15 h) represents early cleavage, during
which the zygote divides basically in a spiral pattern. In
vivo cleavage can only be observed until the third cleavage, after which the opaque yolk granules obscure the
borders of individual blastomeres. During stage 2 (15–
30 h), a small number of yolk-rich, presumably vegetal
blastomeres with large nuclei expand and surround the
other blastomeres (yolk mantle), which form a proliferating mass (here referred to as the embryonic primordium) in the center of the embryo. Stage 3 (30–45 h) is
characterized by the expansion and diversification of the
embryonic primordium. Anteriorly and laterally, cells of
smaller size form the primordium of the body wall and
nervous system (somatic primordium); large, yolk-rich
cells in the center represent the primordium of the gut.
During stage 4 (45–60 h), the outer yolk mantle has become very thin. Definitive primordia of the brain and
pharynx can be distinguished at the anterior pole. Stage 5
(60–75 h) is defined by the onset of tissue and organ
differentiation. Outer cells along the lateral margins dis-
Fig. 3a–d Early stages of Macrostomum sp. development: epiboly
of hull cells (stage 2). a Photograph of living embryo. b Wholemount of embryo stained with basic fuchsin in ventral view. c, d
Cross sections near anterior and posterior pole, respectively. Hull
cells have extended over the surface of the embryo and form the
external yolk mantle (eym). Proliferating blastomeres enclosed by
the external yolk mantle represent the embryonic primordium (emp
in a). Within this mesenchymal cell mass, small cells are clustered
near one pole, and larger, yolk-rich cells near the other. We presume that the smaller cells will give rise to the body wall and
nervous system (somatic primordium; sop in b and c), and the
large-sized cells will form the gut (gut primordium, gp). Arrow in b
points at one of numerous mitotic figures that occur throughout
early development in somatic primordium and gut primordium (nhc
hull cell nucleus; scale bar 20 mm)
place the yolk and form the definitive epidermis. A neuropile forms in the center of the brain primordium;
pharynx and gut become lined by ciliated cells. During
stage 6, the formation of the body wall is completed. The
epidermis spreads around the embryo. Gland cells containing rhabdites are scattered throughout the epidermis.
Myoblasts form a regular, orthogonal grid of fibers underneath the epidermis. During stage 7, the previously
spherical shape of the embryo transforms to an elongated
shape and the embryo bends its caudal end towards the
ventral side of the anterior body half. The brain primordium condenses, and pigmented eyespots form in the
dorsal brain cortex. Stage 8, comprising the final 10–15 h
of embryonic development, resembles the fully elongated
and differentiated freshly hatched juvenile.
Early embryogenesis: cleavage and formation
of the embryonic primordium (stages 1–3)
Our data confirm previous observations (Reisinger 1923;
Bogomolow 1949, 1960; Papi 1953; Seilern-Aspang 1957;
Ax and Borkott 1968a, b) stating that early cleavage
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Fig. 4a–d Early organogenesis in Macrostomum sp.: emergence of
organ primordia (stage 4). a Photograph of living embryo. b
Whole-mount of fuchsin-labeled embryo in dorsal view. c Cross
sections of somatic primordium near anterior pole. d Cross section
at mid-levels of embryo. At the anterior of the embryo the primordium of the brain (br) appears as a bilateral condensation within
the somatic primordium (sop). The somatic primordium forms a
belt around the equator of the embryo. It measures 20–25 cell
diameters in length (AP axis), 10–15 cell diameters in width (DV
axis) and three to four cell diameters in thickness, bringing the
overall cell number up to 2–3,000. Most cells are post-mitotic,
evidenced by the scarcity of mitotic figures from stage 4 onwards,
as well as the prevalence of small, dense nuclei. The somatic primordium is still covered by a thin yolk mantle (eym in c). The gut
primordium (gp) lies in the center of the embryo and contains large,
yolk-rich cells (nhc hull cell nucleus; scale bar 20 mm)
follows a pattern that shows characteristics of quartetspiral cleavage (Fig. 2). Some significant deviations from
spiral cleavage, as seen for instance in polyclads, do occur. Micromeres are not substantially smaller than macromeres (Fig. 2b). At the stage when more than one micromere quartet has been formed, some large, presumably
vegetal cells located at the surface start to flatten and
surround the embryo (Fig. 2d), transforming into the “hull
cells” (Huellzellen), first described by Seilern-Aspang
(1957) for M. appendiculatum and Tyler (1981) for M.
hystricinum. Hull cells form an external yolk mantle
(Dottermantel) that gradually becomes thinner as development progresses (Figs. 1, 3b, d, 4c, d, 5d). The individual hull cells are filled with yolk granules and show
elongated nuclei which are larger than those of most
blastomeres (Fig. 2d).
Once internalized, the cleaving embryonic primordium
forms a mass surrounded by yolk-rich hull cells (stage 2;
Fig. 3). The time course of mitotic divisions and spindle
orientation appears to be quite irregular, and cannot be
followed without special markers or dye injections. Cells
are variable in size and contain yolk granules to a greater
Fig. 5a–d Organogenesis of Macrostomum sp.: onset of organ
differentiation (stage 5). a Photograph of living embryo, anterior
view. b Tilted cross sections of embryo near anterior pole. c, d
Whole-mounts of fuchsin-labeled embryos in ventral view (c) and
lateral view (d). Cells at the outer surface of the somatic primordium have undergone a mesenchymal-epithelial transition and form
the primordium of the epidermis (ep). Deep cells of the somatic
primordium form precursors of muscle cells (sm; arrowheads in b)
and other cell types associated with the body wall. The somatic
primordium does not yet form a complete body wall; dorsally and
ventrally, remnants of the external yolk mantle (eym) still abut the
surface. Neurons of the brain primordium have differentiated and
form a central neuropile (np) surrounded by a cortex (cx) of somata.
Posterior to the brain is the primordium of the pharynx (ph), formed
by a cylindrical array of columnar epithelial cells. Arrow in (d)
points at cells that close the pharynx lumen ventrally. Note the
folding of the eggshell ventral of the arrow, which probably constitutes an artefact related to shrinkage of the preparation. Precursors of muscle cells around the pharynx are indistinct, small,
spindle-shaped cells. The gut primordium (gp) contains large, yolkrich masses, interspersed with small and dense yolk spheres (ys).
The posterior of the embryo contains the primordium of the tail
plate (tp). Scale bars 20 mm
Fig. 6a–j Organogenesis in Macrostomum sp.: ultrastructure of
organ primordia. a–j Electron micrographs of details of cross
sections of a stage 5 embryo. a Overview of somatic primordium,
containing superficial layer of epidermal precursors (ep) and deep
layer of muscle precursors and neural precursors (ne). Epidermal
precursors are cylindrical, ciliated (ci) epithelial cells. Numerous
yolk granules (yg) are still present in the basal portion of the epidermal precursors. b Border between epidermal primordium (ep)
and external yolk mantle (eym). c Magnified views of part of b,
showing details of the junctional complex formed between epidermal precursors. The complex is formed by a pronounced, septate
junction (sj) near the apical pole of the cell. Further apical still is
the zonula adherens (aj) with its characteristic membrane density
and cytoplasmic plaque. An unusual feature also noticed at later
stages is the widening of the intercellular cleft in the zonula ad-
225
herens (arrow). c Magnified views of part of b, showing details of
the junctional complex formed between the epidermal precursor
(ep) and external yolk mantle (eym). The septate junction is similar
to the one between epidermal cells; the adherens junction is not as
wide and lacks a cytoplasmic plaque on the side of the yolk mantle
(arrowhead). e, f Magnified views of part of a, showing details of
bundles of cell processes (cp), probably axons, located at the
border between neural precursors (ne) and epidermal cells (ep).
Myofilaments are not yet formed at this stage. g, j Apical part of
epidermal cell, showing cilia (ci), zonula adherens (aj), septate
junction (sj), and terminal web (tw; microfilaments inserting at the
zonula adherens). Apical of the terminal web is a layer of ultrarhabdites (urh), which are small secretory granules of epidermal
cells. Mitochondria (mit) are clustered underneath the terminal
web. h, I Detail of cell that does not yet form part of the epidermal
primordium but has started formation of cilia (ci). Scale bars: 2 mm
a; 1 mm b, e, g, h
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or lesser extent. Anterior cells, which generally seem
smaller in size than posterior cells (Fig. 3b, c), constitute
the precursors of epidermis, body-wall muscles and nervous system (somatic primordium). During stage 3, the
somatic primordium expands posteriorly, forming two
symmetric plates along the lateral sides of the embryo. It
is unclear whether this expansion involves actual cell
migration, or whether cells at posterior levels which earlier had been large and yolk-rich, decrease in size and
then join the somatic primordium. Blastomeres in the
center of the embryo (gut primordium) remain large and
yolk-rich (Fig. 3d). These cells later transform into the gut
epithelium (Fig. 4d).
The formation of organ primordia (stages 4 and 5)
By mid-embryogenesis (stage 4) proliferation becomes
less pronounced and organ primordia take shape. The
most prominent primordium is that of the brain, forming a
bilaterally symmetric mass of densely packed cells at the
anterior pole of the embryo (Fig. 4a, b). Posterior to the
brain, the somatic primordium consists of intermingled
epidermal, gland, muscle and other precursors that form
plates alongside the lateral surface of the embryo (Fig. 4a,
b). The external yolk mantle still covers the surface, but is
compressed into a thin layer (Fig. 4c, d). The gut primordium fills the posterior-central part of the embryo
(Fig. 4a, b, d). Starting towards the end of stage 4, peculiar spherical structures can be distinguished within
the gut primordium (Figs. 4d, 5d, 7e, 10f). These “yolk
spheres” stand out in histological sections, in electron
micrographs (not shown) and can even be seen in living
material (not shown).
The appearance of definitive organ primordia occurs
concomitantly with the onset of cellular differentiation
during stage 5. Most prominent during this stage are the
epidermis, brain, pharynx and gut. Cells located at the
external surface of the embryonic primordium transform
into a ciliated epithelial epidermal layer that displaces the
external yolk mantle (Figs. 5, 6a–d). The onset of ciliation
of the multiciliated cells has been described in detail by
Tyler (1981). As of yet we do not have data that indicate
the resorption of the yolk mantle by the primary epidermis. During stage 5, the epidermis covers only the flanks
of the embryo, whereas yolk still reaches the periphery of
the embryo at dorsal and ventral levels (Fig. 5a, b). As
development proceeds, the epidermis and underlying cells
of the somatic primordium stretch in the transverse axis
and fully enclose the embryo by the end of stage 6.
Cells located underneath the epidermis differentiate
primarily as muscle and nerve cells. The anterior of the
embryo is dominated by the large brain primordium.
Neurons extend axons towards the center of the primordium, resulting in the formation of a typical invertebrate ganglion, which consists of a cortex of neuronal cell
bodies surrounding a central neuropile (Fig. 5b, c, f; see
also Rieger 1998). The cortex is three to four cell bodies
in thickness, except posteriorly where only a single layer
of cell bodies separates the neuropile from the developing
pharynx (Fig. 5b). At the beginning, the brain primordium is wide (in the transverse axis) and flat (in the AP
axis); as development progresses, the primordium condenses, becoming increasingly narrower and thicker (compare Figs. 5b and 11b).
The pharynx primordium appears postero-ventrally of
the brain primordium as a cylindrical array of cuboidal
cells that develop as the ciliated pharynx epithelium
(Fig. 5b–d, see also later stages, e.g. Figs. 7d, 10f). As has
been described for other flatworm species (see Thomas
1986 and Bagu and Boyer 1990 for summaries), the
pharynx primordium does not evolve by invagination of a
pre-existing epithelium, but develops within the deep
layer of the somatic primordium. The pharynx lumen
initially seems to have no connection to the epidermis, or
to the gut lumen. The connection forms during stage 6
when the pharynx primordium elongates both dorsally
and ventrally, and establishes contact with the ventral
epidermis.
Muscle precursors initially appear as elongated and
flattened cells forming irregular layers underneath the
epidermal primordium (see also Reiter et al. 1996 for M.
hystricinum). Muscle precursors are concentrated in a
bilateral band that extends along the flanks of the embryo
(Figs. 5b, 7b). Fewer muscle precursors populate the
space between epidermis and brain primordium, and
around the pharynx. Muscle differentiation sets in towards
the end of stage 5 with the formation of myofilamentcontaining processes which form a highly regular network
of longitudinal, circular and diagonal fibers (see section
Muscular system).
A conspicuous condensation of cells in the deep layers
of the somatic primordium at the posterior tip of the
embryo demarcates the primordium of the tail plate
(Fig. 5c) The tail plate primordium consists of muscles
and glands of the duo-gland adhesive system (Tyler
1988), that allow the freshly hatched animal to quickly
grab on to the substrate. Cells from this caudal region
later may also give rise to the external genitalia, i.e., the
genital pores and the male copulatory organ. Along with
the gonads, the external genitalia apparently do not differentiate during the embryonic period, and will not be
considered here.
Organ differentiation (stages 6–8)
Final organ formation takes place during the last 2 days of
the 5-day embryonic period (stages 6–8; Figs. 7, 8, 9, 10,
11, 12, 13).
Epidermis
During early stages (5 and 6) the epidermal primordium
consists of cylindrical multiciliated epithelial cells
(Fig. 6a, b). Underneath the apical membrane the terminal
web, a dense layer of microfilaments, is already devel-
227
Fig. 7a–h Organogenesis of
Macrostomum sp.: epiboly and
differentiation of the body wall
(stage 6). a Photograph of living
embryo, ventral view, b, c
Whole-mount of fuchsin-labeled embryo in ventral view
(b) and lateral view (c). d, e
Parasagittal sections of embryo.
f–h Magnified views of part of
brain primordium (f), body wall
(g) and gut primordium (h).
During stage 6, the epidermal
primordium (ep) extends around
the embryo, replacing the external yolk mantle. Coordinated
epidermal ciliary beating causes
the embryo to rotate in the
eggshell. Beside the smooth
epidermal layer, the brain (br,
cx cortex, np neuropile), pharynx (ph) and gut (gp) can be
clearly distinguished in the living embryo. In fixed wholemounts (b, c), the brain and
pharynx primordia stand out.
Nuclei of gut cells are not labeled optimally. The increasing
number of axons leads to a
thickening of the neuropile (np).
Underneath the epidermis, somatic muscle precursors (sm in
b, c, g) have differentiated and
form flat, elongated cells that
produce a regular network of
circular, diagonal and longitudinal fibers. The pharynx has
lengthened (c) and cilia can be
recognized under light microscopy in the lumen of the pharynx (b, d), as well as the gut (e).
The gut primordium has reorganized into a population of
large, yolk-rich epithelial cells
(ge in e) loosely arranged
around a central lumen. Some
of the yolk still forms dense
yolk spheres (ys in e, h). At the
posterior end of the embryo, a
subepidermal condensation of
cells forms the primordium of
the male copulatory apparatus
and the tail plate (tp). Scale
bars: 20 mm a–e; 10 mm f–h
oped (Fig. 6g, j). Ultrarhabdites (or epitheliosomes; for
terminology see Rieger et al. 1991), small vesicles above
the terminal web, are already present between the terminal web and the apical membrane (Fig. 6d, g). Basally,
epidermal cells still contain numerous yolk granules,
which will decrease as development proceeds (Fig. 6a).
The junctional complex in between epidermal cells is well
established by stage 5. It consists of an apical zonula
adherens and a wide belt of septate junctions below
(Fig. 6g, j). Of special significance is that septate junctions and adherens junctions also join neighboring cell
membranes at the boundary between epidermal primordium and hull cells (Fig. 6c). The zonula adherens exhibits the characteristic membrane thickening and sub-
228
Fig. 8a–i Organogenesis in Macrostomum sp.: ultrastructure of the
body wall. a–i Electron micrographs of details of longitudinal
sections of a stage 6 embryo. a Overview of body wall, with cil-
iated epidermal layer (ep), subepidermal muscle fibers (mf), main
longitudinal cord (mln) and gut primordium (gp). Epidermal cells
still contain yolk granules (yg) near their basal pole. b Magnified
229
membraneous plaque. However, the cleft that separates
cells at the level of the zonula adherens is wider, rather
than narrower, than the intercellular cleft at more basal
levels (Fig. 6g, see also below, Fig. 9d). This feature of
the zonula adherens (“open zonula adherens”) has also
been observed in embryos of other flatworm species
(Younossi-Hartenstein et al. 2000) and is generally observed in adult specimens which were anesthetized with
isotonic MgCl2 solutions (Tyler 1984).
Later epidermal cells become cuboidal to squamous,
except for the anterior region (apical plate) and tail plate,
which maintains more cylindrical epidermal cells (Fig. 9a,
d). Microvilli become very prominent. A thin and patchy
ECM separates the epidermis from the underlying muscle
and nerve cells (Fig. 9a, h). It has been observed in other
macrostomids (Rieger et al. 1991) that epidermal cells
form extensive sheath-like processes at their baso-lateral
membrane, which intercalate between neighboring cells,
suggesting that this interdigitation of epidermal cells may
compensate for the absence of a continuous basement
membrane. However, interdigitation of epidermal cells is
not a significant feature of epidermal cells in Macrostomum sp. (Fig. 9a). The apical junctional complex between
epidermal cells remains similar to the one described for
stage 5, with a wide and prominent septate junction and an
open zonula adherens (Fig. 9d). Epidermal nuclei acquire
their characteristic multilobulate shape during stage 7
(Fig. 13a). During this stage, rhabdite-filled gland cells
with subepidermal cell bodies and long excretory ducts
permeating the epidermis can be distinguished by light
(Fig. 11f) and electron microscopy (Fig. 9d). These
rhabdite glands are rather evenly distributed over the
dorso-lateral sides of the body (Ladurner et al. 2004).
Specialized glands are the adhesive glands in the tail plate
(Fig. 9j), as well as a population of rhammite glands
whose cell bodies are located posterior to the brain and
whose long necks cross the brain to terminate in the apical
plate (not shown).
Brain
view of apical part of epidermal cell with cilia (ci), ultrarhabdites
(urh), terminal web (tw), zonula adherens (aj) and septate junction
(sj). Epidermal cells contain prominent Golgi complexes (Ga). c
Oblique section of main longitudinal cord (mln) carrying axons (ax)
from the brain to the periphery. Scattered patches of electron dense
extracellular material (basement membrane, bm) separate nerve and
muscle fibers from overlying epidermis. d, e Muscle cell body (sm),
circular muscle fiber (cf) and longitudinal muscle fiber (lf). Note
close contact between axons (ax; magnified view in e) and muscle
plexus. f Overview of sagittal section reaching from brain (to the
left of panel) and pharynx (right), showing neurons (ne), neuropile
(np), muscle fiber piercing brain cortex (brm), pharynx epithelium
(phe) and pharynx muscle (phm). g High magnification of central
neuropile. h Root of main longitudinal nerve cord (mln) and attached muscle cells (sm) and longitudinal muscle fibers (lf). I High
magnification of deep array of cilia (ci) surrounded by protonephridial precursor cell (pn). Scale bars 1 mm
Main longitudinal nerve cords
and protonephridial system
Brain neurons differentiate during stages 5 and 6 when the
brain primordium forms a broad and flat structure at the
anterior tip of the animal (Figs. 5b, 6b, c). Whole-mounts
labeled with anti-tyrosinated tubulin (tyrTub; Fig. 12)
reveal that the brain cortex is formed by multiple clusters
of five to ten neurons each. These clusters (possibly lineages) extend axons that fasciculate together, forming a
thin tract; several such tracts in turn converge and form a
distinct neuropile compartment. We will describe details
of the anatomy of the juvenile and late embryonic brain
elsewhere (Morris et al., in preparation) and will therefore
keep the description of the nervous system brief.
During stage 5 (Fig. 12a–c), four large systems of
tracts that lay down the brain neuropile can be distinguished. Each system is formed by six to eight converging
axon fascicles; during later stages, as more neurons differentiate, fascicles are added to each system. Medially in
the brain, a dorsal and ventral medial longitudinal system
(MLV, MLD) is laid down. Axon fascicles contributing
to these systems are formed by neuronal clusters located
in the anterior cortex; to a lesser extent, posterior neurons with anteriorly projecting axons also exist. The
medial longitudinal systems can be followed throughout
development into the brain of freshly hatched juveniles
(Fig. 12g–I); at this stage, following brain condensation,
systems of both sides approach each other and are separated only by a narrow cleft. Adjacent to the longitudinal
systems are the medio-dorsal and medio-ventral commissural fiber systems (MCD, MCV). The MCD forms
the largest system in the early embryo. It comprises
commissural axon fascicles emitted by at least ten clusters
of neurons located in the dorsal cortex. Fewer axon fascicles of neuronal clusters located in the ventro-medial
cortex converge to form the ventro-medial commissural
tract (MCV; Fig. 12) Finally, axons of neuronal clusters in
the lateral wings of the brain primordium come together
as the lateral commissural (LC) system. During later
stages, the same subdivision into MCV, MCD, MLD,
MLV and LC remains visible, although the number of
neuron clusters and axon fascicles formed by them goes
up by a factor of 2 and 3.
The main longitudinal nerve cords (MLN) extend from
the posterior surface of the brain towards the tail of the
embryo (Figs. 10c, 11e, 12g). They form one large postpharyngeal commissure (not shown). Densely packed cell
bodies and muscle cells accompany the axons. The protonephridial system extends parallel and dorsal to the
MLN (Figs. 9a, b, 12k, l). In the hatching juvenile, the
MLN (at a level posterior to the post-pharyngeal commissure) contains approximately 100 axons. The MLN
reaches towards the tail where it thickens to form a caudal
ganglion associated with the muscles and glands of the
230
Fig. 9a–j Ultrastructure of hatching juvenile. a–d Cross sections
of body wall, showing epidermis (ep), subepidermal gland cells
(glc) with rhabdites (rhb), muscle fibers (mf), protonephridia (pn)
with ciliated lumen (ci), main longitudinal nerve cord (mln) and gut
epithelium (ge). Epidermal cells are covered apically by microvilli
(mv in d) and cilia (ci). Pronounced junctional complex (jc) interconnects neighboring cells. Microfilaments form terminal web
(tw). e–g Cross section of posterior brain (br) and pharynx (phe).
231
tail plate (not shown). The protonephridial system is
formed by a pair of longitudinal tubes which gives off
several evenly space side branches. The tube and side
branches are lined by a flat epithelium. AcTub and tyrTub
labeling of embryo whole-mounts reveal the cilia in
protonephridia for the first time during late stage 5
(Fig. 12k). Specialized cells called flame cells or cyrtocytes cap the blind ends of the side branches. Protonephridial tubules are readily detectable in electron
micrographs of late embryos (Fig. 9a, b). In cross section
they appear as electron dense cells with a 2–4 mm wide
inner lumen, filled with densely packed cilia. In ventral
view they appear as five to six regularly spaced, cylindrical structures (Fig. 12l) that are easily mistaken for
axons, in particular due to the fact that they are located so
close to the main nerve cord. We assume that the early
acTub labeling corresponds to the short, cyrtocyte-containing branches of the protonephridial system. In hatched
juveniles, a continuous longitudinal trunk that opens anteriorly into the pharynx lumen is labeled in addition to
the cyrtocytes (not shown).
Muscular system
Muscle cells form a grid of extraordinary regularity underneath the epidermis; they also surround the neuropile
of the brain, the pharynx, gut and male copulatory apparatus (see post-embryonic differentiation in Rieger et
al. 1991, 1994 in M. hystricinum). The subepidermal
muscular plexus of the late embryo forms a pattern that
is closely correlated to the pattern of epidermal cells
(Fig. 13). This is particularly obvious in the case of the
longitudinal fibers, which are thicker and more invariant
in number and spacing than the circular and diagonal fibers. As evident from Z-projections of a series of confocal
sections in which both muscle fibers and epidermal nuclei
are labeled, there exists an almost perfect 1:1 relationship
between rows of epidermal nuclei and longitudinal fibers
(Fig. 13a, b). The epidermis in freshly hatched juveniles
measures approximately 30–35 cells in perimeter (at midbody level), and possesses the same number of longitudinal muscle fibers.
Two systems of preferentially longitudinal fibers diverge from the subepidermal muscle grid and extend into
Brain neuropile (np) is separated from pharynx only by a scattered
population of neuronal cell bodies (ne). Muscle fibers (mf) traverse
the neuropile. Densely ciliated, cylindrical pharynx epithelium
(phe) surrounds a circular lumen (phl). Muscle cells form a thin
layer of scattered fibers (phm) at the basal surface of the epithelium.
Necks of gland cells (pgl) terminate bilaterally in pharynx lumen. h
Section of body wall. An elongated muscle cell (sm) gives rise to a
longitudinal fiber (mf). A thin basement membrane (bm) separates
epidermis (ep) and muscle layer. I Cross section of gut with internal
lumen (gtl) and gut epithelial cells (ge) covered by cilia (ci). j
Section of adhesive glands (agl) clustered in the tail plate at posterior tip of animal. Scale bar 1 mm (panels B, C, F, G are twofold
magnifications of areas outlined by rectangles in a and e, respectively)
the interior of the animal where they form a muscle net
around the brain neuropile and the pharynx, respectively
(Fig. 13a, b). The brain-related deep muscle plexus arises
in the anterior third of the animal. It forms a group of
crescent-shaped fibers that in part skirt the outer surface
of the brain, in part pass through the cortex and neuropile
(see also Rieger et al. 1991, Fig. 12, p 144 and Rieger et
al. 1994, Fig. 3c for M. hystricinum marinum). These fibers seem to be invariant in relationship to other components of the brain and can serve as landmarks for
subdividing the brain into anatomically defined compartments (see also unvaried condition in freshly hatched
juvenile of M. hystricinum marinum in Rieger et al.
1994).
The second deep muscle grid branches off the subepidermal grid in the mid body and is organizing the
pharynx, thereby serving as the structural support (“pharyngeal suspensor”) anchoring the pharynx to the body
wall (“pharynx holding apparatus” in freshly hatched juveniles, see Rieger et al. 1994 for M. hystricinum marinum). Ventrally, pharyngeal suspensor fibers extend forward underneath the gut, then continue as longitudinal
fibres on the pharynx wall (Fig. 13c, d). Dorsal suspensor
fibers pass over the dorsal gut surface, then curve ventrally, pass between brain and pharynx, and continue as
longitudinal fibers in the ventro-anterior pharyngeal wall.
Together with an inner layer of circular muscles, these
longitudinal fibers form the intrinsic musculature of the
pharynx (for adults, see also Doe 1981).
Muscle differentiation is initiated during stages 5 and
6. The embryonic muscle pattern has been documented
by Reiter et al. (1996) who used phalloidin to visualize
the actin-rich myofilaments. Our materials can add little
to this description. Muscle precursors initially form a
rather irregular layer underneath the epidermal primordium (Fig. 5b, c). During stage 6, these cells elongate and
send out processes that are preferentially arranged longitudinally. Diagonal and circular fibers appear to develop later. Fibers are 0.5–1 mm in diameter (Figs. 8d, h,
9d, h); based on the findings of Reiter et al. (1996),
longitudinal fibers are much fewer and extend almost
along the entire length of the animal, and circular fibers
surround half of the circumference. Given the close
correlation between muscle fibers and epidermal cells in
the late embryo, it is likely that such correlation may
exist from the very beginning of muscle patterning. This
would imply that inductive interactions between the two
tissue layers control the positioning of muscle fibers and
epidermal cells.
Freshly hatched juveniles possess a thin layer of muscles surrounding the gut (Ch. Seifert, Innsbruck, unpublished results). In preparations of embryos, gutassociated
muscles were undetectable; we therefore assume that
formation of this visceral muscle layer must take place
shortly before hatching. The musculature surrounding the
genitalia develops post-embryonically.
232
Fig. 10a–h Late phase of organogenesis (stage 7). a Photograph of living embryo, ventral
view, b, c Whole-mount of
fuchsin-labeled embryo in ventral view. d, e Parasagittal and
horizontal histological sections
of embryo. f–h Magnified details of section shown in e. a
Embryo has flattened and elongated and moves actively in the
eggshell. A characteristic feature of stage 7 are the pigmented eyespots (eye) embedded in the brain (br). b, c Brain
has condensed in medio-lateral
axis (compare with corresponding views of stage 6 and
stage 5 embryos shown in previous figures). The eyes are
embedded in the dorsal brain
cortex (cx in b). Main longitudinal cords (mln) can be seen
leaving the brain posteriorly.
Lumina of pharynx (ph) and gut
(gtl) have become confluent
(arrow in c). d, e Sections show
epidermis (ep), brain with neuropile (np) and cortex (cx),
pharynx (ph; arrow points at
junction between lumina of gut
and pharynx), gut epithelium
(ge), and tail plate (tp). f Magnified view of body wall with
epidermis (ep), gland cell (glc)
and rhabdite (rh) filling external
secretory duct of gland cell. g
Ciliated pharyngeal epithelium.
h Epidermis with cilia (ci),
darkly staining terminal web
(tw), basement membrane (bm),
gland cell (glc) and body-wall
muscle (sm, ge gut epithelium,
gp gut primordium). Scale bars:
20 mm a–e; 10 mm f–h
Pharynx and gut
During stages 5 and 6, the pharynx primordium appears as
a rosette-shaped structure that is closely attached to the
posterior surface of the brain (Figs. 5b, c, 7b, c). Compared to other flatworms, the number of cells forming part
of the pharynx primordium is not high, and a radial or-
ganization of myoblasts that is so characteristic of the
pharynx primordium of higher flatworms is absent. The
pharynx primordium starts out as a cylinder three to four
cell diameters high and six to eight cell diameters in perimeter. These cells form the epithelial lining of the
pharynx. In the interior of the embryo, the pharyngeal
lumen borders the gut primordium of the early embryo;
233
Fig. 11a–f Late phase of organogenesis (stage 8). a Photograph of living embryo, lateral
view. b, c Whole-mount of
fuchsin-labeled embryo in ventral view. d–f Cross sections of
embryo labeled with acTub. a
Embryo has elongated further
and adopted shape of juvenile.
b, c Brain has continued to
condense in medio-lateral axis.
Eyespots (eye) have come
closer to each other (compared
to stage 7) following brain (br)
condensation. d–f Sections taken at level of anterior brain (d),
pharynx (e) and gut (f). AcTub
labels densely stacked cilia on
epidermis (ep), pharynx (ph),
gut epithelium (ge) and protonephridia (pn). Gland cells
containing elongated rhabdites
(rh) can be recognized. The
terminal web (tw) forms a dark
line along apical surface of
epidermis (cx cortex, mln main
longitudinal nerve cord, np
neuropile, sm body-wall muscles, tp tail plate). Scale bars:
20 mm a, b, d–f; 10 mm c
ventrally, the pharynx lumen initially seems to be closed
off by hull cells or precursors of the epidermis (Fig. 5c,
arrow). During stage 6, the pharynx elongates and forms
an opening at the ventral surface (Fig. 7c, arrow). Pharynx
epithelial cells resemble epidermal cells in their ultrastructural differentiation, including the apical ciliation,
terminal web and junctional complex (Fig. 9e, g). In late
embryos, gland cells with long secretory ducts can be
seen to open into the pharynx lumen (Fig. 9g). A sparse
network of circular muscle fibers, as well as neurons and
axon fascicles, surround the pharynx epithelium (Fig. 9g).
234
235
Formation of the gut is difficult to follow, because
cell boundaries are obscured by the high yolk content of
gut precursors. From late stage 6 onward a narrow central lumen surrounded by cilia can be observed (Fig. 7e).
Initially, the gut lumen is closed on all sides (Fig. 12k);
during stage 7, the inner end of the pharynx connects to
the gut, and the lumina of pharynx and gut become confluent (Fig. 10c, e, arrow). The gut epithelial cells that
surround the lumen and carry cilia on their apical surface
are few in number and reach a large size, compared to
other cells of the late embryo. During intermediate stages
of gut development (stages 4–6), cell borders are difficult to discern light microscopically. Whether the gut
primordium represents a true dynamic syncytium will
remain an interesting problem for future studies.
Discussion
In this paper, we have described the principal steps in
morphogenesis that shape the embryonic development of
the macrostomid flatworm, Macrostomum sp. We have
employed a morphological staging system recently introduced for the rhabdocoel species Mesostoma lingua
(Hartenstein and Ehlers 2000), and thereafter adapted
to other flatworm taxa, including polyclads (YounossiHartenstein and Hartenstein 2000b) and acoels (Ramachandra et al. 2002). The stages facilitate the comparison
of developmental events in different groups, and will add
a useful tool to the description of gene expression patterns
that form part of the molecular-genetic analysis of embryogenesis currently under way.
Fig. 12a–l Organogenesis in Macrostomum sp.: development of the
nervous system and protonephridia. a–l Confocal sections of wholemounts of embryos at stage 5 (a–c), stage 6 (d–f) and stage 8 (g–h)
labeled with tyrTub antibody, which labels microtubular skeleton.
Sections show brain primordium of one side; sections of upper row
(a, d, g) were taken at dorsal level, middle row (b, e, h) at intermediate level, and lower row (c, f, I) at ventral level of developing
neuropile. Labeling shows outline of neuronal cell bodies and axons.
At stage 5 (a–c), several neuropile founder clusters can be distinguished: dorsal medial longitudinal cluster (mld), ventral medial
longitudinal cluster (mlv), dorsal medial commissural cluster (mcd),
ventral commissural cluster (mcv), and lateral commissural cluster
(lc). Axon bundles formed by these clusters are later joined by more
fibers, resulting in the growth and compaction of the neuropile.
Axons forming the main longitudinal cord (mln) that extends into
the trunk can be distinguished from stage 6 onward (not shown) and
are prominent at late stages (g). TyrTub antibody also labels cilia of
epidermis (ep), pharynx (ph) and protonephridia (pn). j, k Wholemount of stage 5 embryo labeled with acTub antibody labeling cilia
of epidermis (j), pharynx (k; ph), gut lumen (gtl) and protonephridia
(pn). l Whole-mount of stage 7 embryo labeled with acTub. Protonephridia (pn) are formed by tubules arranged in a row that extends on either side of the embryo dorsal of the main longitudinal
nerve cord. Scale bars: 10 mm a–l; 20 mm j–l
New aspects of the quartet-spiral development
in platyhelminthes and the evolution of ectolecithal eggs
of the neoophora
Macrostomid development begins with a spiral cleavage
of the entolecithal egg, a feature it shares with all other
archoophoran “turbellarians”, including polyclads and
acoels. Subsequently, during later cleavage (stage 2) the
developmental path of Macrostomum sp. starts to deviate
from that of typical spiralian embryos (as represented by
the polyclads), in that some blastomeres extend around
the embryonic primordium (a process called “inverse
epiboly” by Thomas 1986) and give rise to a layer of
yolk-rich hull cells that form an external yolk mantle (see
Tyler 1981, for the M. hystricinum species group defined
by Rieger 1977; Gehlen and Lochs 1990). In a Macrostomum species, such a yolk mantle originating from hull
cells was first reported by Seilern-Aspang (1957 for M.
appendiculatum) who described the formation of these
cells from early vegetal blastomeres. For a similar species, Papi (1953) remarks that the fate of blastomeres
could not be followed in vivo beyond the eight-cell stage.
In other Macrostomum species hull cells have not been
mentioned, but may have simply been overlooked (see
Reisinger 1923, for M. viride). Blastomeres could be
followed during a typical quartet-spiral cleavage up to the
64-cell stage by Bogomolow (1949, 1960) for M. viride
and M. rossicum). Ax and Borkott (1968a, b, for M. romanicum) refer to a hull membrane that obscures the
embryo after the 16-cell stage.
Based on stylet morphology, all species of the genus
Macrostomum used in these previous embryological
studies appear to be related to the M. hystricinum species
group (see Luther 1960, Fig. 17). Macrostomum sp., on
the other hand, is a member of the M. tuba species group
(Ladurner et al. 2004). Hull cells therefore appear to be a
common feature within the genus Macrostomum. As
mentioned by Ax (1961), the lack of hull cells in Macrostomum species described by Bogomolow (1949, 1960)
warrants further investigation.
Gastrulation in Macrostomum sp. also deviates significantly from the typical spiralian mode that prevails in
other archoophorans. Previous studies had not provided
any details on gastrulation because, as already stated by
Seilern-Aspang (1957) for M. appendiculatum, the mode
and type of gastrulation in living embryos cannot be observed due to the presence of the yolk mantle. Our data
suggest a morphogenetic process that leaves out typical
gastrulation movements and, like the formation of the
external yolk mantle, bears resemblance to early developmental stages in neoophorans (Bresslau 1904; Hartenstein and Ehlers 2000). Thus, following the establishment
of the yolk mantle, cells in the interior of the embryo sort
out into a yolk-rich gut primordium, formed by large cells
located more centrally, and a somatic primordium that
consists of smaller cells located anteriorly. These cells
gradually expand posteriorly and form plate-like mesenchymal cell masses on either side of the gut primordium.
Only during late stages of embryogenesis, long after onset
236
Fig. 13a–d Muscle pattern of
juvenile. Whole-mounts were
labeled with phalloidin (red,
labels myofilaments) and Sytox
(green, labels nuclei); ventral
view, anterior up . a, b Z-projection of five consecutive
confocal sections (1-mm interval) showing pattern of epidermal nuclei (ep) and somatic
muscle fibers (sm) of ventral
body wall. b Magnified view
illustrating one-to-one relationship between columns of epidermal nuclei and longitudinal
muscle fibers. c Z-projection of
12 consecutive confocal sections (1-mm interval) showing
deep systems of muscle fibers,
consisting of brain-related
muscles (brm), pharyngeal suspensors (psm), and internal
pharyngeal muscles (phm). d
Schematic diagram of juvenile,
lateral view, clarifying location
of muscle systems. (cx Cortex,
gt gut, np neuropile, ph pharynx, vm visceral muscle; scale
bar 20 mm)
of cell differentiation, does the somatic primordium expand dorsally and ventrally to enclose the gut primordium. The difference between this peculiar mode of body
wall formation and the corresponding process in polyclads and other spiralians is evident. Thus, in polyclads,
hull cells are absent. The definitive epidermis primordium
is formed at an early stage during gastrulation by animal
micromeres, which by epibolic movements stretch over
the large, yolk-rich cells at the vegetal pole (Surface
1908; Boyer et al. 1996, 1998; Younossi-Hartenstein and
Hartenstein 2000b). The latter include the descendants of
the fourth quartet micromere that form the mesoderm and
the gut, and the macromeres providing yolk that will
come to be located in the lumen of the gut, or (in case of
polyclad flatworms) stay rather small and degenerate at
an early stage (Surface 1908).
The origin of hull cells from blastomeres may have
evolutionary significance for the development of ectolecithal eggs, an issue that clearly needs further investigation. Ax (1961) drew attention to the different ontogenetic origin of hull cells in certain archoophora and
neoophora. In the latter, hull cells have been described as
descendants of yolk cells (vitellocytes; Bresslau 1904;
Thomas 1986; Hartenstein and Ehlers 2000). Giesa (1966)
also describes, for the proseriate Monocelis, a primary
cover of the ectolecithal embryo formed by specialized
yolk cells (“Vitellocytenepithel”). Later, a secondary set
of hull cells that arise from blastomeres displace the
vitellocytes. Such blastomere-derived hull cells have also
been described for other proseriates (Minona, Bothrioplana, Otomesostoma) as well as for the lecithoepitheliate
Xenoprorhynchus by Reisinger et al. (1974a, b).
The formation of an external yolk mantle by hull cells
and the absence of a characteristic gastrulation are two
criteria which would place macrostomids between the
more “basal” archoophorans (e.g., polyclads) and neoophorans. This is in contrast to other criteria, such as the
structure of the pharynx, which, in macrostomids, is of the
pharynx simplex type, whereas poIyclads show the more
advanced pharynx plicatus type. More cytological studies
on the hull cells are needed. In any event, the new data on
the embryonic development of Macrostomum presented
here open new avenues to investigate the origin of the
heterocellular gonad with ectolecithal eggs and highly
derived embryonic development from the basic quartetspiral cleavage of primitive archoophorans.
The inverse epiboly seen in Macrostomum sp. (of the
M. tuba species group) and certain other macrostomid
species (of the M. hystricinum species group) poses an
interesting phenomenon also from a developmental perspective. Spiralian cleavage is commonly seen as a developmental mechanism that generates fixed lineages.
237
Injecting early blastomeres with lineage tracers has revealed a high degree of invariance in regard to what organs and cell types of the hatching larva are derived from
which blastomere (Costello and Henley 1976; Verdonk
and van den Biggelaar 1983; Henry and Martindale 1998;
Boyer et al. 1996, 1998; Henry et al. 2000). Experimental
manipulations and molecular studies have also revealed
intrinsic cell fate determinants that are expressed at an
early stage in a distinct blastomere and are then distributed by means of an invariant pattern of cleavage divisions to the different cells whose fate they control
(Bagu and Boyer 1990). As pointed out above, in
Macrostomum sp., cleavage initially follows the conventional pattern, but then departs from this pattern when
certain blastomeres, most likely vegetal cells, stretch out
around the remaining blastomeres to form the outer yolk
mantle. It is currently not clear exactly which cells contribute to the yolk mantle. Inverse epiboly begins at a
stage when only two to three micromere quartets are
formed. Seilern-Aspang (1957) suggested that either all
four macromeres, or part of the macromeres plus part of
the third quartet micromeres, give rise to the outer yolk
mantle. In either way, the remaining blastomeres that become surrounded by the external yolk need to generate all
of the cell types, including the mesoderm and the gut,
with a reduced number of macromeres or no macromeres
at all. Once surrounded by the yolk mantle, cell divisions
in these inner blastomeres (the embryonic primordium)
are difficult to observe. So far, we have no observations
that any sort of spiral pattern is resumed; mitotic spindles
appear to be random, and cell sizes initially do not suggest
a distinction between macromeres and micromeres. Dye
injection-based lineage studies are required to follow later
cleavage and to reconstruct lineage relationships. It will
also be interesting to establish how the expression of
specific determinants in Macrostomum sp. has adapted to
the novel types of lineages. For example, are endo- and
mesodermal fate determinants, which in conventional
spiralians appear in the macromeres and in specific fourth
quartet micromeres, not expressed in early vegetal blastomeres, but rather come on at a later stage in distinct
locations within the embryonic primordium? We expect
that molecular-genetic studies will shed light on this and
related questions that are important in reconstructing the
evolution of developmental mechanisms.
Organogenesis from organ primordia
The developmental steps that lead from the early embryonic primordium of Macrostomum sp. to the body of the
hatching juvenile correspond closely in most important
aspects to what has been described for other flatworm
taxa. Thus, following the establishment of the undifferentiated embryonic primordium, individual organ primordia appear. The first organ primordia invariably are
those of the brain and the pharynx; in Macrostomum sp.,
as in the dalyellid (rhabdocoel) Gieysztoria superba and
the temnocephalid (rhabdocoel) Craspedella pedum, the
pharynx primordium appears directly posterior of the brain
(Younossi-Hartenstein and Hartenstein 2000a, 2001); in
the typhloplanoid (rhabdocoel) Mesostoma lingua the
pharynx primordium is formed at mid-levels along the
antero-posterior axis (Bresslau 1904; Hartenstein and
Ehlers 2000), and in triclads (e.g., Schmidtea polychroa;
Bennazzi and Gremigni 1982) it defines the posterior pole.
The position of the pharynx primordium at early stages
thus closely corresponds to the position where the pharynx
ends up later. Aside from its location, the later small size
and low complexity of the Macrostomum pharynx is also
reflected in its early appearance. This supports the notion
that the pharynx simplex coronatus, as defined by Doe
(1981) for all Macrostomorpha, may be homologous to the
other pharyngeal differentiations seen in higher rhabditophoran flatworms (see Ehlers 1985; Rieger et al. 1991,
for summaries). Precursors of the massive arrays of radial
muscle fibers characteristic of the pharynx bulbosus, the
most advanced pharynx type, are visible from the very
beginning; by contrast, in Macrostomum sp., pharynx
muscle precursors in the pharynx simplex form only a
thin, inconspicuous layer around the pharynx epithelium.
The primordium of the body wall musculature in
Macrostomum sp. first becomes apparent during stage 5
as an irregular, two to three cell diameters thick cell layer
underneath the epidermis. A series of block-shaped myoblasts have been reported for M. hystricinum marinum
(Reiter et al. 1996). These authors observed a close association between myoblasts and neuroblasts during differentiation, perhaps reflecting the intricate functional
relationship of muscle and nerve cells in the body-wall
musculature. Myoblasts subsequently flatten, elongate
and send out muscle processes. Nothing can be said
currently about the earlier origin of muscle precursors. In
larval polyclads there is a different origin of longitudinal
and circular muscles, the longitudinal fibers deriving from
the vegetal 4d blastomere called ento-mesoderm, the
circular fibers from cells deriving from the 2b micromere,
called ecto-mesoderm or ecto-mesenchyme (Boyer et al.
1996). In Acoela all muscle cells are apparently derived
from the ento-mesoderm (Henry et al. 2000). Dye injection-based lineage tracing experiments will be required to
verify whether a similar distinction also exists for Macrostomum sp. Furthermore, we have recently cloned the
homolog of the muscle specification gene Mef 2 (Morris
et al., in preparation) which, in both vertebrates and Drosophila, is expressed in mesoderm, followed by muscle
cells. Macrostomum sp. Mef 2 is expressed specifically in
the adult musculature, and it is hoped that in situ hybridization to early embryos will provide us with a more
detailed picture of the origin of muscles in this species.
Specific molecular markers will also be needed to
make progress in understanding the origin of the central
nervous system in Macrostomum sp. or any other flatworm. Neuronal precursors can only be distinguished
from myoblasts at the time when cell differentiation sets
in, and neurons send out axons (see Reiter et al. 1996;
Rieger 1998). The tyrTub marker allows one to study late
aspects of neural differentiation (similar to acTub in other
238
species; Younossi-Hartenstein et al. 2000; YounossiHartenstein and Hartenstein 2001), but it does not help to
follow crucial steps in early neurogenesis, because it labels all cells at this early stage. Given the close spatial
relationship between differentiating muscle and nerve
cells (see above), it is highly likely that precursors of
these cells are intermingled at an earlier stage. This would
be in stark contrast to “higher” animals, including arthropods and vertebrates, where these tissues originate apart
from each other in different germ layers.
With the present study on the embryogenesis of Macrostomum sp. we could not resolve the intriguing issue of
the origin of the unique post-embryonic stem cell system
(neoblast system) from embryonic stem cell precursors
(see Ladurner et al. 2000; Peter et al. 2001, 2004). As
summarized in Peter et al. (2001), circumstantial evidence
even suggests that platyhelminths seem not to have a
separate germ line. It is the general assumption in the
literature that the possibly totiponent post-embryonic
stem cell system (the neoblast system) originates from
certain embryonic stem cells (see literature in Peter et al.
2001). Our investigation on Macrostomum sp., similar to
many others on organ differentiation in platyhelminths,
does support the notion that the organ primordia are
clusters of embryonic stem cells that, like post-embryonic
stem cells, are the only cells capable of mitotic proliferation. In analogy to post-embryonic stem cells (neoblasts), embryonic stem cells might either come from a
single, totipotent stem cell pool in each organ primordium
or each organ primordium may have specific subtypes of
embryonic stem cells that differentiate only into specific
tissue types. Which of the two developmental processes
actually occur, will be one of the major challenges of
further research on development and growth of the
platyhelminths, a taxon so important because of its many
human parasites (Xylander 2004).
Acknowledgements We would like to thank Dennis Montoya,
Willi Salvenmoser, and Birgitta Sjostrand for their technical support. This work was supported by NSF grant IBN-0110718 to V.H.,
FWF grants P15204 and 16618 to R.M.R. and the Ruth L.
Kirschstein National Research Service Award GM07185 to J.M.
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