An ancient process in a modern mollusc: early
development of the shell in Lymnaea stagnalis
Hohagen and Jackson
Hohagen and Jackson BMC Developmental Biology 2013, 13:27
http://www.biomedcentral.com/1471-213X/13/27
Hohagen and Jackson BMC Developmental Biology 2013, 13:27
http://www.biomedcentral.com/1471-213X/13/27RESEARCH ARTICLE Open AccessAn ancient process in a modern mollusc: early
development of the shell in Lymnaea stagnalis
Jennifer Hohagen and Daniel J Jackson*Abstract
Background: The morphological variety displayed by the molluscan shell underlies much of the evolutionary
success of this phylum. However, the broad diversity of shell forms, sizes, ornamentations and functions contrasts
with a deep conservation of early cell movements associated with the initiation of shell construction. This process
begins during early embryogenesis with a thickening of an ectodermal, ‘dorsal’ (opposite the blastopore)
population of cells, which then invaginates into the blastocoel to form the shell gland. The shell gland evaginates
to form the shell field, which then expands and further differentiates to eventually become the adult shell-secreting
organ commonly known as the mantle. Despite the deep conservation of the early shell forming developmental
program across molluscan classes, little is known about the fine-scale cellular or molecular processes that underlie
molluscan shell development.
Results: Using modern imaging techniques we provide here a description of the morphogenesis of a gastropod
shell gland and shell field using the pulmonate gastropod Lymnaea stagnalis as a model. We find supporting
evidence for a hypothesis of molluscan shell gland specification proposed over 60 years ago, and present
histochemical assays that can be used to identify a variety of larval shell stages and distinct cell populations in
whole mounts.
Conclusions: By providing a detailed spatial and temporal map of cell movements and differentiation events
during early shell development in L. stagnalis we have established a platform for future work aimed at elucidation
of the molecular mechanisms and regulatory networks that underlie the evo-devo of the molluscan shell.
Keywords: Shell, Mollusc, Biomineralisation, Evolution, Development, Specification, Mantle, Alkaline phosphatase,
PeroxidaseBackground
Molluscs constitute one of the most successful, morpho-
logically diverse and ancient phyla of the animal king-
dom. They posses an extensive fossil record dating back
to the early Cambrian (543+ MYA) and comprise more
than 200,000 extant species occupying various marine
and terrestrial environments from the deep sea to desert
habitats [1,2]. Much of this evolutionary success can be
attributed to the phenotypic plasticity of the external
shell which displays an incredible range of mineralogical
textures [3], pigments [4,5] and ornamentations [6]. This
phenotypic diversity is underscored by a diversity in the
molecular mechanisms responsible for the construction
of the adult shell [7-10].* Correspondence: djackso@uni-goettingen.de
Courant Research Centre Geobiology, Georg-August University of Göttingen,
Goldschmidtstrasse 3, 37077, Göttingen, Germany
© 2013 Hohagen and Jackson; licensee BioMe
Creative Commons Attribution License (http:/
distribution, and reproduction in any mediumDespite the morphological and functional-molecular
diversity of the adult shell, there is deep conservation of
the cellular and morphogenic movements that initiate
larval shell secretion (reviewed in [11]). Importantly,
larval shell forming cells are thought to give rise to the
fully differentiated adult shell forming organ, the mantle,
suggesting that trochophore, veliger and adult gastropod
shells do not have independent evolutionary origins as
previously suggested [12]. Cell lineage studies in dispar-
ate gastropods support a common ontogenetic origin of
embryonic, larval and adult gastropod shells; derivatives
of the 2d and 2c micromeres in Ilyanassa give rise to
the shell gland [13], and the same lineage of cells in
Crepidula fornicata contributes to the mantle of the
veliger [14]. Furthermore, veliger mantle cells expressing
shell forming genes continue to do so following meta-
morphosis in the abalone Haliotis asinina [15,16]. Thed Central Ltd. This is an Open Access article distributed under the terms of the
/creativecommons.org/licenses/by/2.0), which permits unrestricted use,
, provided the original work is properly cited.
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 2 of 13
http://www.biomedcentral.com/1471-213X/13/27histochemical properties of larval and adult shell forming
organs in L. stagnalis also reveal a similar spatial arrange-
ment of enzymatic activities, suggesting that boundaries
of shell forming cell populations established in larval
stages are maintained into adult life [17]. Additionally,
regulatory genes encoding transcription factors and sig-
nalling molecules (such as members of the Hox cluster,
engrailed and decapentaplegic) are expressed in embry-
onic shell forming tissue in disparate molluscan taxa
[18-25]. This raises the possibility that extant shelled
molluscs may all initiate shell formation using the same
developmental program inherited from a distant ances-
tor, and that it is the downstream shell forming programs
operating in the mature mantle which, during evolution,
have generated today’s diversity of shelled adult molluscs.
If such a scenario were true, this would mean that a com-
mon ancestor of the shelled molluscs evolved a develop-
mental program to form a shell which was passed on to
all of its future descendants; a 540+ million year old
innovation that was of great importance to the future
evolutionary success of the phylum.
The pulmonate gastropod Lymnaea stagnalis (Linnaeus,
1758) was once a much used model for understanding
both molluscan development in general [26] and develop-
ment of the shell in particular [27]. Development of
Lymnaea’s shell displays many of the features observed in
other gastropod species. Across Molluscan classes, the
first morphological sign of shell development is a thicken-
ing of the dorsal ectoderm in the post-trochal region of
the embryo following gastrulation (see [11] for a review).
Briefly, these dorsal ectodermal cells elongate and are
often the only ectodermal cells in contact with the under-
lying endoderm, specifically cells at the tip of the archen-
teron (the so called 'small-celled endoderm' due to their
lack of large vacuoles present in other endodermal cells
[26,28]). These elongated dorsal ectodermal cells then
invaginate to form a ‘shell gland’ [11,29]. It is during this
stage that secretion of the first shell-associated insoluble
material takes place. The shell gland subsequently
evaginates to form the ‘shell field’, a process during which
the contact of ectodermal and endodermal cells is lost,
and the first signs of calcification of the previously
secreted insoluble material can be observed (e.g. [13,26,27,
30]). The initial contact between endoderm and dorsal
ectoderm that precedes shell gland invagination has been
observed in representatives of the Gastropoda, Bivalvia,
Scaphopoda and Cephalopoda (reviewed in [11]). This
contact between dorsal ectoderm and endoderm has lead
to the idea that this event is required for the specification
of future shell forming cells, and represents a 'true'
induction event [26].
While Raven’s model of shell gland induction [26]
represents the canonical theory of molluscan shell field
specification, the molecular mechanisms that initiateand underlie this process remain largely unknown.
Molecular analyses that have previously identified
transcription factors and signalling molecules in the shell
gland and the evaginated and expanding shell field
are expressed well after the specification of shell
forming cells [19,21,24,31]. We are therefore developing
L. stagnalis as a model for molecular investigations into
the mechanisms that first specify shell forming cells, and
through comparative studies, to enhance our under-
standing of how the variety of molluscan shells evolved.
Previous cytological studies on the early development
of the shell field in L. stagnalis do not include descrip-
tions of the cellular arrangements preceding the first
contact between the dorsal ectoderm and the small-
celled endoderm at the tip of the archenteron. Here we
employ confocal laser scanning microscopy (CLSM) to
provide a detailed temporal and spatial description of
the morphogenic events associated with development of
the larval shell in L. stagnalis. We have also employed
histochemical assays based on endogenous peroxidase
(PO) and alkaline phosphatase (AP) activity to identify
distinct cell populations within the developing shell
gland, shell field and other larval structures in whole
mounts. These enzymes are known to be active in the
shell forming tissues of several molluscan taxa, including
L. stagnalis [17]. These assays allow us to trace discrete
cell populations in larval shell forming tissues, and may
in the future be employed to characterise the effects of
experiments aimed at the perturbation of normal shell
development. This work represents a platform from
which further studies will investigate the molecular
processes leading to the specification and differentiation
of molluscan shell forming cells.
Methods
Cultivation of adult L. stagnalis
Adult specimens of L. stagnalis were collected from the
Northeimer Seenplatte near Northeim, Germany (51° 43’
26.5368’, 9° 57’ 24.75’) and from a pond on the North
campus of the University of Göttingen, Germany (51°
33’ 23.727’, 9° 57’ 25.617’). Snails were kept in standard
tap water at 25°C, under a 16:8 light dark regime and
fed adlibidum with lettuce and a variety of other
vegetables.
Staging and preparation of embryos of L. stagnalis
Freshly deposited egg masses were collected and their
development monitored. Following the first cleavage, egg
masses were cultured in snail water [32] at 25°C. All
stages are indicated in hours post first cleavage (hpfc)
and days post first cleavage (dpfc). At the desired devel-
opmental time point individual egg capsules were
removed from an egg mass and freed from the jelly by
rolling them over moist filter paper. Embryos were
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 3 of 13
http://www.biomedcentral.com/1471-213X/13/27manually dissected from their capsules using forceps
and needles and fixed according to the subsequent ex-
perimental procedure (see below).
Confocal laser scanning microscopy (CLSM)
29 embryonic stages between 27 and 87 hpfc were fixed
at intervals of one to five hours. For each developmental
stage, 29 to 126 individuals were visualised, and on aver-
age 6 individuals were imaged. To account for fixation
artefacts several fixation treatments were tested on em-
bryos between 27 and 37 hpfc, ranging from no fixation
to extended fixations overnight at room temperature
and in varying amounts of gluteraldehyde in combin-
ation with a paraformaldehyde-based fixation. Fixation
with 4% paraformaldehyde (PFA) in 1X phosphate buff-
ered saline (PBS) for one hour at room temperature, or
overnight at 4°C, was found to be optimal. Fixed speci-
mens were washed three times in PBS and processed im-
mediately or stored at 4°C for up to five weeks. For
cytoplasmic and nuclear staining samples were incu-
bated in a 1/1000 dilution of Sytox Orange (Molecular
probes, S11368) in PBS with 0.1% TritonX for two hours
at room temperature. Samples were then washed three
times in PBS, dehydrated through a graded ethanol
series and embedded in a 1:2 mixture of benzyl benzoate
and benzyl alcohol (BB:BA). Optical sections were cap-
tured using a Zeiss LSM 510 Meta with the following
settings: HeNe 543 laser at a power of 2.9%; pinhole be-
tween 50 μm and 60 μm (0.94 to 1.13 Airy Units); amp-
lifier gain of 1; amplifier offset and gain adjusted to the
sample brightness; stack size 1024 x 1024 with a stack
thickness between 0.81 μm and 0.9 μm; scan speed and
number of scans 7 and 4 or 6 and 8 respectively. For in-
dividual images the stack size was 2048 x 2048 with a
scan speed and number of 6 and 8 respectively. All im-
ages were false-coloured and adjusted for brightness
using Macnification version 2.0.1.
Scanning electron microscopy
Between 55 and 278 embryos for each time point from 27
to 67 hpfc at five hour intervals were fixed in 2.5%
gluteraldehyde in PBS at 4°C overnight. These were then
dehydrated through a graded ethanol series and dried
overnight in hexamethyldisilazane. Samples were mounted
on carbon pads on aluminium stubs and sputter-coated
with a gold-palladium alloy before being imaged with a
scanning electron microscope at 3.8 kV. All SEM images
were edited in Adobe Photoshop CS3 version 10.0.1 by ap-
plying the ‘auto levels’ and ‘auto contrast’ functions.
Histology and Histochemistry
The endogenous alkaline phosphatase (AP) activity of 13
developmental stages ranging from 37 to 117 hpfc at 3
to 5 hour intervals was examined. Between 34 and 154individuals were included in each experiment. Addition-
ally, 14 older larvae (ranging from five days post first
cleavage until hatching from the egg capsule) were also
assayed for AP activity. For each developmental stage,
images of three to 19 individuals were captured. Em-
bryos were fixed for 45 to 60 min in 4% PFA in 1X PBS
containing 0.1% Tween20 (PBTw), and rinsed in 1X PBS
before being incubated in AP reaction buffer (100 mM
Tris, 100 mM NaCl, pH 9.5) for 5 to 20 min. AP reac-
tion buffer was replaced by detection buffer (AP reaction
buffer, 50 mM MgCl2, 175 μg/mL BCIP and 450 μg/mL
NBT). The colour reaction was stopped after 15 to 60
min at an optimal signal to background ratio by re-
placing the detection buffer with 0.1 M Glycine pH 2
containing 0.1% Tween20. Samples were then rinsed in
PBS and post-fixed overnight at room temperature in 4%
PFA in PBS, dehydrated through a graded ethanol series,
embedded in BB:BA and viewed and photographed using
a Zeiss microscope Axio Imager Z1. A fraction of larger,
older (5+ dpfc) larvae were washed twice in PBTw fol-
lowing fixation, and then embedded in 60% glycerol and
imaged under a Zeiss stereo microscope discovery V8.
For developmental stages between 47 hpfc and 5+
dpfc, the endogenous peroxidase (PO) activity of 45 to
139 individuals was examined prior to performing the
AP assay (described above) in order to visualise the ac-
tivity of both enzymes at once. Samples fixed as de-
scribed above were first rinsed twice in 50 mM Tris pH
7.3, and then pre-incubated in 50 mM Tris pH 7.3
containing 1 mg/mL diaminobenzidine (DAB) for 15 to
20 min before supplementing the solution with a 1/3000
dilution of 30% hydrogen peroxide. The colour reaction
was monitored and stopped (usually after one to two mi-
nutes) by rinsing the samples for about 10 min in 1X
PBS. The AP assay (as described above) was then
performed on this material. One to 13 individuals per
developmental stage were photo-documented.
Results
Using CLSM we have studied the cell arrangements and
movements of the embryo from early gastrulation
(which precedes any contact between the dorsal ecto-
derm and the underlying endoderm) until evagination of
the shell gland. At 27 hpfc the ventral ectoderm (oppos-
ite the future site of the shell field) is broadly depressed
representing the initiation of gastrulation (Figures 1A’
and 2A). The proximal (‘basal’) side of the dorsal ecto-
derm faces inwards to a large blastocoel cavity (Figure 2A
arrow). Between 29 hpfc and 32 hpfc the invagination
of the archenteron initiates and completes. Cells of
the archenteron assume an elongated shape from 29
to 35 hpfc (Figure 2B-E). By 30 hpfc in almost all em-
bryos observed, the endodermal cells at the tip of the
archenteron are in contact with the dorsal ectoderm.
sh
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dorsal ventral Figure 1 Early shell development in L. stagnalis illustrated byScanning Electron Microscopy (SEM). A-C’ Gastrulation and
formation of the archenteron. The site of the future shell gland is
marked by white arrowheads. The blastopore is marked by an
asterisk. D-E’ The first outward signs of shell gland invagination are
two shallow depressions at 42 hpfc (arrowheads in D’ and E’).
F-I’ Insoluble material secreted by the shell gland is visible from
52 hpfc onwards. The first asymmetry of the shell is evident at
62 hpfc (highlighted by two dashed ovals in H’). All scale bars are
10 μm. Numbers in the lower left of each panel indicate the age in
hours post first cleavage (hpfc). Panel G is reflected about the
vertical axis for consistency of presentation.
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 4 of 13
http://www.biomedcentral.com/1471-213X/13/27Initially, this contact does not exist over the entire sur-
face of each cell. Rather, each endodermal cell appears
to send out pseudopodia-like projections to the overly-
ing ectoderm (Figure 2B boxed region). This results in
small spaces being observed between the contacting cel-
lular extensions. In other regions of the embryo, the cells
of the invaginating archenteron are separated from the
ectoderm by mesodermal cells or intercellular spaces.
The overall shape of the early 29 to 32 hpfc archen-
teron is slit-like (Figures 1B’ and 2B, C). Between 34 and
37 hpfc the blastopore opening narrows, and the archen-
teron develops a large round lumen (Figures 1C’ and 2D,
E). In most embryos at this stage, a variable number of
cells at the tip of the archenteron are in contact with
dorsal ectodermal cells directly beneath the large head
vesicle cells. By 37 hpfc the contact between endoderm
and dorsal ectoderm appears to be firmly established
(Figure 2F). This is the only region in the embryo where
these two cell layers are in direct contact with each
other, the archenteron is otherwise bordered by meso-
dermal cells or intercellular spaces. At this stage, neither
the ectodermal nor the endodermal cells at the contact
site display an altered cell morphology compared with
their neighbours using Sytox Orange.
The first signs of differentiation of the dorsal ecto-
derm cells as observable by CSLM occur at 39 hpfc. At
this stage, these cells take on a columnar morphology
and are clearly distinguishable from adjacent ectoderm
cells (Figure 2G). The columnar cells are in direct
contact with four to five cells of the tip of the under-
lying archenteron. These endodermal cells in turn
are characterised by a lack of large vacuoles that
are present in adjacent cells of the archenteron (indi-
cated by ‘x”s in Figure 2G). For this reason these
archenteron-tip cells have been referred to as “small-
celled entoderm” [28], here as small celled endoderm.
The number of endodermal cells contacting presump-
tive shell gland cells remains low during the period of
contact, never exceeding six cells. During the next
hours the morphological differentiation of both cell
layers becomes more pronounced with the nuclei
in
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Figure 2 Early shell development in L. stagnalis illustrated by Confocal Laser Scanning Microscopy (CSLM). A-F During the course of
gastrulation the initial invagination of the blastopore (asterisks) deepens to form the archenteron. The initial contact between dorsal ectoderm
and endoderm is loose and is characterised by cellular projections (boxed region in B). G-I Upon contact, ectoderm and endoderm display signs
of differentiation: the dorsal ectodermal cells at the contact zone differentiate into highly columnar shell field cells, and the endodermal cells are
characterised by a lack of large vacuoles which are present in adjacent endodermal cells (indicated in G by white “x”s). J-R The initial bilateral
invagination of the shell gland is visible in J (arrows). During this invagination the margins of the shell gland begin to converge (curved arrows in
K to P). By 62 hpfc the non-invaginated margins of the shell gland have converged and form a closed lumen (white dot in Q). By 67 hpfc cells at
the shell gland margin are highly elongated (arrow in R). Embryos in A-S are oriented with the shell field to the top and the veliger in T is
oriented with the shell field to the left. An asterisk marks the position of the blastopore. Panels A-P are transverse optical sections and Panels Q-T
are sagittal optical sections. All scale bars are 20 μm. Numbers in the lower left of each panel indicate the age in hours post first cleavage (hpfc).
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 5 of 13
http://www.biomedcentral.com/1471-213X/13/27of presumptive shell forming cells assuming a basal
location (Figure 2I-K).
The first external signs of shell gland differentiation
are two lateral slit-like depressions that form directly
beneath the large head vesicle cells at 42 hpfc (Figure 1D,
D'). The first sign of endogenous AP activity can be
detected at the same age in the two lateral depressions
(Figure 3A), eight hours earlier than previously reported
[17]. In transverse CSLM optical sections, the two lateral
depressions of the dorsal ectodermare first observed at
44 hpfc and deepen in the following hours to form an
invaginated shell gland (Figure 2J-Q). During the inva-
gination process columnar cells at the periphery of
the shell gland begin to converge towards each other
(Figure 2K arrows). Between 50 hpfc and 52 hpfc the
shell gland is comprised of two prominent lateral invagi-
nations and a central elevation (Figure 2M-O). This
bifurcated shell gland morphology is easily visualised byintense AP activity (Figure 3C, D). Scanning electron mi-
crographs of this stage show a depression surrounded by
a concentric arrangement of cells representing the non-
invaginated part of the shell gland (Figure 1F). In the
outer-most ring of cells, a second domain of AP activity
can be detected which has not been previously reported
for Lymnaea. This domain is first visible at 50 hpfc as
a semicircle lining the posterior half of the shell gland
(Figure 3C’, D’). During the next seven hours the tips of
the semi-circle steadily extend anteriorly until a closed
ring is formed (white arrows in Figure 3C’, D’ and 4A”').
From 54 hpfc onwards the central elevated part
of the invagination flattens, and the non-invaginated
shell gland margins continue to converge (arrows in
Figure 2P). By 62 hpfc the margins have converged and
the shell gland appears to be a sealed lumen (white dot
in Figure 2Q). All invaginated cells of the shell gland at
this stage are AP positive (white dot in Figure 4A, B),
42
A
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B
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47
C
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50
D
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B’
C’
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B’’
C’’
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*
52
anterior dorsal lateral
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pn pn
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Figure 3 Alkaline phosphatase (AP) activity in the early shell
gland of L. stagnalis. Endogenous alkaline phosphatase activity
(dark blue precipitate in all panels) highlights the development of
distinct cell populations and structures within the shell gland, and
allows for the identification of distinct stages of larval shell
development. A-A”(42 hpfc). The first evidence of AP activity in the
shell gland occurs at 42 hpfc (arrows). B-B” (47 hpfc). Invagination of
the shell gland begins at 47 hpfc. This is visible in AP+ cells which
can be seen just below the outermost level of the dorsal ectoderm
(arrow in B). C-C” (50 hpfc). Non-invaginated AP+ cells at the margin
of the shell gland (white arrows in C) expand in an anterior direction
(curved white arrows in C’). Invaginated AP+ cells (black arrows in C
and C’) intensify their AP activity. The anlage of the protonephridia
(pn) and apical plate also become AP+ at this stage. D-D” (52 hpfc).
The non-invaginated AP+ cells at the margin of the shell gland
continue to migrate in an anterior direction (curved white arrows in
D’). All embryos are oriented with the shell field to the top. An
asterisk marks the position of the blastopore. All scale bars are
20 μm. Numbers in the lower left of each panel row indicate the
age in hours post first cleavage (hpfc).
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 6 of 13
http://www.biomedcentral.com/1471-213X/13/27with AP activity in non-invaginated cells of the shell
gland margin also persisting (white arrows in Figure 4A).
At this stage, more than ten hours earlier than previ-
ously reported [17], the first signs of endogenous PO ac-
tivity in and around the shell gland are evident.
Peroxidase activity can be detected in non-invaginated
cells directly adjacent to the shell gland lumen (arrows
in Figure 4A’) and adjacent to the peripheral AP positive
ring of cells (arrows in Figure 4A”’) which are not
detected in double staining experiments against both en-
zyme’s activities. Also, between 52 hpfc and 57 hpfc, thefirst extra-cellular organic material has been secreted
and is stretched over the entire shell gland (Figures 1F’
and 4A’ arrowhead). By 62 and 67 hpfc the non-
invaginated cells at the periphery of the shell gland are
highly elongated (arrow in Figure 2R). Scanning electron
micrographs reveal the shell gland margin as an elevated
ring (Figure 1H and I’). The secreted insoluble material
now lies loosely on the elevated shell gland margin and
displays PO activity (arrows in Figure 4B'). Peroxidase
and AP activity also persists in adjacent non-invaginated
rings (Figure 4B'' and B”’). The first asymmetry in the
shell gland is also visible at 62 hpfc with the shell gland
slightly shifted to the left side (indicated by the dashed
ovals in Figure 1H’). This asymmetry becomes more
pronounced in subsequent stages.
From 67 hpfc onwards the shell gland evaginates
giving rise to the shell field. First, the non-invaginated
margins of the shell gland diverge, opening up the shell
gland lumen (Figure 2S and curved arrows in Figure 4C,
5A and 6A). Contact between endodermal cells and the
dorsal ectodermal is lost at 77 hpfc, and the shell
field expands in size during subsequent development.
Peripheral cells (formerly non-invaginated cells of the
shell gland) maintain their columnar shape whereas the
central, formerly invaginated cells flatten (Figure 2T).
The relative arrangement of PO and AP activity do-
mains persists from 77 hpfc to 117 hpfc. The centre of
the shell field displays AP activity with increasing inten-
sity towards the shell field margin (Figure 5C, C’, D”).
During the following stages AP activity in the centre of
the shell field gradually decreases (Figure 5C, D). At 117
hpfc AP activity is found in a line of cells proximal to
the shell field margin (Figure 5D). At this stage the
secreted organic material and the highly elongated cells
of the shell field margin continue to exhibit a strong PO
activity (Figure 5C”’ and D”’).
In the periphery of the PO positive shell field mar-
gin, a faint ring of AP+ cells is detectable from 72 hpfc
on (Figure 6). This signal possibly represents the AP+
domain of non-invaginated cells seen in earlier stages
(see white arrows in Figures 3 and 4). During the
course of shell field differentiation, the activities of
both enzymes are continuously located in adjacent,
non-overlapping cell populations within the shell field
(Figure 6A-C”’; see Figure 8 for a schematic summary
of these observations).
Non-shell related AP and PO activities during larval
development
Endogenous AP and PO activities can also be used
to follow the development of larval structures in
L. stagnalis in a more general way. Several structures be-
sides the shell gland and shell field display endogenous
activity of these enzymes (Figure 7). AP activity is
dorsalanterior
SG
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57
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A
B
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B’
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A’’
B’’
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AP
AP +
peroxidase
AP +
peroxidaseAP
Figure 4 Alkaline phosphatase (AP) and peroxidase (PO) activity in the mature shell gland of L. stagnalis. Endogenous AP activity
intensifies as the shell gland matures, and PO activity also becomes detectable. A-A”’ (57 hpfc). The mature shell gland at 57 hpfc is characterised
as a closed lumen with intense AP activity (white dot in A). AP activity has also increased in non-invaginated cells at the margin of the shell
gland (white arrows in A), and in the anlage of the protonephridia (pn). Weak PO activity is also evident at this stage (black arrows in A’ and A”’),
and secreted insoluble material can also be seen in preparations of this age (arrowhead in A’). Non-invaginated AP+ cells at the margin of the
shell gland finish their anterior expansion, meeting at the midline (curved white arrows in A”). B-B”’ (62 hpfc). In 62 hpfc larvae, the shell gland is
maintained as a closed lumen (white dot in ) while PO activity in non-invaginated cells intensifies (black arrows in B’). C-C”’(67 hpfc). Between 62
and 67 hpfc evagination of the shell gland has commenced (transparent white arrows in C) and PO activity in non-invaginated cells of the shell
gland margin has increased (black arrow in C’). The asymmetry of the shell gland is made clearly visible by populations of AP+ and PO+ cells
(dashed ovals in C” and C”’ respectively). All embryos are oriented with the shell gland to the top. An asterisk marks the position of the
blastopore. All scale bars are 20 μm. Numbers in the lower left of each panel row indicate the age in hours post first cleavage (hpfc).
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 7 of 13
http://www.biomedcentral.com/1471-213X/13/27present in most ciliated fields (Figure 7E, I, K and N)
and the protonephridia (Figure 7F-H) [26]. In older 5+
dpfc stages the developing radula (Figure 7J and M) and
cells throughout the foot exhibit AP activity (Figure 7A).
Endogenous PO activity can be found in ectodermally
derived cells scattered over the head and foot region
(Figure 7B and K) and later in the head vesicle cells
anterior from of the apical plate (Figure 7B, C, K and N)
[33]. Both enzymes show adjacent, but non-overlapping
activity in the head region and activity in the velum [33]
(Figure 7C and D).
Discussion
In various molluscan groups, the initial differentiation of
the dorsal shell forming ectoderm has been observed to
coincide with the presence of a tight contact with under-
lying endodermal cells (reviewed in [11]). These observa-
tions raised the possibility that this contact is requiredfor the specification of molluscan shell forming cells in
general. Based on manipulative experiments, first Raven
[26] and later Hess [34,35] concluded that it is this dor-
sal ectoderm/endoderm contact that specifies the shell
field. Raven differentiates between two possibilities of
contact-dependent shell field specification, induction vs.
activation. Raven [26] realised that if the dorsal ecto-
derm is truly induced to become the shell gland by such
a contact (rather than activated as would be the case if a
population of dorsal ectodermal cells were already speci-
fied in someway), two morphogenic preconditions must
be realised. Firstly, only a restricted part of the endo-
derm (i.e. cells at the tip of the archenteron) should be
able to elicit this specification in the overlying ectoderm.
Secondly, the whole ectoderm (or at least the majority)
should be able to respond to this induction by forming a
shell gland. Raven’s observations based on four embryos
(whose gastrulation had been perturbed by lithium and
**
*
*
*
A
77
*
B
87
*
C
97
*
D
A’
B’
C’
D’
A’’
B’’
C’’
D’’
A’’’
B’’’
C’’’
D’’’
117
SG
 e
va
gi
na
tio
n 
an
d 
SF
 e
xp
an
sio
n
anterior lateraldorsal
pn
Figure 5 Alkaline phosphatase (AP) and peroxidase (PO) activity in the evaginating shell gland and expanding shell field of
L. stagnalis. All panels are double labelled for AP and PO activity. A-A”’ (77 hpfc). By 77 hpfc secreted birefringent material (black arrow in A)
overlying the evaginating shell gland is clearly visible. The shell gland is no longer a closed lumen (cf. Figure 4A and 4B) as it continues to
evaginate (indicated by transparent white arrows in A). A ring of intense PO activity now surrounds a field of AP activity (A’). The protonephridia
(pn) remain intensely AP+ (A”). B-B”’ (87 hpfc). By 87 hpfc shell gland evagination appears to be complete and the field of AP+ cells in the shell
gland has expanded in all directions (B’). A sheet of organic material overlies the shell field (arrow in B”’). C-C”’ (97 hpfc). At 97 hpfc the shell
field continues to increase in size and the border between PO+ and PO- cells at the shell field margin sharpens (C”’). D-D”’ (117 hpfc). By 117 hpfc
AP activity in the shell gland is concentrated in cells directly adjacent to PO+ cells. All larvae are oriented with the shell field to the left. An
asterisk marks the position of the stomodaeum. Scale bars are 20 μm (A”’, B”’, C”’ and D”’) or 50 μm (A-A”, B-B”, C-C” and D-D”). Numbers in
the lower left of each panel row indicate the age in hours post first cleavage (hpfc). Panel A'' is reflected about the vertical axis for consistency
of presentation.
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 8 of 13
http://www.biomedcentral.com/1471-213X/13/27which developed an ectopic shell gland) lead him to
conclude that most of the ectoderm is indeed able to
respond to signals from the underlying endoderm, and
that therefore the dorsal ectoderm is truly induced by
the endoderm to become shell forming tissue, rather
than activated. This model of induction-mediated shell
field specification has since been supported [34], modi-
fied [13,35] and contradicted [36-38]. These studies
report a wide capacity of the ectoderm to form a shell
field, but disagree about the origin of the “inductive
cue”. Hess’s observations of partial embryos after blasto-
mere separation in Bythinia and L. stagnalis [34,35]
support the contact dependent model of shell field speci-
fication, but indicate that any endodermal tissue, evensingle cells, is capable of induction. Cell deletion experi-
ments in Ilyanassa [13] support the hypothesis that
there is no cellular specificity in the inducing endoderm:
all combinations of ectoderm and endoderm can gener-
ate a shell field. Furthermore, in Ilyanassa it has been
shown that the tip of the archenteron is never in close
proximity to the dorsal ectoderm [37]. Labordus and van
der Wal [38] extending the studies on Ilyanassa by
Clement [36] and Cather [13], suggest a scenario which
distinguishes between the histogenic and morphogenic
differentiation of the shell gland. Based on observations
of misdeveloped embryos producing internal shell
material, Labordus and van der Wal [38] propose that
the histogenic differentiation necessary to produce
lateral anteriordorsal
AP
AP +
peroxidase
A
72
*
*
B
87
*
lateral
C
A’
B’
C’
A’’
B’’
C’’
A’’’
B’’’
C’’’
97
**
lateral dorsal
Figure 6 Multiple alkaline phosphatase (AP) domains reveal a differentiated and complex organisation of the shell gland. A second
domain of AP activity present within the shell gland and shell field (not described by Timmermans [17]), is located outside of the PO domain and
can not be clearly detected after prior detection of PO activity (see Figure 5 and Figure 6A cf. 6A'). A-A''' (72 hpfc). This second AP+ domain
associated with non-invaginated cells of the shell gland margin can first be detected at 50 hpfc (see white arrow in Figure 3C), and is more
pronounced at 72 hpfc (white arrows in A-A'''). This AP+ domain subsequently decreases in strength. B-B”’ (87 hpfc). At 87 hpfc the distinct
domains of AP activity associated with the evaginating shell gland (black arrows) and the non-invaginated cells of the shell gland margin (white
arrows) are still visible. C-C''' (97 hpfc). By 97 hpfc the domain of AP activity associated with non-invaginated cells of the shell gland marginis
considerably weaker (white arrows). It's position relative to PO+ cells can be seen clearly in C (white arrow). The larvae in A-A”’ are oriented with
the shell field to the top, the larvae in B-C”’ are oriented with the shell field to the left. An asterisk marks the position of the stomodaeum. Scale
bars are 20 μm (A-A’, B-B’ and C-C’) or 100 μm (A”-A”’, B”-B”’ and C”-C”’). Numbers in the lower left of each panel row indicate the age in
hours post first cleavage (hpfc). Panels A”, A”’, B”, C and C”’ are reflected about the vertical axis for consistency of presentation.
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 9 of 13
http://www.biomedcentral.com/1471-213X/13/27such material is independent of inductive interactions,
whereas the correct spatial organisation of shell forming
tissues depends on spatially correct inductive interac-
tions between the D-quadrant macromeres and the over-
lying micromeres earlier in development. Based on that
study, McCain [39] conducted cell deletion experiments
that suggest inductive interactions among the micro-
meres are also required to give rise to the larval shell
forming tissues. The disturbance of these interactions by
the removal of participating cells leads to the internal
deposition of calcium carbonate similar to those ob-
served by earlier workers, supporting the assumption
that the processes leading to the histogenetic vs. the
morphogenetic differentiation of larval shell formingtissues do not depend on each other. This hypothesis is
further corroborated by work in experimental systems
that allow for an artificially induced shell internalisation,
e.g. by exposure to environmental toxins such as plat-
inum [40,41]. In these systems (Marisa cornuarietis and
Planorbis corneus), platinum interferes with the localisa-
tion of shell material and the growth of shell forming
tissues, while the cellular differentiation of these tissues
appears to remain unaffected [40,41].
While Raven’s hypothesis of induction-mediated shell
gland specification still represents the most comprehen-
sive theory of how the future shell forming cells are
initially specified in molluscs, contradictory observations
have been reported for a number of disparate taxa. In
**
A B C D
E F G H
I J K
L M N
*
*
*
*
*
*
*
*
*
Figure 7 Endogenous alkaline phosphatase (AP) and peroxidase (PO) activities as markers of larval development. Other organs and cell
populations not involved in shell formation display AP and PO activity. Endogenous AP activity can be found in most ciliated fields (E and I,
white arrows), including the apical plate (G, I, K, N), the protonephridia (F-H, black arrows), the developing radula (J and M, white open
arrowhead) and dispersed cells throughout the foot (A, black arrowhead). Peroxidase positive structures include the head vesicle cells (B, C, K and
N, black open arrowheads), the velum (C, white arrowhead) and scattered cells throughout the foot and head tissue (B, black arrowhead, and K
respectively). N is a detailed view of the boxed area in K. L is an SEM from a lateral/ventral perspective of a 5+ dpfc staged larva illustrating the
ciliated fields of the foot and head (white arrows) and the velum (white arrow head). A-D, H, J and M show larvae of 5+dpfc, F shows a 77 hpfc
old larva, I shows a 97 hpfc old larva and E, G, K and N show 117 hpfc old larvae. The position of the stomodaeum is marked by an asterisk.
Scale bars are 20 μm (F-I, M and N) or 100 μm (A-E, J, K, L). Panels B, G, I, K, and N are reflected about the vertical axis for clarity of presentation.
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 10 of 13
http://www.biomedcentral.com/1471-213X/13/27the pulmonate taxa Bradybaena and Achatina marginata
the shell field is differentiated before any contact
with the underlying endoderm is established, in the
Caenogastropod Marisa this contact is interrupted by
intermingled cells, and in other gastropod species
(Ilyanassa obsoleta and Achatina fulica) as well as
bivalves (Cyclas and Sphaerium) no contact is present
(reviewed in [11]). Unfortunately, none of these previous
studies utilised high resolution imaging techniques such
as CLSM that are available today, or conducted their in-
vestigations with high temporal resolution. Nonethelesswe must acknowledge that there does exist the possibility
of an alternative, contact-independent shell gland specifi-
cation mechanism, leaving open the question as to
whether the dorsal ectoderm/endoderm contact event
represents the ancestral molluscan mode of shell gland
specification.
While the present study was not intended to clarify
the molecular mechanisms of shell gland specification,
nor to differentiate between scenarios of induction vs.
activation as proposed by Raven [26], it does clarify the
nature of the cellular interactions between endoderm
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 11 of 13
http://www.biomedcentral.com/1471-213X/13/27and ectoderm prior to and during shell gland specifica-
tion, and also provides an accurate framework for the
timing of these events in L. stagnalis at 25°C. Using
CSLM we could reconstruct the cellular arrangements
and movements during contact between dorsal ectoderm
and endoderm. Despite its importance, former studies
do not include a description of how this contact is initially
established. If, as Raven [26] proposes, the ‘small-celled
endoderm’ truly induces the overlying dorsal ectoderm in
Lymnaea, these endodermal cells should have acquired
their inductive capacity prior to contact. Such prior
differentiation is not revealed by Sytox Orange staining in
our study (both dorsal ectoderm and the ‘small-celled
endoderm’ show the first signs of differentiation after
contact establishment; see Figure 2F and G), nor by Raven
using standard histological stains [26]. Raven concluded
that acquisition of an inductive capacity by the endoderm
is not revealed by any histological differentiation. Indeed,
a molecular differentiation of the contacting endoderm
could be expected to precede any visible histological
differentiation. Identification of such molecular markers
would provide great insight into the evolution of the
molluscan shell.
Our study also reveals a pronounced bilateral organi-
sation of early shell gland development; the invagination
of the shell gland begins when two lateral points of the
thickened dorsal ectoderm form two lateral depressions
(Figure 1D-E’, 2J-O). This bilateral organisation persists
until the margins of the shell gland converge above the
lumen of the invaginated shell gland and the bifurcated
lumen rounds up (Figures 1F’, 2P-R). The formation of
this shell gland lumen coincides with the secretion of the~32 hpfc~27 hpfc ~42 hpfc ~47 hpfc
ectoderm endoderm mesoderm AP positi
PO positive cells PO positive extra-cellular materia
Figure 8 A schematic representation of the major events during early
Uppermost row are all dorsal views except for ~87 hpfc which is a lateral v
through the developing shell gland and shell field. By approximately 32 ho
archenteron, the so called“small celled endoderm” for their lack of large va
the overlying cells of the dorsal ectoderm. These are the only endodermal
contact with the endoderm have thickened and some cells display alkaline
extra-cellular material. A bilateral invagination of the shell gland has also co
deepened and non-invaginated, posterior shell gland cells at the periphery
invaginations have fused, and the lumen of the shell gland displays intense
sealed with intense AP activity. The first peroxidase (PO) activity is visible at
material. By 87 hpfc the form of the juvenile snail has been established andfirst insoluble shell material. None of the invaginated shell
gland cells appear to participate in the secretion of this
first water insoluble material which emerges from the per-
ipheral non-invaginated shell gland cells [27] (Figure 1F’).
This observation has raised the hypothesis that the
process of shell gland invagination is required in order to
bring cells at the periphery of the shell gland into close
contact, and to thereby initiate the secretion of an insol-
uble shell forming matrix without a central hole above the
shell gland lumen [42,43]. Shortly afterwards, the first
signs of asymmetry in the shell gland appear. The invagi-
nated part of the shell gland shifts to the left side which
generates a larger distance between the centre of the
lumen and the peripheral secreting cells on the right side
than on the left side (Figures 1H’, 4C” , C”’). This early
asymmetry presumably reflects the future coiling direction
of the mature shell.
While shell gland formation is a deeply conserved fea-
ture of molluscan development, there is considerable di-
versity in its ontogeny within and between all molluscan
groups. For those species with internal or reduced shells,
the formation and further differentiation of the shell
gland differs from that seen in L. stagnalis and other
externally shelled molluscs (reviewed in [11]). In shell-
less cephalopods for example, shell gland development
ceases during dorsal ectoderm invagination, and an
evaginated shell field never forms. In cephalopods with
an internal shell, the shell gland is internalised and
characterised by a closed pore, and is therefore referred
to as a “shell sac”. This structure is not thought to be
formed by an invagination of the central part of the
thickened dorsal ectoderm. Instead, the peripheral cells~87 hpfc~52 hpfc ~62 hpfc ~67 hpfc
ve cells AP positive extra-cellular material
l (periostracum)
development of the shell gland and shell field in L. stagnalis.
iew. Lowermost row are the corresponding transverse sections
urs post first cleavage (hpfc) endodermal cells at the tip of the
cuoles (indicated by black ovals and circles), have made contact with
cells to make contact with ectoderm. By 42 hpfc cells that are in
phosphatase (AP) activity. The strongest AP activity is apparently in
mmenced at this age. By 47 hpfc the bilateral invagination has
of the shell gland also display AP activity. At 52 hpfc the bilateral
AP activity. By 62 hpfc the lumen of the shell gland appears to be
this time and is evident in cells and in the secreted periostracum
the non-overlapping zones of AP and PO activity are maintained.
Hohagen and Jackson BMC Developmental Biology 2013, 13:27 Page 12 of 13
http://www.biomedcentral.com/1471-213X/13/27of the thickened dorsal ectoderm bulge upwards and
overgrow the central cells. An internalised shell gland or
“shell sac” is also found in shell-less terrestrial slugs, but
is formed by a different mechanism. Here, the dorsal
ectoderm invaginates as it does in shelled gastropods,
but continues inwards leading to a complete internalisa-
tion of all shell gland cells, and consequently a closure of
the shell gland. These ontogenetic events in secondarily
shell-less slugs also illustrate their common ancestry with
shelled snails such as Lymnaea.
Endogenous enzyme activities as a tool to illustrate larval
development
Our study demonstrates the usefulness of endogenous
AP and PO activity as markers to map molluscan devel-
opment. During the course of shell gland and shell field
differentiation both enzymes are continuously located
in distinct shell forming cells (summarised in Figure 8).
Larval structures such as ciliated fields and the protonephridia
are also AP positive, and the head vesicles in older em-
bryos display PO activity (Figure 7). Previous studies of
AP activity during shell field development in L. stagnalis
were based on acetone-fixed and paraffin-embedded
sections, a procedure that results in a significant loss of
enzyme activity [17]. Using the methods we describe
here, we can detect both earlier and novel domains of
AP and PO activity, and can also simultaneously detect
AP and PO activities. AP and PO activity in shell
forming tissues has been shown for a number of
gastropod and bivalve taxa (summarised in [17]). Tran-
scripts encoding these enzyme families derived from
developmental stages and/or adult shell-secreting tis-
sues can be found in sequence databases for divergent
molluscan taxa, suggesting that these enzymes might
have conserved functions during molluscan shell devel-
opment. While the precise function of AP in molluscan
shell forming tissues has not been described, AP activ-
ity in vertebrate bone (hydroxy apatite) forming tissues
is known to regulate levels of inorganic pyrophosphate,
a potent inhibitor of mineralisation [44,45]. PO activity
is displayed by non-invaginated cells at the periphery of
the shell gland. These cells appear to be intimately as-
sociated with the production of the periostracum which
is itself PO+ (Figures 4, 5, 6). It has been suggested that
peroxidases in the periostracum may assist in the
crosslinking of periostracal proteins, rendering them
insoluble and resistant to abrasion [17,27]. These
simple histochemical assays provide a tool not only to
identify and trace functionally distinct cell populations
within the developing shell gland and shell field, but
also to simply assist with the orientation of the mollus-
can embryo. In the future, such assays could be used to
assess the effects of manipulative experiments such as
shell-specific gene knock-down assays.Conclusions
This work represents a platform from which analyses
aimed at the identification of the molecular regulators
responsible for shell development in L. stagnalis can be
conducted. We have described the timing of develop-
mental events critical to specification of shell forming
cells, and the movements of cells that take part in these
processes. We also highlight the use of histochemical as-
says that allow for the detection of endogenous alkaline
phosphatase and peroxidase activity within shell forming
cells. Understanding the molecular basis of shell devel-
opment from a range of molluscan representatives will
provide deep insight into the evolutionary events that
supported the generation of much of today’s molluscan
diversity. The work presented here is a first step
towards the development of L. stagnalis as a model to
understand how this diversity arose.
Competing interests
The authors declare that they have no competing interests.
Author’s contributions
JH carried out the experimental procedures. DJJ conceived and supervised
the study. Both authors participated in the design of the study and drafted
the manuscript. Both authors read and approved the final manuscript.
Acknowledgements
We gratefully acknowledge Luciana Macis for technical assistance and
maintenance of snail lines, and Dorothea Hause-Reitner for capturing the
SEM images. Nora Glaubrecht assisted with initial Sytox Orange CLSM
experiments. This work was supported by Deutsche Forschungsgemeinschaft
(DFG) funding to DJJ through the CRC for Geobiology and the German
Excellence Initiative, and DFG project number JA 2108/1-1.
Received: 4 April 2013 Accepted: 8 July 2013
Published: 12 July 2013
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doi:10.1186/1471-213X-13-27
Cite this article as: Hohagen and Jackson: An ancient process in a
modern mollusc: early development of the shell in Lymnaea stagnalis.
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