Contributions to Zoology, 71 (1/3) 37-45 (2002)
SPB Academic Publishing bv, The Hague
Newly-discovered muscle in the larva of Patella coerulea (Mollusca,
the of larval Gastropoda) suggests presence a extensor
Peter Damen and Wim+J.A.G. Dictus
Department ofExperimental Zoology, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands,
Key words: Patella, Gastropoda, Mollusca, development, larva, muscle, antagonist, cell-lineage
Abstract and Dictus and Damen (1997). The formation of
the 3D-macromere, which acts as an organizer and
The developmentofthe muscular system ofthe gastropod mollusc is responsible for the correct dorsoventral pattern- Patella has been thoroughly studied. As a result, two larval of the has been described ing embryo, by e.g. van the main and larval had been retractors, accessory retractor, den Biggelaar and Guerrier (1979), Arnolds et al. described in ofPatella. the larva These muscles were supposed (1983), Verdonk and van den Biggelaar (1983), to be responsible for the retraction of the larval body into the Martindale et al. and Damen and Dictus shell.Previously no larval extensors, which would be responsible (1985), The of function for the extension of the larval body out ofthe shell, have been (1996). analysis gene during early
described. Using cell-lineage tracer injection and phalloidin development has been described by van der Kooij
staining ofmuscles, anewly-discovered muscle is herein identi- et al. (1996, 1998), Damen et al. (1994), Damen fied in the larva of Patella coerulea. A functional model is and Loon Klerkx van (1996), et al. (2001), Lartillot presented in which two muscles areresponsible for the extension et al. (2002), Lespinet et al. (2002), Nederbragt et of the larva, the newly-discovered muscle together with the al. and and but named larval (2002a, b, c), Wanninger Haszprunar previously recognized inaccurately accessory The formation ofthe musculature retractor. In our model,the latter muscle does not seem to function (2001). has been
as a retractor but rather as an extensor. described by Smith (1935), Crofts (1955), and Wan-
ninger et al. (1999).
In the first half of the previous century, Smith
Contents (1935) and Crofts (1955) studied muscle morpho-
in genesis Patella with the use of a traditional
Introduction 27 technique, i.e. microscopical observation of serial
Materials and Methods 39 sections. Much later, Wanninger etal. (1999) studied Results 40 the formation of the musculature in the larva of Discussion 41 Patella using a much more recently introduced Acknowledgements 44 stained larvae of Patella with fluor- References 44 technique. They escently-labeled phalloidin, which binds to F-actin,
in order to visualize the musculature using confocal
Introduction laser scanning microscopy (CLSM). With this tech-
nique, muscle development can be analyzed at a For many decades, the gastropod mollusc Patella much higher resolution and even enables a three- has been used as a model system to study various dimensionalanalysis of muscle localization. Using
aspects of early development. For instance, the cell- CLSM, Wanninger et al. (1999) showed that the
lineage has been described by Patten (1886), Wil- formation of the principal larval and adult muscles
son den in (1904), Smith (1935), van Biggelaar (1977), Patella takes place between about 28 and 52 h
Downloaded from Brill.com10/07/2021 11:07:18AM via free access - 38 P. Damen & W.J.A.G. Dictus Newly-discovered muscle in the larva ofPatella
Fig. I. Post-torsional, 50 h old relaxed larva in which the muscles that are visible at this stage are shown. Right-lateral view. (A)
Stereo image. Phalloidin labeling. (B) Schematic drawing ofA. In this schematic drawing and the schematic drawings that follow the
muscles are drawn with different colors. For clarity, one part of the muscles is drawn with saturated colors and the other part of the
muscles with the the larval and the velum muscle which correspondingpastel colors, except accessory retractor, pedal plexus ring, are
drawn in saturated colors, air = larval Ism = left adult shell muscle = mantle accessory retractor (yellow); (pastel brown); m.c. cavity;
mlr = main larval retractor = = = = adult shell muscle (red); oper. operculum; pp pedal plexus (green); prot. prototroch; rsm right
= velum muscle Scale bar: 50 All should be with (saturated brown); vmr ring (pink). pm. stereo images examined red-green stereo
glasses in order to see the three dimensional localization of the structures shown.
first after cleavage. The muscles they described are Patella coerulea were injected with a fluorescent
main their the larval retractor, the accessory larval re- marker to study cell-lineage. Injection of one
tractor, the velum muscle ring, the pedal plexus of the blastomeres, the 2b-micromere, resulted in and the anlagen of the left and right adult shell labeling of an unknown bundle of fibers in 48 to
muscles Detailed of both 52 h old larvae. After of (Fig. 1). comparison ap- post-torsional injection
proaches demonstrates that the traditional approach the 3a- and 3b-micromeres small parts of this bundle
Smith and labeled of (1935) Crofts (1955) is quite prone of fibers were as well. Since this unknown
and result in miss- bundle of fibers to misinterpretations even may strongly resembled a muscle, 48
52 larvae with ing certain details of muscle formation. to h old post-torsional were stained
The 48 to 52 h old post-torsional larva of Pa- fluorescently-labeled phalloidin. In a number of
tella is a well-developed larva with a head, a foot, these larvae, besides the muscles described above,
a shell and a prototroch (Fig. 1). It is capable of the unknown bundle of fibers was labeled as well,
retraction into its larval shell. The main larval re- demonstrating that the unknown fibers are indeed
tractor and the accessory larval retractor are sup- muscle fibers.
posed to be responsible for this process, although Based on its location and orientation, together
this has not been stated explicitly for Patella (Wan- with the fact that no explanation for the extension
ninger et al., 1999). Besides retraction, the animal of the larva out of its shell is given in the litera-
must be able to extend out of its larval shell. De- ture, it is hypothesized that the newly-discovered
spite that the larva is obviously able to do so, nei- bundle of muscle fibers is involved in the exten-
the ther muscles nor other mechanisms responsible for sion of the larval body out of shell. A hypothetical
this action have been described in the literature. model is presented in which the newly-discovered
As “retrac- part of a larger study (Damen and Dictus, muscle, together with the accessory larval
submitted) various early cleavage-stage blastomeres, tor”, are responsible for the extension of the larval
viz., 2b, 3a, 3b, 3c, 3d, 3A, 3B, 3C, 4d and 4D, of body out of the shell. As a result, these two muscles
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form the of main larval determined and in antagonists the retractor. embryos which the 2b-micromere was injected
identified. The 3a- and 3b-micromeres data were were at Reanalysis of literature suggests that a struc- injected the 32-cell stage. Since at about 60 min after fifth cleavage ture similar to the previously undescribed muscle, still at the embryos are 32-cell stage, and the quadrants can be and thus a potential larval extensor, is present in at denominated at this stage, 3a- and 3b-micromeres could be least one other and that the gastropod previously identified before injection. undescribed muscle is homologous to the circular muscle fibers in the Mullers larva of the platyhel- Selection andfixation minth Hoploplana.
Abnormal larvae at 48 to 52 h after first cleavage, i.e., larvae
that do not possess eyes, a prototroch, a shell, a foot and an
that operculum or are not able to retract into their shell, were Materials and methods
discarded. The muscles ofnormal larvae of 48 to 52 h after first
cleavage were relaxed by adding drops of 0.75 M MgCl to the 2 Obtaining embryos MPFSW. Subsequently,the larvae were fixed in 4% formaldehyde
in 0.1 M phosphate buffer (pH 7.4) for 1 h and rinsed in buffer
Adult specimens of the limpet Patella coerulea (Mollusca, (3 times for 10 min).
Gastropoda) were obtained from the Mediterraneancoast near
and Trieste (Italy) near Banyuls-sur-Mer were (France). Embryos Phalloidin staining and mounting obtained as described before (van den Biggelaar, 1977; Damen and Dictus, 1996; Dictus and Damen, 1997). Before injection In order to visualize the musculature, most ofthe fixed larvae of cell-lineage tracer, embryos were treated with acidified were stained with fluorescently-labeledphalloidin (Alexa Fluor MPFSW (pH 3.9) for 2-3 min to remove the surrounding jelly 488 phalloidin, 300 U, A-12379, Molecular Probes Europe, layer. Embryos that were not injected but only culturedto stain Leiden, Netherlands), thatbinds to F-actin (modified after Wan- the musculature with phalloidin were also dejellied. To reduce et ninger al, 1999). Laryae were permeabilized for 1 h in 0.1 M microbial infection, embryos were usually transferred to anti- phosphate buffer (pH 7.4) to which 0.2% (v/v) Triton X-100 MPFSW G and 50 biotic-containing (60 mg penicillin mg strepto- was added to allow penetration of phalloidin. A stock solution liter about 6 7 h after mycin per MPFSW) to first cleavage. of phalloidin was prepared by dissolving onevial ofAlexa Fluor Some embryos were cultured in the absence ofantibiotics. During 488 phalloidin (300 U) in 1.5 ml methanol. This stock solution culturing and all experimental procedures embryos and larvae at -20°C. Some was kept 5 or 10 pi ofthe stock solution were were kept at 17 to 18°C. in small dish and the methanol put a glass petri was allowed to
evaporate. Larvae were taken in 200 pi of the 0.1 M phosphate and Cell-lineage, quadrant identification cell-lineage buffer 7.4) to (pH which 0.2% Triton X-100 was added and put tracer on the The dish injection dry phalloidin. petri was gently shakento dissolve the dried phalloidin after which the larvae were incubated for 1
h. Subsequently, the larvae were washed in 0.1 M phosphate The early development and cell-lineage of Patella have been
buffer times for 10 and in series of described before (3 min) dehydrated a graded (see e.g., Wilson, 1904; Smith, 1935; van den ethanol (70%, 80%, 90%, 96%, 100%, 100%, 100%; each Biggelaar 1977; Dictus and Damen, 1997). The nomenclature step
5 min). After transferring the larvae to a 1:1 mixture of employed is that of Wilson (1892)and Conklin (1897). For clarity, (v/v) ethanol and Murray’s (1:2 v/v benzylalcohol and benzylbenzoate), figure 2 shows some stages of the development ofPatella.
The they were mounted in convex polished slides in Murray’s to cell-lineage tracer tetramethylrhodamine dextran (MW which 0.1% ofthe antifading agent St. 10,000 Da, D-1868, Molecular Probes Europe, Leiden, Nether- n-propyl gallate (Sigma,
Louis, Mo, USA) was added. • lands; TMR-dextran) was injectedusing high pressure-injection as described previously (Damen and Dictus, 1994; Dictus and
Damen, was 1997). TMR-dextran dissolved at a concentration Confocal laser scanning microscopy (CLSM) °f 10% in dest in buffer mM (w/v) aqua or injection (10 Hepes, 150 mM KC1, 1 mM CaCI 10 mM EGTA, 7.0). , pH 2 All larvae were observed in a Leica upright confocal laser Blastomeres, viz., 2b, 3a, 3b, 3c, 3d, 3A, 3B, 3C, 4d and 4D, scanning microscope [Leica DM-RBE (microscope), Leica were injected at specific stages of development. For instance, TCSNT (confocal laser), Leica, Heidelberg, Germany], One, the 2b-micromere was injected at the 16-cell of stage. Embryos in or most cases two or even three z-series were recorded at Patella are radially symmetrical up to about 60 min after different All z-series angles. consisted of72 images and were the filth cleavage (32-cell stage). Therefore, quadrant identity transformed into stereo images that were analyzed with a pair
- (A B-, or * C-, D-quadrant) cannot be determined until after ofred-green stereo glasses. this moment den (van Biggelaar 1977; van den Biggelaar and Guerrier, 1979;Damen and Dictus, 1996). By analyzing injected larvae retrospectively, the identity of injected blastomeres was
Downloaded from Brill.com10/07/2021 11:07:18AM via free access 40 P. Damen & W.J.A. G. Dictus - Newly-discovered muscle in the larva ofPatella
Results labeledwas a post-trochally located transverse band
of fibers that was connectedto the lateral shell walls.
Fluorescent cell-lineage tracer injection This band of fibers was U-shaped with the base of
the U at the ventral side of the larva. This trans-
Early cleavage-stage blastomeres, viz., 2b, 3a, 3b, verse-oriented band of fibers strongly resembled
3c, 3d, 3A, 3B, 3C, 4d and 4D, were injected as muscle fibers. After injection of cell-lineage tracer
into the 3a- 3b-micromeres in part of a large study to trace the cell-lineage of the and (yellow Fig. 2B),
in left of musculature in Patella coerulea (Damen and Dictus, small areas the right, respectively the part
submitted). Figure 2 shows drawings of early cleav- the U-shaped transverse bandof fibers were labeled
age-stage embryos in which the localization of some as well (results not shown).
injected blastomeres is indicated. Injection into one of these blastomere, the 2b-micromere (yellow in Phalloidin staining
Fig. 2A), resulted in a complex pattern of labeling
in 48 to 52 h old post-torsional larvae (Fig. 3). In order to verify whether the transverse-oriented
with and the formed from the of the 2b-mi- Analysis CLSM subsequent analysis structure progeny of z-series revealed two large, ciliated prototroch cromere is indeed a muscle, 48 to 52 h old larvae
cells and a ring of fluorescent cells that bordered were stained with phalloidin and analyzed. The
the the side fluorescence of the main larval prototroch at post-trochal (Fig. 3A). retractor, accessory
This Medially, a strand of cells was labeled. strand larval retractor, pedal plexus, velum muscle ring,
the left adult shell ran from a ventral position, between the two la- and anlagen of the and right beled main prototroch cells, in a dorsal direction muscles was clearly visible. In addition, an unknown
towards the dorsal side of the prototroch and curved muscle was detected (Fig. 4). The morphology of
in that a posterior direction towards a position near the this muscle was exactly like of the other muscles,
visceral mass (Fig. 3). Adjacent to this strand, other although the phalloidin staining of this unknown
cells were labeled at the level of the prototroch. In muscle was weaker. This newly-discovered muscle
addition, some labeling was seen on the inside of is located between the two lateral shell walls and
The base the right-lateral side of the shell. Most prominently is U-shaped. of the “U” is located near
Fig. 2. Schematic drawings ofseveral stages in the developmentofPatella,modifiedafter van den Biggelaar (1977). (A) Lateral view
of a 16-cell stage embryo. The first quartetmicromeres (la-Id) have divided and the second quartetmicromeres are formed (2a-2d).
Lateral view of (B) a 32-cell stage embryo. In this view the first (la-!d progeny), second (2a-2d progeny) and third (3a-3d) quartet
micromeres as well as the third generation macromeres (3A-3D) are visible.
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3. Stereo Fig- images and schematic drawings of a 48 h old post-torsional larva that is retracted in its shell and in which the 2b- progeny are labeled. (A) Stereo image showing the fluorescence ofthe tracer. Animal view. cell-lineage Amongst other structures, a post-trochally located transverse band offibers (new) is labeled that is connected to the lateral shell walls (*). (B) Schematic drawing ofA. (C) Stereo image ofthe same larva showing the fluorescence ofthe cell-lineage tracer. Left-lateral view. (D) Schematic drawing
* °f C. = 0 attachment ofthe transverse band offibers to the shell; = ciliated = large, prototroch cells; x labeling on the inside ofthe
side of # = ofcells right-lateral the = shell; ring bordering the prototroch at the post-trochal side; + medially located strand of cells;
ant. = = For rest of Scale bar: 50 anterior; post. posterior. legend see figure 1. pm.
the hinge of the operculum. The. fluorescence of Discussion this muscle co-localized with the fluorescence of
the transverse-oriented band of fibers observed after Newly-discovered muscle
tracing the 2b-progeny with TMR-dextran.
The results above describe a hitherto unknownstruc-
ture in the 48 to 52 h old post-torsional larva of
'Patella coerulea that strongly resembles a muscle.
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Fig. 4. Stereo images and schematic drawings of 48 to 52 h old larvae stained with fluoresccntly-labeled phalloidin. ( A) Stereo
image. Ventral view ofa 56 h old post-torsional larva. Phalloidin staining. The unknown muscle is clearly visible (new). In the right
side of the the four fibers of the larval visible. Schematic larva, accessory retractor (air) are (B) drawing of A. (C) Stereo image.
Animal view of a 48 h old post-torsional larva. Phalloidin staining. The unknown muscle (new) is located perpendicular to and
the ventrally of the main larval retractor. The four fibers of the accessory larval retractor, located in right side of the larva, are
ofC. For 1. Scale bar: 50 indicated as well. (D) Schematic drawing legend see figure pm.
the small the In Based on morphology, the positive staining with form parts of transverse muscle. con-
phalloidin, and the resemblance to muscle fibers, trast to the other muscles in phalloidin-stained lar-
it is muscle. muscle exhibited weaker concluded that the structure is indeed a vae, the transverse staining.
Based on its location, this muscle has been named This indicates that these muscle fibers are not so
transverse muscle. heavily filled with F-actin containing myofibrils,
Analysis of cell-lineage data demonstrates that and this may explain why the transverse muscle
the transverse muscle is derived from the 2b-mi- has not been observed before by Smith (1935), Crofts
cromere. In addition, the 3a- and 3b-micromeres (1955), and Wanninger et al. (1999).
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Hypothetical modelfor larval extension the shell. Since the body of the larva functions as
a ‘hydrostatic skeleton’, this will result in extru-
The discovery ofan undescribed muscle, the trans- sion of the larval body out of the shell (see Fig. 5).
allows The function of the transverse muscle be verse muscle, us to present a hypothetical may com- model of to explain the extension of the larval body pared to squeezing meat out a sausage that is out of the larval shell (Fig. 5). Fig. 5A shows a opened on one side. In addition to the transverse
retracted larva closed The think that known fully with a operculum. muscle, we the muscle previously larva retracts into its shell and closes the opercu- as the accessory larval retractor (air) may also be lum when it is the involved in the extension disturbed, e.g. by “teasing” larva. process. We observed that
the so-called Fig. 5B shows a larva that is partly retracted. This accessory larval retractor consists of
the 1A and All is an intermediate state between fully extended four fibers (see Fig. 4C). four fibers and the fully retracted state that is also obtained reached far into the mantle and did not reach the when larvae are treated with MgCl, to relax the pedal region or the velum. Since the fibers of the muscles. When larvae left undisturbed for larval retractor all end in the the are some accessory mantle,
become extended larval cannot be involved in the time, they can fully (Fig. 5C). accessory retractor
with Because of its Since treatment MgCl, immobilizes the mus- retraction process of the larva. loca-
culature, the situation of Fig. 5B represents the tion and insertion area, we propose the opposite;
situation in the larval is resting which no muscular activity is accessory retractor involved in the present. extension process of the larva. Observation of nu-
In the 48 to 52 h old post-torsional larva of Patella merous partly and fully extended larvae demon-
coerulea, contractionof the transverse muscle, which strated that in these larvae the greater part of the inserts on the two lateral shell walls, will decrease shell is empty, i.e.,.not filled with the larva body.
the volume that the larval within This can also be in various body can occupy seen papers, e.g., Patten
Fig- 5- Hypothetical model explaining the extension ofthe larval body out of the larval shell. In this model, the main larval retractor
(mlr) the larval back into the shell. The larval with the muscle (new), the pulls body accessory retractor (air), together transverse are
of the main larval and for antagonists retractor are responsible the extension of the larval body out of the shell. Note that in the
schematic the muscles involved in retraction and extension are The the larval that drawings only depicted. gray area represents body
functions as a skeleton’. retracted larva. The main larval retractor (mlr) is contracted. The ‘hydrostatic (A) Fully accessory larval retractor (air) and the transverse muscle (new) are relaxed (extended). (B) Partly retracted larva. The main larval retractor (mlr).
(air), the (new) all accessory larval retractor and transverse muscle are partly contracted. In this intermediate state, the main larval retractor the and the larval retractor (air) and the transverse (mlr) on one hand, accessory muscle (new) on the other hand, are in
equilibrium. extended larva. The main larval (mlr) is relaxed The (C) Fully retractor (extended). accessory larval retractor (air) and the transverse muscle (new) are contracted. For see 1.'Scale bar: 50 legend figure pm.
Downloaded from Brill.com10/07/2021 11:07:18AM via free access 44 P. Damen & W.J.A.G. Dictus — Newly-discovered muscle in the larva ofPatella
(1886), Crofts (1955), and Wanninger et al. (1999). vestigations will have to provide evidence that the
In Fig. 5 [compare to e.g., Fig. 3A in the paper of transverse muscle is indeed a larval extensor and
Wanninger et al. (1999)], the insertion area of the whether a transverse muscle is a universal muscle
larval the larval shell is vis- in is in accessory retractor at gastropods or only present some species.
ible. Since the fibers of the accessory larval retrac- In Patella coerulea, the transverse muscle is
tor insert ventrally on the shell and project into the almost entirely formed from the progeny of the 2b-
mantle, and the mantle is connected to the rim of micromere. The circular muscle fibers in the Mullers
the contraction of the larval larva of the also shell, accessory retractor platyhelminth Hoploplana are
will result in extrusion of the larval body out of the derived from the 2b-micromere (Henry et ah, 2000).
circular muscle fibers homolo- shell. Since we expect that the accessory larval Possibly these are
is for the the muscle of Patella coerulea. retractor responsible major displacement gous to transverse of the larval body out of the shell, this muscle ful- Further ontogenetic research, especially in species
fills the function of a main larval extensor. The located in a phylogenetic position between the platy-
transverse muscle, which is expected to play a helminths and the gastropods, can shed more light
in the ofthe moderate role displacement larval body, on the possible homology of the circular muscle
muscle fulfills the function of an accessory larval exten- fibers in Hoploplana and the transverse in
sor. As a result, these two muscles form the an- Patella coerulea.
tagonists of the main larval retractor. According to
this the larval should be model, accessory retractor Acknowledgements renamed the main larval extensor, whereas the trans-
muscle also be called the lar- verse may accessory The authors wish to thank H.A. Wagemaker for his technical val extensor. Since no accessory larval retractor is assistance and the stafffrom the aquarium for taking care ofthe in this model, the main larval retractor present may is limpets. Last but not least, Mr. W.J. Hage thanked for his be renamed larval retractor. assistance with the CLSM. P.D. was supported by the Earth Until for the now, no satisfactorily explanation and Life Sciences foundation (ALW), which is subsidized by extension of the larval body out of the shell has the Netherlands Organisation for Scientific Research (NWO), the Netherlands. This is publication number 19 of the Dutch been given in the literature. The model presented in evolution and national research program development, grant above, which incorporates a completely different num. 805.33.430. function of the larval accessory retractor, together
with a function of the transverse muscle, solves
this problem. References
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