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1 Michigan State University, East Lansing, MI
2 Union College, Schenectady, NY
* Corresponding author: hillss{at}msu.edu
Capitella sp. I is an opportunistic polychaete that is widespread and is among the early colonizers of disturbed areas that are high in organic content. Larvae are lecithotrophic and are competent to settle within a few moments of emerging from the brood tube, although they can remain viable in the water column without feeding for two or three days if environmental cues for settling are not present. Under experimental conditions larvae demonstrate a notable ability to select the appropriate environment for settlement and metamorphosis (1, 2).
Fertilized eggs develop in a brood tube, emerging after eight or nine days as segmented, motile metatrochophores. The muscular framework that will be used by the juvenile worm after metamorphosis is already in place (3), but swimming is powered by two bands of cilia, an anterior prototroch, and a posterior telotroch. A large cluster of cilia, the neurotroch, is apparent on the ventral surface in scanning electron micrographs (4). These ciliary bands are not only important in locomotion, but may also have a chemosensory role in settlement and metamorphosis (5).
This study is part of an ongoing investigation of the interaction between nerves and muscles during development and its role in locomotion. We used a monoclonal antibody against HNK-1/N-CAM, which is expressed on neuroepithelial cells in early embryos (6), as a tool to follow neural development; we have found that it is also a marker of ciliary pattern formation in capitellid larvae. In the present study, HNK-1 antibody is used to visualize the sequence of ciliary development in Capitella sp. 1.
Capitellids were cultured in filtered seawater and mud in finger bowls (J. P. Grassle, pers. comm.), and brood tubes were collected. Larvae at different developmental stages were removed from the brood tubes and fixed in 4% formaldehyde in PBS. They were then permeabilized in acetone, treated with blocking serum, and incubated in anti-HNK-1/N-CAM (Molecular Probes). This was followed by incubation in a Texas red-labeled secondary antibody. Whole mount preparations were made and observed with an Olympus BX60 fluorescence microscope and imaged with an Olympus Magnifier digital camera (model S99860).
There is no evidence of HNK-1 reactivity during cleavage or gastrulation. If larvae are removed from the brood tube immediately after gastrulation, no locomotion or distinct morphological characteristics are visible. For a brief period before they begin to swim, the larvae are able to glide slowly over the substrate. Most of the changes in patterns of fluorescence occur as the larvae acquire swimming ability.
A positive reaction is first observed in nonmotile, postgastrula larvae several days prior to emergence from the brood tube. The label is seen in the developing prototrochal region in the form of a row of sparse fluorescent dots. This is followed by a small amount of punctate fluorescence in the perianal region and in the episphere, the region anterior to the prototroch, which will become the prostomium (Fig. 1a).
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The width of the prototrochal band of fluorescence next increases significantly, although the posterior edge remains less defined than the anterior. The amount of label in the episphere continues to increase. Antibody labeling reveals a distinct perianal ring and beginning organization of the pygidial region. In contrast to the labeled prototroch, there is not yet a distinct band of telotrochal label (Fig. 1b). At this stage the larvae are able to glide on the substrate.
After reaching its maximum width, the prototrochal fluorescence begins to recede towards the initial band, which is now prominently labeled. Concurrently the neurotroch becomes evident as a midventral mass of punctate fluorescence, with the densest reactivity just posterior to the stomodeum. Label in the pygidium is becoming dense and organized except in the dorsal region where there is no fluorescence. This reflects the ciliary patterns seen in scanning electron micrographs in which the dorsal area of the pygidium in the hatched larva is devoid of cilia (4). The telotroch is now prominently labeled, forming a band of fluorescence several dots wide (Fig. 1c). Although they have not yet emerged from the brood tube, the larvae are now capable of swimming.
Next, the anterior boundary of the prototroch remains lightly labeled, and some scattered fluorescence persists in the episphere. The neurotrochal labeling is greatly diminished, while the pygidial region and telotroch are heavily labeled (Fig. 1d). By this stage, the ciliary patterning appears to be complete.
The patterns of cilia in newly emerged metatrochophores of Capitella sp. I have been described by Eckelbarger and Grassle (4). These include well-developed prototrochal and telotrochal bands, and a distinct neurotroch—a midventral cluster of cilia beginning posterior to the prototroch and terminating a short distance anterior to the telotroch. There is no apical tuft, although there are scattered cilia and mucous glands in the episphere. Discrete rays of cilia radiate from the perianal ring toward the telotroch except in the dorsal area, which is free of cilia. Our labeling, which proceeds from anterior to posterior and corresponds to these patterns, indicates that the HNK-1/N-CAM protein is being expressed sequentially as the ciliary organization is laid down. We suggest that anti-HNK-1/N-CAM labeling may prove to be an important tool for studying the development of cilia.
This work was supported in part by Michigan State University and the Union College Faculty Research Fund. The authors gratefully acknowledge the generous assistance of Dr. William Eckberg in creating the figure.
Literature Cited
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