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For postmetamorphic specimens of amphioxus (Cephalochordata), serial block-face scanning electron microscopy (SBSEM) is used to describe the long-ignored Rohde-like cells (RLCs) at the extreme posterior end of the dorsal nerve cord. These cells, numbering about three dozen in all, are divisible into a group with larger diameters running near the dorsal side of the cord and a more ventral group with smaller diameters closely associated with the central canal of the neurocoel. It is possible that the smaller ventral cells might be generated at the ependymal zone of the dorsal nerve cord and later migrate to a dorsal position, although a functional reason for this remains a mystery. All the RLCs have conspicuous regions of microvilli covering as much as 40% of their surface; limited data (by others) on the more anterior bona fide Rohde cells also indicate an extensive microvillar surface. Thus, both the RLCs and the better-known Rohde cells appear to be rhabdomeric photoreceptors, although a specific function for this feature is currently unknown. Even more perplexingly, although the Rohde cells are quintessential neurons extending giant processes, each RLC comprises a perikaryon that does not bear any neurites.

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Lancelets (Phylum Chordata, subphylum Cephalochordata) readily regenerate a lost tail. Here, we use light microscopy and serial blockface scanning electron microscopy (SBSEM) to describe tail replacement in the Bahamas lancelet, Asymmetron lucayanum. One day after amputation, the monolayered epidermis has migrated over the wound surface. At 4 days, the regenerate is about 3% as long as the tail length removed. The re-growing nerve cord is a tubular outgrowth of ependymal cells, and the new part of the notochord consists of several degenerating lamellar cells anterior to numerous small vacuolated cells. The cut edges of the mesothelium project into the regenerate as tubular extensions. These tubes anastomose with each other and with midline mesodermal canals beneath the regenerating edges of the dorsal and ventral fins. SBSEM did not reveal a blastema-like aggregation of undifferentiated cells anywhere in the regenerate. At 6 days, the regenerate (10% of the amputated tail length) includes a notochord in which the small vacuolated cells mentioned above are differentiating into lamellar cells. At 10 days, the regenerate is 22% of the amputated tail length: myocytes have appeared in the walls of the myomeres, and sclerocoels have formed. By 14 days, the regenerate is 35% the length of the amputated tail, and the new tissues resemble smaller versions of those originally lost. The present results for A. lucayanum, a species regenerating quickly and with little inter-specimen variability, provide the morphological background for future cell-tracer, molecular genetic, and genomic studies of cephalochordate regeneration.

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BACKGROUND: The cellular basis of adult growth in cephalochordates (lancelets or amphioxus) has received little attention. Lancelets and their constituent organs grow slowly but continuously during adult life. Here, we consider whether this slow organ growth involves tissue-specific stem cells. Specifically, we focus on the cell populations in the notochord of an adult lancelet and use serial blockface scanning electron microscopy (SBSEM) to reconstruct the three-dimensional fine structure of all the cells in a tissue volume considerably larger than normally imaged with this technique. RESULTS: In the notochordal region studied, we identified 10 cells with stem cell-like morphology at the posterior tip of the organ, 160 progenitor (Müller) cells arranged along its surface, and 385 highly differentiated lamellar cells constituting its core. Each cell type could clearly be distinguished on the basis of cytoplasmic density and overall cell shape. Moreover, because of the large sample size, transitions between cell types were obvious. CONCLUSIONS: For the notochord of adult lancelets, a reasonable interpretation of our data indicates growth of the organ is based on stem cells that self-renew and also give rise to progenitor cells that, in turn, differentiate into lamellar cells. Our discussion compares the cellular basis of adult notochord growth among chordates in general. In the vertebrates, several studies implied that proliferating cells (chordoblasts) in the cortex of the organ might be stem cells. However, we think it is more likely that such cells actually constitute a progenitor population downstream from and maintained by inconspicuous stem cells. We venture to suggest that careful searches should find stem cells in the adult notochords of many vertebrates, although possibly not in the notochordal vestiges (nucleus pulposus regions) of mammals, where the presence of endogenous proliferating cells remains controversial.

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Serial blockface scanning electron microscopy (SBSEM) is used to describe the sensory peripheral nervous system (PNS) in the tail of a cephalochordate, Asymmetron lucayanum. The reconstructed region extends from the tail tip to the origin of the most posterior peripheral nerves from the dorsal nerve cord. As peripheral nerves ramify within the dermis, all the nuclei along their course belong to glial cells. Invaginations in the glial cell cytoplasm house the neurites, an association reminiscent of the nonmyelinated Schwann cells of vertebrates. Peripheral nerves pass from the dermis to the epidermis via small fenestrae in the sub-epidermal collagen fibril layer; most nerves exit abruptly, but a few run obliquely within the collagen fibril layer for many micrometers before exiting. Within the epidermis, each nerve begins ramifying repeatedly, but the branches are too small to be followed to their tips with SBSEM at low magnification (previous studies on other cephalochordates indicate that the branches end freely or in association with epidermal sensory cells). In Asymmetron, two morphological kinds of sensory cells are scattered in the epidermis, usually singly, but sometimes in pairs, evidently the recent progeny of a single precursor cell. The discussion considers the evolution of the sensory PNS in the phylum Chordata. In cephalochordates, Retzius bipolar neurons with intramedullary perikarya likely correspond to the Rohon-Beard cells of vertebrates. However, extramedullary neurons originating from ventral epidermis in cephalochordates (and presumably in ancestral chordates) contrast with vertebrate sensory neurons, which arise from placodes and neural crest.

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BACKGROUND: For early larvae of amphioxus, Kaji et al. (Zool Lett 2:2, 2016) proposed that mesoderm cells are added to the rim of the forming mouth, giving it the quality of a coelomoduct without homology to the oral openings of other animals. They depended in part on non-serial transmission electron microscopic (TEM) sections and could not readily put fine structural details into a broader context. The present study of amphioxus larvae is based largely on serial blockface scanning electron microscopy (SBSEM), a technique revealing TEM-level details within an extensive anatomical volume that can be reconstructed in three dimensions. RESULTS: In amphioxus larvae shortly before mouth formation, a population of compact mesoderm cells is present at the posterior extremity of the first left somite. As development continues, the more dorsal of these cells give rise to the initial kidney (Hatschek's nephridium), while the more ventral cells become interposed between the ectoderm and endoderm in a localized region where the mouth will soon penetrate. SBSEM reveals that, after the mouth has opened, a majority of these mesoderm cells can still be detected, sandwiched between the ectoderm and endoderm; they are probably myoblasts destined to develop into the perioral muscles. CONCLUSIONS: SBSEM has provided the most accurate and detailed description to date of the tissues at the anterior end of amphioxus larvae. The present study supports the finding of Kaji et al. (2016) that the more dorsal of the cells in the posterior region of the first left somite give rise to the initial kidney. In contrast, the fate of the more ventral cells (called here the oral mesoderm) is less well understood. Although Kaji et al. (2016) implied that all of the oral mesoderm cells joined the rim of the forming mouth, SBSEM reveals that many of them are still present after mouth penetration. Even so, some of those cells go missing during mouth penetration and their fate is unknown. It cannot be ruled out that they were incorporated into the rim of the nascent mouth as proposed by Kaji et al. (2016). On the other hand, they might have degenerated or been shed from the larva during the morphogenetic interaction between the ectoderm and endoderm to form the mouth. The present SBSEM study, like Kaji et al. (2016), is based on static morphological data, and dynamic cell tracer experiments would be needed to decide among these possibilities.