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  1. Abstract

    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 forA. 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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  2. Serial block-face scanning electron microscopy of the tail tip of post-metamorphic amphioxus (Branchiostoma floridae) revealed some terminal myomeres never been seen before with other techniques. The morphology of these myomeres differed markedly from the chevron shapes of their more anterior counterparts. Histologically, these odd-shaped myomeres ranged from empty vesicles bordered by undifferentiated cells to ventral sacs composed of well-developed myotome, dermatome, and sclerotome. Strikingly, several of these ventral sacs gave rise to a nipple-like dorsal projection composed either entirely of sclerotome or a mixture of sclerotome and myotome. Considered as a whole, from posterior to anterior, these odd-shaped posterior myomeres suggested that their more substantial ventral part may represent the ventral limb of a chevron, while the delicate projection represents a nascent dorsal limb. This scenario contrasts with formation of chevron-shaped myomeres along most of the antero-posterior axis. Although typical chevron formation in amphioxus is surprisingly poorly studied, it seems to be attained by a dorso-ventral extension of the myomere accompanied by the assumption of a V-shape; this is similar to what happens (at least superficially) in developing fishes. Another unusual feature of the odd-shaped posterior myomeres of amphioxus is their especially distended sclerocoels. One possible function for these might be to protect the posterior end of the central nervous system from trauma when the animals burrow into the substratum. 
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    Free, publicly-accessible full text available January 1, 2025
  3. AbstractWithin phylum Chordata, the subphylum Cephalochordata (amphioxus and lancelets) has figured large in considerations of the evolutionary origin of the vertebrates. To date, these discussions have been predominantly based on knowledge of a single cephalochordate genus (Branchiostoma), almost to the exclusion of the other two genera (Asymmetron and Epigonichthys). This uneven pattern is illustrated by cephalochordate hematology, until now known entirely from work done on Branchiostoma. The main part of the present study is to describe hemocytes in the dorsal aorta of a species of Asymmetron by serial block-face scanning electron microscopy. This technique, which demonstrates three-dimensional fine structure, showed that the hemocytes have a relatively uniform morphology characterized by an oval shape and scanty cytoplasm. Ancillary information is also included for Branchiostoma hemocytes, known from previous studies to have relatively abundant cytoplasm; our serial block-face scanning electron microscopy provides more comprehensive views of the highly variable shapes of these cells, which typically extend one or several pseudopodium-like protrusions. The marked difference in hemocyte morphology found between Asymmetron and Branchiostoma was unexpected and directs attention to investigating comparable cells in the genus Epigonichthys. A broader knowledge of the hemocytes in all three cephalochordate genera would provide more balanced insights into the evolution of vertebrate hematopoiesis. 
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  4. For neurula embryos of amphioxus (chordate subphylum Cephalochordata), the anterior region of the neural tube was studied with transmission electron microscopy. This survey demonstrated previously unreported cells, each characterized by a cilium bearing on its shaft a protruding lateral bubble packed with vesicles. Such cilia resemble those known from immature coronet cells in other chordates-namely, fishes in the Vertebrata and ascidians and appendicularians in the Tunicata. This wide occurrence of coronet-like cells raises questions about their possible homologies within the phylum Chordata. When considered at the level of the whole cell, such homology is not well supported. For example, the fish cells are generally thought to be glia, while the tunicate cells are considered to be neurons; moreover, cytoplasmic smooth endoplasmic reticulum, which is predominant in the former, is undetectable in the latter. In contrast, a more convincing case for homology can be made by limiting comparisons to the cell apices with their modified cilia. In addition to the fine-structural similarities between fishes and tunicates already mentioned, nonvisual opsins have been found associated with the vesicles in the modified cilia of both groups. Such opsins are thought to link photoreception to endocrine output controlling behavior. Further work would be needed to test the idea that the amphioxus diencephalic cells with lateral bubble cilia might similarly be opsin rich and could provide insights into the evolutionary history of the coronet cells within the phylum Chordata. 
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  5. Cephalochordates (amphioxus) are invertebrate chordates closely related to vertebrates. As they are evolving very slowly, they are proving to be very appropriate for developmental genetics studies aimed at understanding how vertebrates evolved from their invertebrate ancestors. To date, techniques for gene knockdown and overexpression have been developed, but methods for continuous breeding cultures and generating germline mutants have been developed only recently. Here we describe methods for continuous laboratory breeding cultures of the cephalochordate Branchiostoma floridae and the TALEN and Tol2 methods for mutagenesis. Included are strategies for analyzing the mutants and raising successive generations to obtain homozygotes. These methods should be applicable to any warm water species of cephalochordates with a relatively short generation time of 3-4 months and a life span of 3 years or more. 
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  6. Abstract 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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