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2016
Thomsen, J, Himmerkus N, Holland N, Sartoris FJ, Bleich M, Tresguerres M.  2016.  Ammonia excretion in mytilid mussels is facilitated by ciliary beating. Journal of Experimental Biology.   10.1242/jeb.139550   Abstract

The excretion of nitrogenous waste products in the form of ammonia (NH3) and ammonium (NH4 (+)) is a fundamental process in aquatic organisms. For mytilid bivalves, little is known about the mechanisms and sites of excretion. This study investigated the localization and the mechanisms of ammonia excretion in mytilid mussels. An Rh protein was found to be abundantly expressed in the apical cell membrane of the plicate organ, which was previously described as a solely respiratory organ. The Rh protein was also expressed in the gill, although at significantly lower concentrations, but was not detectable in mussel kidney. Furthermore, NH3/NH4 (+) was not enriched in the urine, suggesting that kidneys are not involved in active NH3/NH4 (+) excretion. Exposure to elevated seawater pH of 8.5 transiently reduced NH3/NH4 (+) excretion rates, but they returned to control values following 24 h acclimation. These mussels had increased abundance of V-type H(+)-ATPase in the apical membranes of plicate organ cells; however, NH3/NH4 (+) excretion rates were not affected by the V-type H(+)-ATPase specific inhibitor concanamycin A (100 nmol l(-1)). In contrast, inhibition of ciliary beating with dopamine and increased seawater viscosity significantly reduced NH3 excretion rates under control pH (8.0). These results suggest that NH3/NH4 (+) excretion in mytilid mussels takes place by passive NH3 diffusion across respiratory epithelia via the Rh protein, facilitated by the water current produced for filter feeding, which prevents accumulation of NH3 in the boundary layer. This mechanism would be energy efficient for sessile organisms, as they already generate water currents for filter feeding.

Yue, JX, Holland ND, Holland LZ, Deheyn DD.  2016.  The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus). Scientific Reports. 6   10.1038/srep28350   AbstractWebsite

Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

2015
Annona, G, Holland ND, D'Aniello S.  2015.  Evolution of the notochord. Evodevo. 6   10.1186/s13227-015-0025-3   AbstractWebsite

A notochord is characteristic of developing chordates (which comprise amphioxus, tunicates and vertebrates), and, more arguably, is also found in some other animals. Although notochords have been well reviewed from a developmental genetic point of view, there has heretofore been no adequate survey of the dozen or so scenarios accounting for their evolutionary origin. Advances in molecular phylogenetics and developmental genetics have, on the one hand, failed to support many of these ideas (although, it is not impossible that some of these rejects may yet, at least in part, return to favor). On the other hand, current molecular approaches have actually stimulated the revival of two of the old proposals: first that the notochord is a novelty that arose in the chordates, and second that it is derived from a homologous structure, the axochord, that was present in annelid-like ancestors. In the long term, choosing whether the notochord is a chordate novelty or a legacy from an ancient annelid (or perhaps an evolutionary derivative from precursors yet to be proposed) will probably require descriptions of gene regulatory networks involved in the development of notochords and notochord-like structures in a wide spectrum of animals. For now, one-way forward will be studies of all aspects of the biology of enteropneust hemichordates, a group widely thought to be the key to understanding the evolutionary origin of the chordates.

Mansfield, JH, Haller E, Holland ND, Brent AE.  2015.  Development of somites and their derivatives in amphioxus, and implications for the evolution of vertebrate somites. Evodevo. 6   10.1186/s13227-015-0007-5   AbstractWebsite

Background: Vertebrate somites are subdivided into lineage compartments, each with distinct cell fates and evolutionary histories. Insights into somite evolution can come from studying amphioxus, the best extant approximation of the chordate ancestor. Amphioxus somites have myotome and non-myotome compartments, but development and fates of the latter are incompletely described. Further, while epithelial to mesenchymal transition (EMT) is important for most vertebrate somitic lineages, amphioxus somites generally have been thought to remain entirely epithelial. Here, we examined amphioxus somites and derivatives, as well as extracellular matrix of the axial support system, in a series of developmental stages by transmission electron microscopy (TEM) and in situ hybridization for collagen expression. Results: The amphioxus somite differentiates medially into myotome, laterally into the external cell layer (a sub-dermal mesothelium), ventrally into a bud that forms mesothelia of the perivisceral coelom, and ventro-medially into the sclerotome. The sclerotome forms initially as a monolayered cell sheet that migrates between the myotome and the notochord and neural tube; subsequently, this cell sheet becomes double layered and encloses the sclerocoel. Other late developments include formation of the fin box mesothelia from lateral somites and the advent of isolated fibroblasts, likely somite derived, along the myosepta. Throughout development, all cells originating from the non-myotome regions of somites strongly express a fibrillar collagen gene, ColA, and thus likely contribute to extracellular matrix of the dermal and axial connective tissue system. Conclusions: We provide a revised model for the development of amphioxus sclerotome and fin boxes and confirm previous reports of development of the myotome and lateral somite. In addition, while somite derivatives remain almost entirely epithelial, limited de-epithelialization likely converts some somitic cells into fibroblasts of the myosepta and dermis. Ultrastructure and collagen expression suggest that all non-myotome somite derivatives contribute to extracellular matrix of the dermal and axial support systems. Although amphioxus sclerotome lacks vertebrate-like EMT, it resembles that of vertebrates in position, movement to surround midline structures and into myosepta, and contribution to extracellular matrix of the axial support system. Thus, many aspects of the sclerotome developmental program evolved prior to the origin of the vertebrate mineralized skeleton.

Holland, ND, Holland LZ, Holland PWH.  2015.  Scenarios for the making of vertebrates. Nature. 520:450-455.   10.1038/nature14433   AbstractWebsite

Over the past 200 years, almost every invertebrate phylum has been proposed as a starting point for evolving vertebrates. Most of these scenarios are outdated, but several are still seriously considered. The short-range transition from ancestral invertebrate chordates (similar to amphioxus and tunicates) to vertebrates is well accepted. However, longer-range transitions leading up to the invertebrate chordates themselves are more controversial. Opinion is divided between the annelid and the enteropneust scenarios, predicting, respectively, a complex or a simple ancestor for bilaterian animals. Deciding between these ideas will be facilitated by further comparative studies of multicellular animals, including enigmatic taxa such as xenacoelomorphs.

Holland, ND, Holland LZ, Heimberg A.  2015.  Hybrids between the Florida amphioxus (Branchiostoma floridae) and the Bahamas lancelet (Asymmetron lucayanum): Developmental morphology and chromosome counts. Biological Bulletin. 228:13-24. AbstractWebsite

The cephalochordate genera Branchiostoma and Asymmetron diverged during the Mesozoic Era. In spite of the long separation of the parental clades, eggs of the Florida amphioxus, B. floridae, when fertilized with sperm of the Bahamas lancelet, A. lucayanum (and vice versa), develop through embryonic and larval stages. The larvae reach the chordate phylotypic stage (i.e., the pharyngula), characterized by a dorsal nerve cord, notochord, perforate pharynx, and segmented trunk musculature. After about 2 weeks of larval development, the hybrids die, as do the A. lucayanum purebreds, although all were eating the same algal diet that sustains B. floridae purebreds through adulthood in the laboratory; it is thus unclear whether death of the hybrids results from incompatible parental genomes or an inadequate diet. The diploid chromosome count in A. lucayanum and B. floridae purebreds is, respectively, 34 and 38, whereas it is 36 in hybrids in either direction. The hybrid larvae exhibit several morphological characters intermediate between those of the parents, including the size of the preoral ciliated pit and the angles of deflection of the gill slits and anus from the ventral midline. Based on the time since the two parent clades diverged (120 or 160 million years, respectively, by nuclear and mitochondrial gene analysis), the cross between Branchiostoma and Asymmetron is the most extreme example of hybridization that has ever been unequivocally demonstrated among multicellular animals.

Mansfield, JH, Holland ND.  2015.  Amphioxus tails: source and fate of larval fin rays and the metamorphic transition from an ectodermal to a predominantly mesodermal tail. Acta Zoologica. 96:117-125.   10.1111/azo.12058   AbstractWebsite

It was previously discovered that tail fin rays of larval amphioxus are long ciliary rootlets in posterior epidermal cells. This work describes the heretofore unknown origin and fate of these organelles in the Florida amphioxus (Branchiostoma floridae), In late embryos, epidermal cells at the posterior end of the body increase in height, thus producing a tail fin. One ciliary rootlet in each cell elongates and also rotates through about 90 degrees, soon becoming oriented parallel to the long axis of the cell and running continuously from the apical to the basal plasma membrane. During the subsequent growth of the larval tail, the rootlets and epidermal cells housing them reach lengths up to 120 mu m. At metamorphosis, the rootlets become vacuolated and rapidly decrease in length along with the height of the tail epidermis. Contemporaneously, abundant extracellular dermal matrix accumulates in the sagittal plane of the body to produce a predominantly dermal tail fin. Throughout postmetamorphic life, the posterior epidermal cells, now without ciliary rootlets, thinly cover a largely dermal tail flange. Thus, the specialized morphology of the arnphioxus tail fin is generated by two different cellular mechanisms, involving different cell populations (ectoden-nal and mesodermal), at different life-history stages

Holland, ND.  2015.  Nervous systems and scenarios for the invertebrate-to-vertebrate transition. Philosophical Transactions of the Royal Society B-Biological Sciences. 371   10.1098/rstb.2015.0047   AbstractWebsite

Older evolutionary scenarios for the origin of vertebrates often gave nervous systems top billing in accordance with the notion that a big-brained Homo sapiens crowned a tree of life shaped mainly by progressive evolution. Now, however, tree thinking positions all extant organisms equidistant from the tree's root, and molecular phylogenies indicate that regressive evolution is more common than previously suspected. Even so, contemporary theories of vertebrate origin still focus on the nervous system because of its functional importance, its richness in characters for comparative biology, and its central position in the two currently prominent scenarios for the invertebrate-to-vertebrate transition, which grew out of the markedly neurocentric annelid and enteropneust theories of the nineteenth century. Both these scenarios compare phyla with diverse overall body plans. This diversity, exacerbated by the scarcity of relevant fossil data, makes it challenging to establish plausible homologies between component parts (e.g. nervous system regions). In addition, our current understanding of the relation between genotype and phenotype is too preliminary to permit us to convert gene network data into structural features in any simpleway. These issues are discussed here with special reference to the evolution of nervous systems during proposed transitions from invertebrates to vertebrates.

2013
Holland, ND, Osborn KJ, Gebruk AV, Rogacheva A.  2013.  Rediscovery and augmented description of the HMS 'Challenger' acorn worm (Hemichordata, Enteropneusta), Glandiceps abyssicola, in the equatorial Atlantic abyss. Journal of the Marine Biological Association of the United Kingdom. 93:2197-2205.   10.1017/s0025315413000684   AbstractWebsite

A 2009 oceanographic expedition of the Russian Academy of Sciences collected the anterior region of a single acorn worm (phylum Hemichordata, class Enteropneusta) by trawling at a depth of 5560 m in the Romanche Trench (equatorial Atlantic). The specimen was a ripe female with numerous, relatively small oocytes in each ovary. Phylogenetic analysis of rDNA sequences robustly placed the worm in the family Spengelidae. In addition, morphological features of the proboscis, collar, and anterior trunk region indicated that the worm was Glandiceps abyssicola, a species previously represented solely by the holotype, which had been dredged from the equatorial Atlantic in 1873 by the HMS 'Challenger' and subsequently sent to Germany for description by Spengel (1893). The holotype was presumably destroyed by World War II bombing; therefore, we here designate the Romanche Trench specimen as the neotype of G. abyssicola and supply an augmented species diagnosis.

Osborn, KJ, Gebruk AV, Rogacheva A, Holland ND.  2013.  An externally brooding acorn worm (Hemichordata, Enteropneusta, Torquaratoridae) from the Russian arctic. Biological Bulletin. 225:113-123. AbstractWebsite

A single specimen of a previously undescribed acorn worm in the family Torquaratoridae was trawled from a bottom depth of about 350 m in the Kara Sea (Russian Arctic). The new species is the shallowest of the exclusively deep- sea torquaratorids found to date, possibly an example of high- latitude emergence. On the basis of ribosomal DNA sequences and morphology, the worm is described here as the holotype of Coleodesmium karaensis n. gen., n. sp. It is most similar in overall body shape to the previously described enteropneust genus Allapasus, but is uniquely characterized by a tubular component of the proboscis skeleton ensheathing the collar nerve cord. Additionally, within the proboscis, the sparseness of the musculature of C. karaensis clearly distinguishes it from the much more muscular members of Allapasus. The holotype is a female bearing about a dozen embryos on the surface of her pharyngeal region, each recessed within a shallow depression in the dorsal epidermis. The embryos, ranging from late gastrula to an early stage of coelom formation, are a little more than 1 mm in diameter and surrounded by a thin membrane. Each embryo comprises an external ectoderm of monociliated cells (not arranged in obvious ciliated bands) and an internal endo- mesoderm; the blastopore is closed. In the most advanced embryos, the anterior coelom is starting to constrict off from the archenteron. Coleodesmium karaensis is the first enteropneust (and indeed the first hemichordate) found brooding embryos on the surface of the mother's body.

Mallatt, J, Holland N.  2013.  Pikaia gracilens Walcott: stem chordate, or already specialized in the Cambrian? Journal of Experimental Zoology Part B-Molecular and Developmental Evolution. 320B:247-271.   10.1002/jez.b.22500   AbstractWebsite

For the past 35 years, the Cambrian fossil Pikaia gracilens was widely interpreted as a typical basal chordate based on short descriptions by Conway Morris. Recently, Conway Morris and Caron (CMC) (2012, Biol Rev 87:480512) described Pikaia extensively, as a basis for new ideas about deuterostome evolution. This new Pikaia has characters with no clear homologues in other animals, so they could be phylogenetically uninformative autapomorphies. These characters include a dorsal organ, posterior ventral area, posterior fusiform structure, and anterior dorsal unit. Yet CMC interpret most of the unusual characters as primitive for chordates, thereby interpreting Pikaia as an even more convincing stem chordate than before. Moreover, they claim that segment (myomere) shape is a reliable guide for defining a chordate and even for assigning animals to their correct place in deuterostome phylogeny. By defining sigmoidal segments as a basal chordate character, they situate Pikaia at the base of the chordates and banish fossil yunnanozoans (which have straight segments) to a position deep within the deuterostomes. In addition, they consider amphioxus, with its conspicuously chevron-shaped segments, to be so highly derived that it is of little use for reconstructing the first chordates. We question their overemphasis on the phylogenetic value of segment shape and their marginalizing of amphioxus. We deduce that Pikaia, not amphioxus, is specialized. We performed a cladistic analysis that showed the character interpretations of CMC are consistent with their wide-ranging evolutionary scenario, but that these interpretations leave unresolved the position of Pikaia within chordates. J. Exp. Zool. (Mol. Dev. Evol.) 320B:247271, 2013. (c) 2013 Wiley Periodicals, Inc.

Holland, ND.  2013.  Digestive system. Sea urchins: biology and ecology. 38( Lawrence JM, Ed.).:119-133., Amsterdam: Elsevier Abstract
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2012
Holland, ND, Osborn KJ, Kuhnz LA.  2012.  A new deep-sea species of harrimaniid enteropneust (Hemichordata). Proceedings of the Biological Society of Washington. 125:228-240. AbstractWebsite

Ninety-two individuals of a deep-sea harrimaniid enteropneust were imaged between 1675 m and 3225 m off the California coast. Of these, about three-fourths were positioned with their posterior regions buried in sediment or hidden by rocks, and the rest were completely exposed on the substratum. When visible, the posterior end of each worm was typically associated with a dense tangle of fecal strands. One specimen was captured and is described here as the holotype of Saxipendium implication. In life, it was 22 cm long, and the color of its dome-shaped proboscis, narrow collar, and anterior trunk was medium orange. No wing-like folds of the body wall protruded anywhere along the length of the worm. The proboscis complex included a stomochord and glomeruli, but neither a heart nor a pericardial cavity could be detected. Most of the dorsal collar nerve runs along an open invagination in the dorsal midline of the collar and is only roofed over very briefly at the posterior extremity of the collar. Another unusual feature is the exaggerated posterior extension of the horns of the proboscis skeleton, which projected into the anterior extremity of the trunk. The trunk commenced anteriorly with a pharyngeal/esophageal region that included a tract of ovaries on either side of the dorsal midline. The ripest ovaries contained a single oocyte approximately 700 pm in diameter (presumably this species is gonochoric, although no males have yet been collected). The gill skeleton lacked synapticles. More posteriorly, the trunk housed a long, darkly pigmented hepatic intestine without sacculations and a short, lightly pigmented post-hepatic intestine. The geographic range of S. implicatum appears to be restricted to the Davidson, Guide, and Taney Seamounts region in the eastern Pacific offshore of Central California.

Holland, ND, Kuhnz LA, Osborn KJ.  2012.  Morphology of a new deep-sea acorn worm (class Enteropneusta, phylum Hemichordata): A part-time demersal drifter with externalized ovaries. Journal of Morphology. 273:661-671.   10.1002/jmor.20013   AbstractWebsite

Ten individuals of an enteropneust in the family Torquaratoridae were videotaped between 2,900 and 3,500 m in the Eastern Pacificone drifting a few centimeters above the bottom, two exposed on the substrate, and seven partly burrowed, reflecting a bentho-pelagic life style. Here, we describe a captured specimen (26 cm living length) as the holotype of Allapasus aurantiacus n. gen., n. sp. The small proboscis is dome-shaped, and the collar is only slightly wider than deep; both of these body regions are more muscular than in other torquaratorids, which presumably facilitates burrowing. The proboscis complex, in contrast to that of shallow-living enteropneusts, lacks a pericardial sac and is located relatively posteriorly in the proboscis stalk. The stomochord is separated from the main course of the gut by the intervention of a small, plate-like proboscis skeleton lacking posterior horns. The most anterior region of the trunk houses the pharynx, in which the pharyngeal skeletal bars are not connected by synapticles. The postpharyngeal trunk comprises three intestinal regions: prehepatic, hepatic (with conspicuous sacculations), and posthepatic. On either side of the worm, a flap of body wall (lateral wing) runs the entire length of the trunk. The two lateral wings can wrap the body so their edges meet in the dorsal midline, although they often gape open along the pharyngeal region. The holotype is a female (presumably the species is gonochoric) with numerous ovaries located in the lateral wings along the pharyngeal region. Each larger ovary contains a single primary oocyte (up to 1,500 mu m in diameter) and bulges outwards in an epidermal pouch attached to the rest of the body by a slender stalk. Such externalized ovaries are unprecedented in any animal, and nothing is yet known of their role in the reproductive biology of A. aurantiacus. J. Morphol. 2012. (c) 2012 Wiley Periodicals, Inc.

Osborn, KJ, Kuhnz LA, Priede IG, Urata M, Gebruk AV, Holland ND.  2012.  Diversification of acorn worms (Hemichordata, Enteropneusta) revealed in the deep sea. Proceedings of the Royal Society B-Biological Sciences. 279:1646-1654.   10.1098/rspb.2011.1916   AbstractWebsite

Enteropneusts (phylum Hemichordata), although studied extensively because of their close relationship to chordates, have long been considered shallow-water, burrowing animals. The present paper more than doubles the number of enteropneust species recorded in the deep sea based on high-resolution imaging and sampling with remotely operated vehicles. We provide direct evidence that some enteropneusts are highly mobile-using changes in posture and currents to drift between feeding sites-and are prominent members of deep, epibenthic communities. In addition, we provide ecological information for each species. We also show that despite their great morphological diversity, most deep-living enteropneusts form a single clade (the rediagnosed family Torquaratoridae) on the basis of rDNA sequences and morphology of the proboscis skeleton and stomochord. The phylogenetic position of the torquaratorids indicates that the group, after evolving from near-shore ancestors, radiated extensively in the deep sea.

Priede, IG, Osborn KJ, Gebruk AV, Jones D, Shale D, Rogacheva A, Holland ND.  2012.  Observations on torquaratorid acorn worms (Hemichordata, Enteropneusta) from the North Atlantic with descriptions of a new genus and three new species. Invertebrate Biology. 131:244-257.   10.1111/j.1744-7410.2012.00266.x   AbstractWebsite

Enteropneusts in the family Torquaratoridae were imaged using still and video cameras in the deep North Atlantic and then collected by remotely operated vehicles. From this material, we describe Yoda purpurata n. gen, n. sp., Tergivelum cinnabarinum n. sp., and Allapasus isidis n. sp. Individuals of the first two species were browsing completely exposed on the sea floor, whereas the specimen of the last species was encountered floating similar to 1 similar to m above the sea floor. Living specimens of Y. purpurata were 1219 similar to cm long and had a dark reddish-purple proboscis, collar, and genital wings (folded dorsally over the anterior region of the trunk). Members of this species were hermaphrodites (the first ever discovered in the phylum Hemichordata), with numerous separate testes and ovaries in the genital wings. Living specimens of T. cinnabarinum were 1226 similar to cm long and had a cinnabar-colored proboscis, collar, and back veils (arising from the anterior region of the trunk); sexes were separate, and body shape and internal morphology closely resemble those of its brown congener, T. baldwinae, from the eastern Pacific. The only specimen of A. isidis collected was a male 13 similar to cm long and pale yellow when alive. Its body shape was proportionally shorter and broader than that of its orange congener, A. aurantiacus, from the eastern Pacific, but the internal anatomy of the two species is virtually identical. [Correction made after online publication August 21, 2012 to correct species name in preceding sentence.]

2011
Koop, D, Holland LZ, Setiamarga D, Schubert M, Holland ND.  2011.  Tail regression induced by elevated retinoic acid signaling in amphioxus larvae occurs by tissue remodeling, not cell death. Evolution & Development. 13:427-435.   10.1111/j.1525-142X.2011.00501.x   AbstractWebsite

The vitamin A derived morphogen retinoic acid (RA) is known to function in the regulation of tissue proliferation and differentiation. Here, we show that exogenous RA applied to late larvae of the invertebrate chordate amphioxus can reverse some differentiated states. Although treatment with the RA antagonist BMS009 has no obvious effect on late larvae of amphioxus, administration of excess RA alters the morphology of the posterior end of the body. The anus closes over, and gut contents accumulate in the hindgut. In addition, the larval tail fin regresses, although little apoptosis takes place. This fin normally consists of columnar epidermal cells, each characterized by a ciliary rootlet running all the way from an apical centriole to the base of the cell and likely contributing substantial cytoskeletal support. After a few days of RA treatment, the rootlet becomes disrupted, and the cell shape changes from columnar to cuboidal. Transmission electron microscopy (TEM) shows fragments of the rootlet in the basal cytoplasm of the cuboidal cell. A major component of the ciliary rootlet in amphioxus is the protein Rootletin, which is encoded by a single AmphiRootletin gene. This gene is highly expressed in the tail epithelial cells of control larvae, but becomes downregulated after about a day of RA treatment, and the breakup of the ciliary rootlet soon follows. The effect of excess RA on these epidermal cells of the larval tail in amphioxus is unlike posterior regression in developing zebrafish, where elevated RA signaling alters connective tissues of mesodermal origin. In contrast, however, the RA-induced closure of the amphioxus anus has parallels in the RA-induced caudal regression syndrome of mammals.

Holland, ND.  2011.  Walter Garstang: a retrospective. Theory in Biosciences. 130:247-258.   10.1007/s12064-011-0130-3   AbstractWebsite

Although, Walter Garstang died over 60 years ago, his work is still cited-sometimes praised, but sometimes belittled. On the negative side, he often appropriated ideas of others without attribution, ignored earlier studies conflicting with his theories, and clung to notions like inheritance of acquired characters, progressive evolution, and saltation after many of his contemporaries were advancing toward the modern synthesis. Moreover, his evolutionary scenarios-especially his derivation of vertebrates from a sessile ascidian-have not been well supported by recent work in developmental genetics and molecular phylogenetics. On the positive side, Garstang firmly established several points of view that remain useful in the age of evolutionary development (evo-devo). He popularized the valid idea that adaptive changes in larvae combined with shifts in developmental timing (heterochrony) could radically change adult morphology and provide an escape from overspecialization. Moreover, his re-statement of the biogenetic law is now widely accepted: namely, that recapitulation results when characters at one stage of development are required for the correct formation of other characters at subsequent stages (his stepping stone model). In other words, ontogeny creates phylogeny because some developmental features are constraints, favoring particular evolutionary outcomes while excluding others. This viewpoint is a useful basis for advancing concepts of homology and for comparing the phylogeny of ontogenies across a series of animals to ascertain the timing and the nature of the underlying ontogenetic changes.

Holland, ND.  2011.  Spawning periodicity of the lancelet, Asymmetron lucayanum (Cephalochordata), in Bimini, Bahamas. Italian Journal of Zoology. 78:478-486.   10.1080/11250003.2011.594097   AbstractWebsite

Developmental genetic studies of cephalochordates, which give evolutionary insights into the origin of the vertebrates from the invertebrates, require a supply of embryos and larvae. To date, such studies have been limited to the genus Branchiostoma. My purpose here is to establish a practical method for obtaining developmental stages of Asymmetron, a second and relatively inaccessible cephalochordate genus. Reproductive periodicity of Asymmetron lucayanum was studied for a total of 1626 specimens in 22 collections made between November 2008 and December 2010 in the lagoon at Bimini, Bahamas. Water temperatures at the collection site were also measured. The lancelets were collected by sieve at early afternoon low tides and transported to the laboratory for determination of sex, body length, gonad index, and capacity to spawn (by dark stimulation on the evening of collection). The sex ratio did not differ significantly from 1: 1, and the minimum length at sexual maturity was about 10 mm. During spring and autumn, when water temperatures were moderate (mid to high 20s degrees C), lancelets placed in the dark would spawn predominantly 1 day before the date of the new moon. In contrast, during the winter and summer, dark stimulation did not induce spawning, regardless of the moon phase. It is likely that spawning is depressed in months when water temperatures are near their annual maxima and minima. The linkage of temperature and moon phase to spawning raises the possibility that cultures of A. lucayanum maintained in the laboratory under appropriate environmental conditions could provide a year-round, on-demand source of cephalochordate embryos and larvae.

2010
Holland, ND, Holland LZ.  2010.  Laboratory spawning and development of the Bahama lancelet, Asymmetron lucayanum (Cephalochordata): fertilization through feeding larvae. Biological Bulletin. 219:132-141. AbstractWebsite

Here we report on spawning and development of the Bahama lancelet, Asymmetron lucayanum. Ripe adults collected in Bimini spawned the same evening when placed in the dark for 90 minutes. The developmental morphology is described from whole mounts and histological sections. A comparison between development in A symmetron and the better known cephalochordate genus Branchiostoma reveals similarities during the early embryonic stages but deviations by the late embryonic and early larval stages. Thus, the initial positions of the mouth, first gill slit, and anus differ between the two genera. Even more strikingly, Hatschek's right and left diverticula, which arise by enterocoely at the anterior end of the pharynx in Branchiostoma, never form during Asymmetron development. In Branchiostoma, these diverticula become the rostral coelom and preoral pit. In Asymmetron, by contrast, homologs of the rostral coelom and preoral pit form by schizocoely within an anterior cell cluster of unproven (but likely endodermal) origin. Proposing evolutionary scenarios to account for developmental differences between Asymmetron and Branchiostoma is currently hampered by uncertainty over which genus is basal in the cephalochordates. A better understanding of developmental diversity within the cephalochordates will require phylogenetic analyses based on nuclear genes and the genome sequence of an Asymmetron species.

Koop, D, Holland ND, Semon M, Alvarez S, de Lera AR, Laudet V, Holland LZ, Schubert M.  2010.  Retinoic acid signaling targets Hox genes during the amphioxus gastrula stage: Insights into early anterior-posterior patterning of the chordate body plan. Developmental Biology. 338:98-106.   10.1016/j.ydbio.2009.11.016   AbstractWebsite

Previous studies of vertebrate development have shown that retinoic acid (RA) signaling at the gastrula stage strongly influences anterior-posterior (A-P) patterning of the neurula and later stages. However, much less is known about the more immediate effects of RA signaling on gene transcription and developmental patterning at the gastrula stage. To investigate the targets of RA signaling during the gastrula stage, we used the basal chordate amphioxus, in which gastrulation involves very minimal tissue movements. First, we determined the effect of altered RA signaling on expression of 42 genes (encoding transcription factors and components of major signaling cascades) known to be expressed in restricted domains along the A-P axis during the gastrula and early neurula stage. Of these 42 genes, the expression domains during gastrulation of only four (Hox1, Hox3, HNF3-1 and Wnt3) were spatially altered by exposure of the embryos to excess RA or to the RA antagonist BMS009. Moreover, blocking protein synthesis with puromycin before adding RA or BMS009 showed that only three of these genes (Hox1, Hox3 and HNF3-1) are direct RA targets at the gastrula stage. From these results we conclude that in the amphioxus gastrula RA signaling primarily acts via regulation of Hox transcription to establish positional identities along the A-P axis and that Hox1, Hox3, HNF3-1 and Wnt3 constitute a basal module of RA action during chordate gastrulation. (C) 2009 Elsevier Inc. All rights reserved.

2009
Holland, ND, Paris M, Koop D.  2009.  The club-shaped gland of amphioxus: export of secretion to the pharynx in pre-metamorphic larvae and apoptosis during metamorphosis. Acta Zoologica. 90:372-379.   10.1111/j.1463-6395.2008.00379.x   AbstractWebsite

In amphioxus larvae, the club-shaped gland is a tube connecting the pharyngeal lumen with the external environment. The functions of the gland and its fate during the larva-to-juvenile metamorphosis have long been controversial. Here we use a fixative including ruthenium red to preserve extracellular secretions (presumably glycoproteins) in late pre-metamorphic larvae. This procedure reveals reddish, fibrogranular material in the lumen of the club-shaped gland and in the pharynx adjacent to the gland's inner opening. This finding strengthens the idea that secretions of the club-shaped gland are exported to the pharyngeal lumen to help form a mucous trap for capturing food particles entering the mouth. We also use the terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay to study apoptosis in the tissues of metamorphosing larvae. One of the earliest events of metamorphosis is the massive apoptotic destruction of the club-shaped gland. Therefore, despite some previous opinions to the contrary, the cells of the gland do not survive to participate in the genesis of the definitive endostyle or any other post-larval structures.

Holland, ND, Wilson NG.  2009.  Molecular identification of larvae of a tetraphyllidean tapeworm (Platyhelminthes: Eucestoda) in a razor clam as an alternative intermediate host in the life cycle of Acanthobothrium brevissime. Journal of Parasitology. 95:1215-1217.   10.1645/ge-1946.1   AbstractWebsite

Dwarf razor clams (Ensis minor) in the Gulf of Mexico lire known to be infected with plerocercoid larvae of it tetraphyllidean tapeworm. Here, we show that these larvae live unencysted in the intestinal lumen of the clam. Morphologically, the larvae lire similar to (although significantly larger than) tapeworm larvae previously described living in the gut of amphioxus (Branchiostoma floridae) from the same habitat. Sequence data from the D2 region of the 28S rDNA from clam-infecting larvae were identical to the sequence of Acanthobothrium brevissime isolated as larvae from amphioxus and its adults from a stingray (Dasyatis say). The sequence data leave little doubt that the dwarf razor clam and the amphioxus are alternative intermediate hosts in the life cycle of A. brevissime.

Kaltenbach, SL, Yu JK, Holland ND.  2009.  The origin and migration of the earliest-developing sensory neurons in the peripheral nervous system of amphioxus. Evolution & Development. 11:142-151.   10.1111/j.1525-142X.2009.00315.x   AbstractWebsite

In amphioxus embryos, the early development of the type 1 sensory neurons was followed by Tlx gene expression and scanning electron microscopy. At the early neurula stage, AmphiTlx transcription is first detectable in a ventral zone of epidermis. In later embryos, expression is also seen in differentiating type 1 receptor cells of the peripheral nervous system, in a few cells in the central nervous system (CNS), and in anterior notochord cells. The type 1 receptors originate from the ventral epidermis by delaminating individually into the subepidermal space, where they can conveniently be visualized because of their strong expression of AmphiTlx. Within the subepidermal space, the apex of each delaminated receptor loses its cilium and microvilli whereas the base of the cell extends lamellate pseudopodia on the surface of the deeper tissues. The pseudopodia become oriented toward the dorsal side of the embryo, the direction of cell migration. After reaching the flanks of the embryo, the receptor cell stops migrating, loses its pseudopodia, and produces an axon that extends to the CNS. As the receptor finishes differentiating, it downregulates Tlx expression and evidently re-inserts its perikaryon into the epidermis. The discussion considers the possible homology of the type 1 receptor neurons of amphioxus with migrating lateral line primordia and hair cells of vertebrates.

Kaltenbach, SL, Holland LZ, Holland ND, Koop D.  2009.  Developmental expression of the three iroquois genes of amphioxus (BfIrxA, BfIrxB, and BfIrxC) with special attention to the gastrula organizer and anteroposterior boundaries in the central nervous system. Gene Expression Patterns. 9:329-334.   10.1016/j.gep.2009.02.003   AbstractWebsite

Here we describe the developmental expression of the three iroquois genes (BfIrxA, BfIrxB, and BfIrxC) of amphioxus. BfIrxB transcription is first detected at the gastrula stage in mesendoderm just within the dorsal lip of the blastopore (a probable homolog of Spemann's organizer) and in ectoderm. In early neurulae, expression begins in presumptive pharyngeal endoderm, somitic mesoderm, and neural plate. Mid-neurulae express BfIrxB throughout the hindbrain, posterior somites, pharyngeal endoderm, and notochord. In early larvae, expression is largely downregulated in the nerve cord, somites and notochord, but remains strong in the pharyngeal endoderm associated with the forming gill slits; also, a late expression domain appears in the ciliary tuft ectoderm. BfIrxA and BpIrxC, are not as widely expressed as BfIrxB. Both are first expressed in the presumptive hindbrain and presumptive pharyngeal endoderm at the early neurula stages. In the mid-neurula, additional expression domains appear in the extremities of the notochord. Neural expression is downregulated by late neurula. In the early larva, expression is chiefly limited to pharyngeal endoderm associated with the forming gill slits, excepting a small new domain of BfIrxC (not BfIrxA) expression in the ciliary tuft ectoderm. In comparison to developing vertebrates, embryos and larvae of amphioxus express iroquois genes in fewer tissues. Thus, iroquois genes of the proximate ancestor of the vertebrates evidently assumed numerous new roles during vertebrate evolution. including the division of the central nervous system into several sub-regions along its anteroposterior axis. (C) 2009 Published by Elsevier B.V.