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Holland, ND, Holland LZ.  2017.  The ups and downs of amphioxus biology: a history. International Journal of Developmental Biology. 61:575-583.   10.1387/ijdb.160395LH   AbstractWebsite

Humans (at least a select few) have long known about the cephalochordate amphioxus, first as something to eat and later as a subject for scientific study. The rate of publication on these animals has waxed and waned several times. The first big surge, in the late nineteenth century, was stimulated by Darwin's evolutionary ideas and by Kowalevsky's embryologic findings suggesting that an amphioxus-like creature might have bridged the gap between the invertebrates and the vertebrates. Interest declined sharply in the early twentieth century and remained low for the next 50 years. An important contributing factor (in addition to inhibition by two world wars and the Great Depression) was the indifference of the new evolutionary synthesis toward broad phylogenetic problems like the origin of the vertebrates. Then, during the 1960s and 1970s, interest in amphioxus resurged, driven especially by increased government support for basic science as well as opportunities presented by electron microscopy. After faltering briefly in the 1980s (electron microscopists were running out of amphioxus tissues to study), a third and still-continuing period of intensive amphioxus research began in the early 1990s, stimulated by the advent of evolutionary developmental biology (evo-devo) and genomics. The volume of studies peaked in 2008 with the publication of the genome of the Florida amphioxus. Since then, although the number of papers per year has dropped somewhat, sequencing of additional genomes and transcriptomes of several species of amphioxus (both in the genus Branchiostoma and in a second genus, Asymmetron) is providing the raw material for addressing the major unanswered question of the relationship between genotype and phenotype.

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.

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.

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.

Holland, ND, Campbell TG, Garey JR, Holland LZ, Wilson NG.  2009.  The Florida amphioxus (Cephalochordata) hosts larvae of the tapeworm Acanthobothrium brevissime: natural history, anatomy and taxonomic identification of the parasite. Acta Zoologica. 90:75-86.   10.1111/j.1463-6395.2008.00343.x   AbstractWebsite

Holland, N. D., Campbell, T. G., Garey, J. R., Holland, L. Z. and Wilson, N. G. 2009. The Florida amphioxus (Cephalochordata) hosts larvae of the tapeworm Acanthobothrium brevissime: natural history, anatomy and taxonomic identification of the parasite. - Acta Zoologica (Stockholm) 90: 75-86. Plerocercoid larvae of a tapeworm are frequently found in the hindgut lumen of the Florida amphioxus (Branchiostoma floridae) in central west Florida. About three-quarters of the adult amphioxus are parasitized. On average, each adult amphioxus hosts about five tapeworm larvae. The residence time of the parasites in the amphioxus gut appears to be in the order of several months, which is considerably shorter than the potential lifetime of the host. The living larvae range in length (when fully extended) from 300 to 850 mu m and are approximately cone-shaped, tapering to a point posteriorly and bearing a single large sucker anteriorly. Toward the anterior end of the body are four hookless bothridia, each indented by three loculi plus an inconspicuous accessory sucker. The larvae initiate the early stages of hook formation when they are cultured for a few days in urea-saline (mimicking the gut fluid of the definitive host, which is an elasmobranch). The tapeworm larvae are identifiable to genus and species on the basis of correspondences between their nuclear ribosomal DNA genes and those of adult specimens of Acanthobothrium brevissime recovered from the spiral valve of a stingray from the same environment.

Holland, LZ, Short S.  2008.  Gene Duplication, Co-Option and Recruitment during the Origin of the Vertebrate Brain from the Invertebrate Chordate Brain. Brain Behavior and Evolution. 72:91-105.   10.1159/000151470   AbstractWebsite

The brain of the basal chordate amphioxus has been compared to the vertebrate diencephalic forebrain, midbrain, hindbrain and spinal cord on the basis of the cell architecture from serial electron micrographs and patterns of developmental gene expression. In addition, genes specifying the neural plate and neural plate border as well as Gbx and Otx, that position the midbrain/hindbrain boundary (MHB), are expressed in comparable patterns in amphioxus and vertebrates. However, migratory neural crest is lacking in amphioxus, and although it has homologs of the genes that specify neural crest, they are not expressed at the edges of the amphioxus neural plate. Similarly, amphioxus has the genes that specify organizer properties of the MHB, but they are not expressed at the Gbx/Otx boundary as in vertebrates. Thus, the genetic machinery that created migratory neural crest and an MHB organizer was present in the ancestral chordate, but only co-opted for these new roles in vertebrates. Analyses with the amphioxus genome project strongly support the idea of two rounds of whole genome duplication with subsequent gene losses in the vertebrate lineage. Duplicates of developmental genes were preferentially retained. Although some genes apparently acquired roles in neural crest prior to these genome duplications, other key genes (e. g., FoxD3 in neural crest and Wnt1 at the MHB) were recruited into the respective gene networks after one or both genome duplications, suggesting that such an expansion of the genetic toolkit was critical for the evolution of these structures. The toolkit has also increased by alternative splicing. Contrary to the general rule, for at least one gene family with key roles in neural crest and the MHB, namely Pax genes, alternative splicing has not decreased subsequent to gene duplication. Thus, vertebrates have a much larger number of proteins available for mediating new functions in these tissues. The creation of new splice forms typically changes protein structure more than evolution of the protein after gene duplication. The functions of particular isoforms of key proteins expressed at the MHB and in neural crest have only just begun to be studied. Their roles in modulating gene networks may turn out to rival gene duplication for facilitating the evolution of structures such as neural crest and the MHB. Copyright (c) 2008 S. Karger AG, Basel

Kozmik, Z, Holland ND, Kreslova J, Oliveri D, Schubert M, Jonasova K, Holland LZ, Pestarino M, Benes V, Candiani S.  2007.  Pax-Six-Eya-Dach network during amphioxus development: Conservation in vitro but context specificity in vivo. Developmental Biology. 306:143-159.   10.1016/j.ydbio.2007.03.009   AbstractWebsite

The Drosophila retinal determination gene network occurs in animals generally as a Pax-Six-Eyes absent-Dachshund network (PSEDN). For amphioxus, we describe the complete network of nine PSEDN genes, four of which-AmphiSix1/2, AmphiSix4/5, AmphiSix3/6, and AmphiEya-are characterized here for the first time. For amphioxus, in vitro interactions among the genes and proteins of the network resemble those of other animals, except for the absence of Dach-Eya binding. Amphioxus PSEDN genes are expressed in highly stage- and tissue-specific patterns (sometimes conspicuously correlated with the local intensity of cell proliferation) in the gastrular organizer, notochord, somites, anterior central nervous system, peripheral nervous system, pharyngeal endoderm, and the likely homolog of the vertebrate adenohypophysis. In this last tissue, the anterior region expresses all three amphioxus Six genes and is a zone of active cell proliferation, while the posterior region expresses only AmphiPax6 and is non-proliferative. In summary, the topologies of animal PSEDNs, although considerably more variable than originally proposed, are conserved enough to be recognizable among species and among developing tissues; this conservation may reflect indispensable involvement of PSEDNs during the critically important early phases of embryology (e.g. in the control of mitosis, apoptosis, and cell/tissue motility). (C) 2007 Elsevier Inc. All rights reserved.

Beaster-Jones, L, Schubert M, Holland LZ.  2007.  Cis-regulation of the amphioxus engrailed gene: Insights into evolution of a muscle-specific enhancer. Mechanisms of Development. 124:532-542.   10.1016/j.mod.2007.06.002   AbstractWebsite

To gain insights into the relation between evolution of cis-regulatory DNA and evolution of gene function, we identified tissue-specific enhancers of the engrailed gene of the basal chordate amphioxus (Branch iostoma floridae) and compared their ability to direct expression in both amphioxus and its nearest chordate relative, the tunicate Ciona intestinalis. In amphioxus embryos, the native engrailed gene is expressed in three domains - the eight most anterior somites, a few cells in the central nervous system (CNS) and a few ectodermal cells. In contrast, in C. intestinalis, in which muscle development is highly divergent, engrailed expression is limited to the CNS. To characterize the tissue-specific enhancers of amphioxus engrailed, we first showed that 7.8 kb of upstream DNA of amphioxus engrailed directs expression to all three domains in amphioxus that express the native gene. We then identified the amphioxus engrailed muscle-specific enhancer as the 1.2 kb region of upstream DNA with the highest sequence identity to the mouse en-2 jaw muscle enhancer. This amphioxus enhancer directed expression to both the somites in amphioxus and to the larval muscles in C intestinalis. These results show that even though expression of the native engrailed has apparently been lost in developing C intestinalis muscles, they express the transcription factors necessary to activate transcription from the amphioxus engrailed enhancer, suggesting that gene networks may not be completely disrupted if an individual component is lost. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Lin, HC, Holland LZ, Holland ND.  2006.  Expression of the AmphiTcf gene in amphioxus: Insights into the evolution of the TCF/LEF gene family during vertebrate evolution. Developmental Dynamics. 235:3396-3403.   10.1002/dvdy.20971   AbstractWebsite

T-cell factor (TCF) and lymphoid enhancer factors (LEF) genes encode proteins that are transcription factors mediating beta-catenin/Wnt signaling. Whereas mammals have four such genes, the Florida amphioxus (Branchiostoma floridae) apparently has only one such gene (AmphiTcF). From cleavage through early gastrula, cytoplasmic maternal transcripts of this gene are localized toward the animal pole. In gastrulae, AmphiTcf expression begins in the mesendoderm. In neurulae, there is expression in the pharynx, hindgut, anterior notochord, somites, and at the anterior end of the neural plate. In early larvae, expression is detectable in the floor of the diencephalon, notochord, tail bud, forming somites, pharynx, and ciliated pit (a presumed homolog of the vertebrate adenohypophysis). Phylogenetic analysis of TCF/LEF proteins placed AmphiTcf as the sister group of a clade comprising vertebrate Tcf1, Lef1, Tcf3, and Tcf4. Comparison of developmental expression for amphioxus AmphiTcf and vertebrate TCF/LEF genes indicates that this gene family has undergone extensive subfunctionalization and neofunctionalization during vertebrate evolution.

Schubert, M, Yu JK, Holland ND, Escriva H, Laudet V, Holland LZ.  2005.  Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus. Development. 132:61-73.   10.1242/dev.01554   AbstractWebsite

In the invertebrate chordate amphioxus, as in vertebrates, retinoic acid (RA) specifies position along the anterior/posterior axis with elevated RA signaling in the middle third of the endoderm setting the posterior limit of the pharynx. Here we show that AmphiHox1 is also expressed in the middle third of the developing amphioxus endoderm and is activated by RA signaling. Knockdown of AmphiHox1 function with an antisense morpholino oligonucleotide shows that AmphiHox1 mediates the role of RA signaling in setting the posterior limit of the pharynx by repressing expression of pharyngeal markers in the posterior foregut/midgut endoderm. The spatiotemporal expression of these endodermal genes in embryos treated with RA or the RA antagonist BMS009 indicates that Pax1/9, Pitx and Notch are probably more upstream than Otx and Nodal in the hierarchy of genes repressed by RA signaling. This work highlights the potential of amphioxus, a genomically simple, vertebrate-like invertebrate chordate, as a paradigm for understanding gene hierarchies similar to the more complex ones of vertebrates.

Yu, JK, Holland ND, Holland LZ.  2003.  AmphiFoxQ2, a novel winged helix/forkhead gene, exclusively marks the anterior end of the amphioxus embryo. Development Genes and Evolution. 213:102-105.   10.1007/s00427-003-0302-3   AbstractWebsite

A full-length FoxQ-related gene (AmphiFoxQ2) was isolated from amphioxus. Expression is first detectable in the animal/anterior hemisphere at the mid blastula stage. The midpoint of this expression domain coincides with the anterior pole of the embryo and is offset dorsally by about 20degrees from the animal pole. During the gastrula stage, expression is limited to the anterior ectoderm. By the early neurula stage, expression remains in the anterior ectoderm and also appears in the adjacent anterior mesendoderm. By the early larval stages, expression is detectable in the anteriormost ectoderm and in the rostral tip of the notochord. AmphiFoxQ2 is never expressed anywhere except at the anterior tip of amphioxus embryos and larvae. This is the first gene known that exclusively marks the anterior pole of chordate embryos. It may, therefore, play an important role in establishing and/or maintaining the anterior/ posterior axis.

Holland, ND, Venkatesh TV, Holland LZ, Jacobs DK, Bodmer R.  2003.  AmphiNk2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart. Developmental Biology. 255:128-137.   10.1016/s0012-1606(02)00050-7   AbstractWebsite

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express ArnphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization. (C) 2003 Elsevier Science (USA). All rights reserved.

Escriva, H, Holland ND, Gronemeyer H, Laudet C, Holland LZ.  2002.  The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest. Development. 129:2905-2916. AbstractWebsite

Amphioxus, the closest living invertebrate relative of the vertebrates, has a notochord, segmental axial musculature, pharyngeal gill slits and dorsal hollow nerve cord, but lacks neural crest. In amphioxus, as in vertebrates, exogenous retinoic acid (RA) posteriorizes the embryo. The mouth and gill slits never form, AmphiPax1, which is normally downregulated where gill slits form, remains upregulated and AmphiHox1 expression shifts anteriorly in the nerve cord. To dissect the role of RA signaling in patterning chordate embryos, we have cloned the single retinoic acid receptor (AmphiRAR), retinoid X receptor (AmphiRXR) and an orphan receptor (AmphiTR2/4) from amphioxus. AmphiTR2/4 inhibits AmphiRAR-AmphiRXR-mediated transactivation in the presence of RA by competing for DR5 or IR7 retinoic acid response elements (RAREs). The 5' untranslated region of AmphiTR2/4 contains an IR7 element, suggesting possible auto- and RA-regulation. The patterns of AmphiTR2/4 and AmphiRAR expression during embryogenesis are largely complementary: AmphiTR2/4 is strongly expressed in the cerebral vesicle (homologous to the diencephalon plus anterior midbrain), while AmphiRAR expression is high in the equivalent of the hindbrain and spinal cord. Similarly, while AmphiTR2/4 is expressed most strongly in the anterior and posterior thirds of the endoderm, the highest AmphiRAR expression is in the middle third. Expression of AmphiRAR is upregulated by exogenous RA and completely downregulated by the RA antagonist BMS009. Moreover, BMS009 expands the pharynx posteriorly; the first three gill slit primordia are elongated and shifted posteriorly, but do not penetrate, and additional, non-penetrating gill slit primordia are induced. Thus, in an organism without neural crest, initiation and penetration of gill slits appear to be separate events mediated by distinct levels of RA signaling in the pharyngeal endoderm. Although these compounds have little effect on levels of AmphiTR2/4 expression, RA shifts pharyngeal expression of AmphiTR2/4 anteriorly, while BMS009 extends it posteriorly. Collectively, our results suggest a model for anteroposterior patterning of the amphioxus nerve cord and pharynx, which is probably applicable to vertebrates as well, in which a low anterior level of AmphiRAR (caused, at least in part, by competitive inhibition by AmphiTR2/4) is necessary for patterning the forebrain and formation of gill slits, the posterior extent of both being set by a sharp increase in the level of AmphiRAR.

Schubert, M, Holland LZ, Stokes MD, Holland ND.  2001.  Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: The evolution of somitogenesis in chordates. Developmental Biology. 240:262-273.   10.1006/dbio.2001.0460   AbstractWebsite

The amphioxus tail bud is similar to the amphibian tail bud in having an epithelial organization without a mesenchymal component. We characterize three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) and show that their early expression around the blastopore can subsequently be traced into the tail bud; in vertebrate embryos, there is a similar progression of expression domains for Wnt3, Wnt5, and Wnt6 genes from the blastopore lip (or its equivalent) to the tail bud. In amphioxus, AmphiWnt3, AmphiWnt5, and AmphiWnt6 are each expressed in a specific subregion of the tail bud, tentatively suggesting that a combinatorial code of developmental gene expression may help generate specific tissues during posterior elongation and somitogenesis. In spite of similarities within their tail buds, vertebrate and amphioxus embryos differ markedly in the relation between the tail bud and the nascent somites: vertebrates have a relatively extensive zone of unsegmented mesenchyme (i.e., presomitic mesoderm) intervening between the tail bud and the forming somites, whereas the amphioxus tail bud gives rise to new somites directly. It is likely that presomitic mesoderm is a vertebrate innovation made possible by developmental interconversions between epithelium and mesenchyme that first became prominent at the dawn of vertebrate evolution. (C) 2001 Academic Press.

Holland, LZ, Rached LA, Tamme R, Holland ND, Inoko H, Shiina T, Burgtorf C, Lardelli M.  2001.  Characterization and developmental expression of the amphioxus homolog of notch (AmphiNotch): Evolutionary conservation of multiple expression domains in amphioxus and vertebrates. Developmental Biology. 232:493-507.   10.1006/dbio.2001.0160   AbstractWebsite

Notch encodes a transmembrane protein that functions in intercellular signaling. Although there is one Notch gene in Drosophila, vertebrates have three or more with overlapping patterns of embryonic expression. We cloned the entire 7575-bp coding region of an amphioxus Notch gene (AmphiNotch), encoding 2524 amino acids, and obtained the exon/intron organization from a genomic cosmid clone. Southern blot and PCR data indicate that AmphiNotch is the only Notch gene in amphioxus. AmphiNotch, like Drosophila Notch and vertebrate Notch1 and Notch2, has 36 EGF repeats, 3 Notch/lin-12 repeats, a transmembrane region, and B ankyrin repeats. Phylogenetic analysis places it at the base of all the vertebrate genes, suggesting it is similar to the ancestral gene from which the vertebrate Notch family genes evolved. AmphiNotch is expressed in all three embryonic perm layers in spatiotemporal patterns strikingly similar to those of all the vertebrate homologs combined. In the developing nerve cord, AmphiNotch is first expressed in the posteriormost part of the neural plate, then it becomes more broadly expressed and later is localized dorsally in the anteriormost part of the nerve cord corresponding to the diencephalon. In late embryos and larvae, AmphiNotch is also expressed in parts of the pharyngeal endoderm, ill the anterior gut diverticulum, and, like AmphiPax2/5/8, in the rudiment of Hatschek's kidney. A comparison with Notch1 and Pax5 and Pax8 expression in the embryonic mouse kidney helps support homology of the amphioxus and vertebrate kidneys. AmphiNotch is also an early marker for presumptive mesoderm, transcripts first being detectable at the gastrula stage in a ring of mesendoderm just inside the blastopore and subsequently in the posterior mesoderm, notochord, and somites. As in sea urchins and vertebrates, these domains of AmphiNotch expression overlap with those of several Wnt genes and brachyury. These relationships suggest that amphioxus shares with other deuterostomes a common mechanism for patterning along the anterior/posterior axis involving a posterior signaling center in which the Notch and Wnt pathways and brachyury interact. (C) 2001 Academic Press.

Holland, LZ, Holland ND, Schubert M.  2000.  Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily. Development Genes and Evolution. 210:522-524.   10.1007/s004270000089   AbstractWebsite

A full-length Wnt1 gene (AmphiWnt1) was isolated from amphioxus. Expression is first detectable in the gastrula around the lip of the blastopore. By the early neurula, transcription is in the mesendoderm near the closed blastopore, but is down-regulated in the overlying ectoderm. In the late neurula, expression is limited to the posterior wall of the neurenteric canal. Later in development, AmphiWnt1 transcripts can no longer be detected. AmphiWnt1 has no counterpart of the predominant expression domains of vertebrate Wnt1 genes in the neural tube, but its expression may be more comparable to that of wingless in the invaginating hindgut primordium of insects.

Schubert, M, Holland LZ, Holland ND.  2000.  Characterization of an amphioxus Wnt gene, AmphiWnt11, with possible roles in myogenesis and tail outgrowth. Genesis. 27:1-5. AbstractWebsite

The full-length sequence and developmental expression of an amphioxus Wnt gene (AmphiWnt11) are described. A phylogenetic analysis of all known full-length Wnt11 sequences indicates that a gene duplication occurred at the base of the vertebrate Wnt11 clade. The developmental expression domains of AmphiWnt11 resemble those of Wnt11 homologs in vertebrates. The earliest detectable expression is transiently associated with the dorsal lip of the blastopore. At the neurula stage, AmphiWnt11 is expressed in myotomal muscle cells; however, AmphiWnt11 transcription is not associated with metameric pre-patterning prior to morphological segmentation. Finally, in amphioxus and the vertebrates, Wnt11 homologs are expressed in anteroventral ectoderm and in association with the tailbud and the tail fin. Thus, in amphioxus and lower vertebrates, the posterior expression of Wnt11 may be involved in tail fin outgrowth, and this ancient genetic program might have been cc-opted at least in part for lateral appendage development during vertebrate evolution. genesis 27: 1-5, 2000, (C) 2000 Wiley-Liss, Inc.

Schubert, M, Holland LZ, Holland ND.  2000.  Characterization of two amphioxus Wnt genes (AmphiWnt4 and AmphiWnt7b) with early expression in the developing central nervous system. Developmental Dynamics. 217:205-215.   10.1002/(sici)1097-0177(200002)217:2<205::aid-dvdy7>;2-f   AbstractWebsite

Full-length sequences and developmental expression patterns of two amphioxus Wnt genes (AmphiWnt4 and AmphiWnt7b) are described for the first time. The dynamic expression pattern of AmphiWnt4 suggests roles in the development of the posterior mesoderm, central nervous system, muscular somites, heart, and endostyle (a homolog of the vertebrate thyroid). The less diverse expression domains of AmphiWnt7b indicate that this gene may be involved only in the development of the central nervous system and the endostyle, In contrast to amphioxus, vertebrate embryos do not express Wnt4 homologues in the posterior mesoderm, somites, or heart; instead, Wnt genes of other subfamilies are expressed in these developing vertebrate organs, Because the developmental genetic programs of amphioxus may approximate those in the invertebrate chordate ancestor of the vertebrates, it is possible that some developmental functions of an ancestral Wnt4 gene may have been assumed by genes of other Wnt subfamilies during vertebrate evolution, possibly as a result of functional redundancy among Wnt subfamilies. (C) 2000 Wiley-Liss, Inc.

Schubert, M, Holland LZ, Holland ND, Jacobs DK.  2000.  A phylogenetic tree of the Wnt genes based on all available full-length sequences, including five from the cephalochordate amphioxus. Molecular Biology and Evolution. 17:1896-1903. AbstractWebsite

The Wnt gene family is large, and new members are still being discovered. We constructed a parsimony tree for the Wnt family based on all 82 of the full-length sequences currently available. The inclusion of sequences from the cephalochordate amphioxus is especially useful in comprehensive gene trees, because the amphioxus genes in each subfamily often mark the base of the vertebrate diversification. We thus isolated full-length cDNAs of five amphioxus War genes (AmphiWnt1, AmphiWnt4, AmphiWnt7, AmphiWnt8, and AmphiWnt11) for addition to the overall War family tree. The analysis combined amino acid and nucleotide sequences (excluding third codon positions), taking into account 97% of the available data for each sequence. This combinatorial method had the advantage of generating a single most-parsimonious tree that was trichotomy-free. The reliability of the nodes was assessed by both jackknifing and Bremer support (decay index). A regression analysis revealed that branch length was strongly correlated with branch support, and possible reasons for this pattern are discussed. The tree topology suggested that in amphioxus, at least an AmphiWnt5 and an AmphiWnt10 have yet to be discovered.

Venkatesh, TV, Holland ND, Holland LZ, Su MT, Bodmer R.  1999.  Sequence and developmental expression of amphioxus AmphiNk2-1: insights into the evolutionary origin of the vertebrate thyroid gland and forebrain. Development Genes and Evolution. 209:254-259. AbstractWebsite

We characterized an amphioxus NK-2 homeobox gene (AmphiNk2-1), a homologue of vertebrate Nkx2-1, which is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2-1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented posteriorly and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2-1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions: this pattern is comparable to Nkx2-1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homologue of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain.

Holland, LZ, Holland ND.  1998.  Developmental gene expression in amphioxus: New insights into the evolutionary origin of vertebrate brain regions, neural crest, and rostrocaudal segmentation. American Zoologist. 38:647-658. AbstractWebsite

Amphioxus is widely held to be the closest invertebrate relative of the vertebrates and the best available stand-in for the proximate ancestor of the vertebrates. The spatiotemporal expression patterns of developmental genes can help suggest body part homologies between vertebrates and amphioxus, This approach is illustrated using five homeobox genes (AmphiHox1, AmphiHox2, AmphiOtx, AmphiDll, and AmphiEn) to pro,ide insights into the evolutionary origins of three important vertebrate features: the major brain regions, the neural crest, and rostrocaudal segmentation. During amphioxus development, the neural expression patterns of these genes are consistent with the presence of a forebrain (detailed neuroanatomy indicates that the forebrain is all diencephalon without any telencephalon) and an extensive hindbrain; the possible presence of a midbrain requires additional study. Further, during neurulation, the expression pattern of AmphiDll as web as migratory cell behavior suggest that the epidermal cells bordering the neural plate may represent a phylogenetic precursor of the vertebrate neural crest. Finally, when the paraxial mesoderm begins to segment, the earliest expression of AmphiEn is detected in the posterior part of each nascent and newly formed somite, This pattern recalls the expression of the segment-polarity gene engrailed during establishment of the segments of metameric protostomes. Thus, during animal evolution, the role of engrailed in establishing and maintaining metameric body plans may have arisen in a common segmented ancestor of both the protostomes and deuterostomes.

Schubert, M, Holland ND, Holland LZ.  1998.  Amphioxus AmphiDRAL encoding a LIM-domain protein: expression in the epidermis but not in the presumptive neuroectoderm. Mechanisms of Development. 76:203-205.   10.1016/s0925-4773(98)00120-8   AbstractWebsite

The sequence and developmental expression have been determined for amphioxus AmphiDRAL, which encodes a homolog of vertebrate DRAL (down-regulated in rhabdomyosarcoma LIM-protein). This is the first clear example of a DRAL homolog in an invertebrate. Detectable developmental expression begins at the gastrula stage in the epidermis, but not in the neuroectoderm; thus the early stages of AmphiDRAL expression indicate the neural/non-neural boundary. During subsequent embryonic stages, expression continues in the epidermis (but not in the developing central nervous system) until it fades during the later larval stages. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Holland, ND, Holland LZ.  1991.  The Fine-Structure of the Growth Stage Oocytes of a Lancelet (= Amphioxus), Branchiostoma-Lanceolatum. Invertebrate Reproduction & Development. 19:107-122.   10.1080/07924259.1991.9672164   AbstractWebsite

Oocytes of the European lancelet (Branchiostoma lanceolatum) were fixed for transmission electron microscopy at Banyuls (French Mediterranean) in mid spring, which is just before the spawning season. Special attention is given to the growth stage (= diplotene) oocytes and to their relations with non-germinal cells of the ovary. At the time of year studied, the ovaries contain both relatively small (7-57-mu-m) and relatively large (97-127-mu-m) oocytes, but none of medium-size. In addition to the usual cell organelles (like free ribosomes, mitochondria, and endoplasmic reticulum), the oocytes include a number of more specialized structures. Nuage is present throughout the growth stage, both adjacent to the nucleus and scattered elsewhere in the cytoplasm. Other specialized structures do not begin to appear until the oocytes attain a certain diameter. The following structures are first detected at the following oocyte diameters: cortical granules, always in close association with Golgi complexes (20-mu-m); vacuoles containing presumed precursors of the vitelline layer (30-mu-m); yolk granules (35-mu-m); and a central vacuole within the nucleolus (50-mu-m). The cytoplasm of the largest oocytes includes a few annulate lamellae and some very unusual striated fibers resembling ciliary rootlets. These fibers, which are banded with a 70-nm periodicity (but are evidently not associated with centrioles), occur only at the animal pole of the oocyte, and their functional significance is unknown. The smaller oocytes have relatively smooth plasma membranes, except where endocytotic pits are abundant, whereas the larger oocytes bear abundant microvilli and are covered by a vitelline layer of dense, granular material. During oocyte growth, areas of contact between the oocytes and neighboring non-germinal cells shrink progressively. In the large oocytes, such contacts are limited to the animal pole, where a few processes from non-germinal cells pass through the vitelline layer and terminate on the oocyte plasma membrane at adhaerens type junctions (perhaps mixed with gap junctions). The possible functions and phylogenetic significance of such junctions are discussed.