Publications

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2017
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.

2016
Yue, JX, Kozmikova I, Ono H, Nossa CW, Kozmik Z, Putnam NH, Yu JK, Holland LZ.  2016.  Conserved noncoding elements in the most distant genera of cephalochordates: The Goldilocks principle. Genome Biology and Evolution. 8:2387-2405.   10.1093/gbe/evw158   AbstractWebsite

Cephalochordates, the sister group of vertebrates + tunicates, are evolving particularly slowly. Therefore, genome comparisons between two congeners of Branchiostoma revealed so many conserved noncoding elements (CNEs), that it was not clear how many are functional regulatory elements. To more effectively identify CNEs with potential regulatory functions, we compared noncoding sequences of genomes of the most phylogenetically distant cephalochordate genera, Asymmetron and Branchiostoma, which diverged approximately 120-160 million years ago. We found 113,070 noncoding elements conserved between the two species, amounting to 3.3% of the genome. The genomic distribution, target gene ontology, and enriched motifs of these CNEs all suggest that many of them are probably cis-regulatory elements. More than 90% of previously verified amphioxus regulatory elements were re-captured in this study. A search of the cephalochordate CNEs around 50 developmental genes inseveral vertebrate genomes revealed eight CNEs conserved between cephalochordates and vertebrates, indicating sequence conservation over > 500 million years of divergence. The function of five CNEs was tested in reporter assays in zebrafish, and one was also tested in amphioxus. All five CNEs proved to be tissue-specific enhancers. Taken together, these findings indicate that even though Branchiostoma and Asymmetron are distantly related, as they are evolving slowly, comparisons between them are likely optimal for identifying most of their tissue-specific cis-regulatory elements laying the foundation for functional characterizations and a better understanding of the evolution of developmental regulation in cephalochordates.

2015
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, LZ.  2015.  Evolution of basal deuterostome nervous systems. Journal of Experimental Biology. 218:637-645.   10.1242/jeb.109108   AbstractWebsite

Understanding the evolution of deuterostome nervous systems has been complicated by the by the ambiguous phylogenetic position of the Xenocoelomorpha (Xenoturbellids, acoel flat worms, nemertodermatids), which has been placed either as basal bilaterians, basal deuterostomes or as a sister group to the hemichordate/echinoderm clade (Ambulacraria), which is a sister group of the Chordata. None of these groups has a single longitudinal nerve cord and a brain. A further complication is that echinoderm nerve cords are not likely to be evolutionarily related to the chordate central nervous system. For hemichordates, opinion is divided as to whether either one or none of the two nerve cords is homologous to the chordate nerve cord. In chordates, opposition by two secreted signaling proteins, bone morphogenetic protein (BMP) and Nodal, regulates partitioning of the ectoderm into central and peripheral nervous systems. Similarly, in echinoderm larvae, opposition between BMP and Nodal positions the ciliary band and regulates its extent. The apparent loss of this opposition in hemichordates is, therefore, compatible with the scenario, suggested by Dawydoff over 65 years ago, that a true centralized nervous system was lost in hemichordates.

2013
Holland, LZ, Carvalho JE, Escriva H, Laudet V, Schubert M, Shimeld SM, Yu JK.  2013.  Evolution of bilaterian central nervous systems: a single origin? Evodevo. 4   10.1186/2041-9139-4-27   AbstractWebsite

The question of whether the ancestral bilaterian had a central nervous system (CNS) or a diffuse ectodermal nervous system has been hotly debated. Considerable evidence supports the theory that a CNS evolved just once. However, an alternative view proposes that the chordate CNS evolved from the ectodermal nerve net of a hemichordate-like ancestral deuterostome, implying independent evolution of the CNS in chordates and protostomes. To specify morphological divisions along the anterior/posterior axis, this ancestor used gene networks homologous to those patterning three organizing centers in the vertebrate brain: the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer, and subsequent evolution of the vertebrate brain involved elaboration of these ancestral signaling centers; however, all or part of these signaling centers were lost from the CNS of invertebrate chordates. The present review analyzes the evidence for and against these theories. The bulk of the evidence indicates that a CNS evolved just once - in the ancestral bilaterian. Importantly, in both protostomes and deuterostomes, the CNS represents a portion of a generally neurogenic ectoderm that is internalized and receives and integrates inputs from sensory cells in the remainder of the ectoderm. The expression patterns of genes involved in medio/lateral (dorso/ventral) patterning of the CNS are similar in protostomes and chordates; however, these genes are not similarly expressed in the ectoderm outside the CNS. Thus, their expression is a better criterion for CNS homologs than the expression of anterior/posterior patterning genes, many of which (for example, Hox genes) are similarly expressed both in the CNS and in the remainder of the ectoderm in many bilaterians. The evidence leaves hemichordates in an ambiguous position - either CNS centralization was lost to some extent at the base of the hemichordates, or even earlier, at the base of the hemichordates + echinoderms, or one of the two hemichordate nerve cords is homologous to the CNS of protostomes and chordates. In any event, the presence of part of the genetic machinery for the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer in invertebrate chordates together with similar morphology indicates that these organizers were present, at least in part, at the base of the chordates and were probably elaborated upon in the vertebrate lineage.

2011
Wu, HR, Chen YT, Su YH, Luo YJ, Holland LZ, Yu JK.  2011.  Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Developmental Biology. 353:147-159.   10.1016/j.ydbio.2011.02.014   AbstractWebsite

The origin of germline cells was a crucial step in animal evolution. Therefore, in both developmental biology and evolutionary biology, the mechanisms of germline specification have been extensively studied over the past two centuries. However, in many animals, the process of germline specification remains unclear. Here, we show that in the cephalochordate amphioxus Branchiostoma floridae, the germ cell-specific molecular markers Vasa and Nanos become localized to the vegetal pole cytoplasm during oogenesis and are inherited asymmetrically by a single blastomere during cleavage. After gastrulation, this founder cell gives rise to a cluster of progeny that display typical characters of primordial germ cells (PGCs). Blastomeres separated at the two-cell stage grow into twin embryos, but one of the twins fails to develop this Vasa-positive cell population, suggesting that the vegetal pole cytoplasm is required for the formation of putative PGCs in amphioxus embryos. Contrary to the hypothesis that cephalochordates may form their PGCs by epigenesis, our data strongly support a preformation mode of germ cell specification in amphioxus. In addition to the early localization of their maternal transcripts in the putative PGCs, amphioxus Vasa and Nanos are also expressed zygotically in the tail bud, which is the posterior growth zone of amphioxus. Thus, in addition to PGC specification, amphioxus Vasa and Nanos may also function in highly proliferating somatic stem cells. (C) 2011 Elsevier Inc. All rights reserved.

2010
Holland, LZ, Short S.  2010.  Alternative Splicing in Development and Function of Chordate Endocrine Systems: A Focus on Pax Genes. Integrative and Comparative Biology. 50:22-34.   10.1093/icb/icq048   AbstractWebsite

Genome sequencing has facilitated an understanding of gene networks but has also shown that they are only a small part of the answer to the question of how genes translate into a functional organism. Much of the answer lies in epigenetics-heritable traits not directly encoded by the genome. One such phenomenon is alternative splicing, which affects over 75% of protein coding genes and greatly amplifies the number of proteins. Although it was postulated that alternative splicing and gene duplication are inversely proportional and, therefore, have similar effects on the size of the proteome, for ancient duplications such as occurred in the Pax family of transcription factors, that is not necessarily so. The importance of alternative splicing in development and physiology is only just coming to light. However, several techniques for studying isoform functions both in vitro and in vivo have been recently developed. As examples of what is known and what is yet to be discovered, this review focuses on the evolution and roles of the Pax family of transcription factors in development and on alternative splicing of endocrine genes and the factors that regulate them.

2009
Holland, LZ.  2009.  Chordate roots of the vertebrate nervous system: expanding the molecular toolkit. Nature Reviews Neuroscience. 10:736-746.   10.1038/nrn2703   AbstractWebsite

The vertebrate brain is highly complex with millions to billions of neurons. During development, the neural plate border region gives rise to the neural crest, cranial placodes and, in anamniotes, to Rohon-Beard sensory neurons, whereas the boundary region of the midbrain and hindbrain develops organizer properties. Comparisons of developmental gene expression and neuroanatomy between vertebrates and the basal chordate amphioxus, which has only thousands of neurons and lacks a neural crest, most placodes and a midbrain-hindbrain organizer, indicate that these vertebrate features were built on a foundation already present in the ancestral chordate. Recent advances in genomics have provided insights into the elaboration of the molecular toolkit at the invertebrate-vertebrate transition that may have facilitated the evolution of these vertebrate characteristics.

2008
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

2007
Rasmussen, SLK, Holland LZ, Schubert M, Beaster-Jones L, Holland ND.  2007.  Amphioxus AmphiDelta: Evolution of delta protein structure, segmentation, and neurogenesis. Genesis. 45:113-122.   10.1002/dvg.20278   AbstractWebsite

The amphioxus genome has a single Delta gene (AmphiDelta) encoding a protein 766 amino acids long. Comparison of Delta proteins of amphioxus and other animals indicates that AmphiDelta retains features of a basal bilaterian Delta protein-in having nine epidermal growth factor (EGF) repeats and also in having char acteristic numbers of amino acids separating successive cysteines between and within EGF repeats. During development, AmphiDelta is expressed in the forming somites, in some regions of pharyngeal endoderm, and in cells (presumably differentiating neurons) scattered in both the neural plate and ectoderm. Expression is strongly associated with cells initiating movements to separate themselves from parent epithelia, either en masse by evagination (endoderm and mesoderm) or by delamination as isolated cells (ectoderm). The AmphiDelta-expressing cells delaminating from the ectoderm apparently migrate beneath it as they begin differentiating into probable sensory neurons, suggesting a scenario for the evolutionary origin of the placode-derived neurons of vertebrate cranial ganglia. genesis 45:113-122, 2007. Published 2007 Wiley-Liss, lnc.(dagger)

Yu, JK, Satou Y, Holland ND, Shin-I T, Kohara Y, Satoh N, Bronner-Fraser M, Holland LZ.  2007.  Axial patterning in cephalochordates and the evolution of the organizer. Nature. 445:613-617.   10.1038/nature05472   AbstractWebsite

The organizer of the vertebrate gastrula is an important signalling centre that induces and patterns dorsal axial structures. Although a topic of long-standing interest, the evolutionary origin of the organizer remains unclear. Here we show that the gastrula of the cephalochordate amphioxus expresses dorsal/ventral (D/V) patterning genes (for example, bone morphogenetic proteins (BMPs), Nodal and their antagonists) in patterns reminiscent of those of their vertebrate orthlogues, and that amphioxus embryos, like those of vertebrates, are ventralized by exogenous BMP protein. In addition, Wnt-antagonists (for example, Dkks and sFRP2-like) are expressed anteriorly, whereas Wnt genes themselves are expressed posteriorly, consistent with a role for Wnt signalling in anterior/posterior (A/P) patterning. These results suggest evolutionary conservation of the mechanisms for both D/V and A/P patterning of the early gastrula. In light of recent phylogenetic analyses placing cephalochordates basally in the chordate lineage, we propose that separate signalling centres for patterning the D/V and A/P axes may be an ancestral chordate character.

2006
Holland, ND, Holland LZ.  2006.  Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development. Evolution & Development. 8:142-149.   10.1111/j.1525-142X.2006.00085.x   AbstractWebsite

The distribution of dividing cells is described for embryos and larvae of amphioxus (Branchiostoma floridae) pulse labeled with bromodeoxyuridine. Because cell division is assessed for all of the developing tissues, this is the first comprehensive study of developmental cell proliferation for an animal lacking a stereotyped cell lineage. In amphioxus, cell divisions are virtually synchronous during cleavage, but become asynchronous at the blastula stage. Starting at the neurula stage, after the origin of the mesoderm, the proportion of dividing cells progressively declines in the somitic mesoderm and notochord. Other tissues, however, deviate from this pattern. For example, in the mid-neurula, there is a brief, intense burst of mitosis at the anterior end of the neural plate. Also, from the neurula through the early larval stage, all of the ectoderm cells cease dividing and develop cilia that propel the animal through the water; subsequently, in the epidermis of later larvae, mitosis resumes and the proportion of ciliated cells declines as muscular undulation gradually replaces ciliation for swimming. Finally, in the early larvae, there is a terminal arrest of cell division in three cell types that differentiate early to participate in feeding as soon as the mouth opens-namely the ciliated pharyngeal cells that produce the feeding current and the secretory cells of the club-shaped gland and endostyle that export food-trapping mucus into the pharynx. In sum, these stage- and tissue-specific changes in cell proliferation intensity illustrate how the requirements of embryonic and larval natural history can shape developmental programs.

Schubert, M, Holland ND, Laudet V, Holland LZ.  2006.  A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus. Developmental Biology. 296:190-202.   10.1016/j.ydbio.2006.04.457   AbstractWebsite

Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS). However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebrate chordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain, midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normally limited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1, 3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes is controlled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrain and in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatments with an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNS and indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification of neuronal identity. (c) 2006 Elsevier Inc. All rights reserved.

2003
Mazet, F, Yu JK, Liberles DA, Holland LZ, Shimeld SM.  2003.  Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria. Gene. 316:79-89.   10.1016/s0378-1119(03)00741-8   AbstractWebsite

The Forkhead or Fox gene family encodes putative transcription factors. There are at least four Fox genes in yeast, 16 in Drosophila melanogaster (Dm) and 42 in humans. Recently, vertebrate Fox genes have been classified into 17 groups named FoxA to FoxQ [Genes Dev. 14 (2000) 142]. Here, we extend this analysis to invertebrates, using available sequences from D. melanogaster, Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), the sea squirt Ciona intestinalis (Ci) and amphioxus Branchiostoma floridae (Bf), from which we also cloned several Fox genes. Phylogenetic analyses lend support to the previous overall subclassification of vertebrate genes, but suggest that four subclasses (FoxJ, L, N and Q) could be further subdivided to reflect their relationships to invertebrate genes. We were unable to identify orthologs of Fox subclasses E, H, I, J, M and Q1 in D. melanogaster, A. gambiae or C. elegans, suggesting either considerable loss in ecdysozoans or the evolution of these subclasses in the deuterostome lineage. Our analyses suggest that the common ancestor of protostomes and deuterostomes had a minimum complement of 14 Fox genes. (C) 2003 Elsevier B.V. All rights reserved.

Gibson-Brown, JJ, Osoegawa K, McPherson JD, Waterston RH, de Jong PJ, Rokhsar DS, Holland LZ.  2003.  A proposal to sequence the amphioxus genome submitted to the joint genome institute of the US department of energy. Journal of Experimental Zoology Part B-Molecular and Developmental Evolution. 300B:5-22.   10.1002/jez.b.00042   Website
2002
Yu, JK, Holland ND, Holland LZ.  2002.  An amphioxus winged helix/forkhead gene, AmphiFoxD: Insights into vertebrate neural crest evolution. Developmental Dynamics. 225:289-297.   10.1002/dvdy.10173   AbstractWebsite

During amphioxus development, the neural plate is bordered by cells expressing many genes with homologs involved in vertebrate neural crest induction. However, these amphioxus cells evidently lack additional genetic programs for the cell delaminations, migrations, and differentiations characterizing definitive vertebrate neural crest. We characterize an amphioxus winged helix/forkhead gene (AmphiFoxD) closely related to vertebrate FoxD genes. Phylogenetic analysis indicates that the AmphiFoxD is basal to vertebrate FoxD1, FoxD2, FoxD3, FoxD4, and FoxD5. One of these vertebrate genes (FoxD3) consistently marks neural crest during development. Early in amphioxus development, AmphiFoxD is expressed medially in the anterior neural plate as well as in axial (notochordal) and paraxial mesoderm; later, the gene is expressed in the somites, notochord, cerebral vesicle (diencephalon), and hindgut endoderm. However, there is never any expression in cells bordering the neural plate. We speculate that an AmphiFoxD homolog in the common ancestor of amphioxus and vertebrates was involved in histogenic processes in the mesoderm (evagination and delamination of the somites and notochord); then, in the early vertebrates, descendant paralogs of this gene began functioning in the presumptive neural crest bordering the neural plate to help make possible the delaminations and cell migrations that characterize definitive vertebrate neural crest. (C) 2002 Wiley-Liss, Inc.

Kreslova, J, Holland LZ, Schubert M, Burgtorf C, Benes V, Kozmik Z.  2002.  Functional equivalency of amphioxus and vertebrate Pax258 transcription factors suggests that the activation of mid-hindbrain specific genes in vertebrates occurs via the recruitment of Pax regulatory elements. Gene. 282:143-150.   10.1016/s0378-1119(01)00840-x   AbstractWebsite

Pax genes encode transcription factors that control key developmental decisions in various animal phyla. The Pax2/5/8 subfamily plays a key role in specification and/or maintenance of vertebrate mid-hindbrain boundary (MHB) region by directly regulating expression of other genes, most notably En2. In the invertebrate chordate amphioxus, expression of AmphiPax2/5/8 is found in many sites that are homologous to the regions of the vertebrate embryo expressing orthologous genes Pax2, Pax5 or Pax8. However, no co-expression of AmphiPax2/5/8 and AmphiEn is detected in the region of the neural tube that might correspond to the vertebrate MHB. Based on this observation and the absence of AmphiWnt expression in this region it appears that amphioxus does not have a MHB. Here we investigated the possibility that the AmphiPax2/5/8, as a key component of MHB development, has lost some of the properties of its vertebrate counterparts. We have analyzed both the DNA-binding and transactivation properties of AmphiPax2/5/8 as well as its ability to interact with the groucho co-repressor. In all these assays AmphiPax2/5/8 is indistinguishable from the human Pax5. In addition, we found two alternatively spliced AmphiPax2/5/8 isoforms that function similarly to the alternatively spliced isoforms of human Pax8. Analysis of the AmphiEn regulatory region provided no evidence for AmphiPax2/5/8 binding and transactivation. Therefore, in amphioxus, AmphiPax2/5/8, although capable of performing all the necessary functions has not been recruited for a developmental mechanism which usually sets up MHB development in vertebrates. (C) 2002 Elsevier Science B.V. All rights reserved.

2001
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.

Kozmik, Z, Holland LZ, Schubert M, Lacalli TC, Kreslova J, Vlcek C, Holland ND.  2001.  Characterization of amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm. Genesis. 29:172-179.   10.1002/gene.1021   AbstractWebsite

Structure and developmental expression are described for amphioxus AmphiVent, a homolog of vertebrate Vent genes. In amphioxus, AmphiVent-expressing ventral mesoderm arises at midneurula by outgrowth from the paraxial mesoderm, but in vertebrates, Vent-expressing ventral mesoderm originates earlier, at the gastrula stage. In other embryonic tissues (nascent paraxial mesoderm, neural plate, endoderm, and tail-bud), AmphiVent and its vertebrate homologs are expressed in similar spatiotemporal domains, indicating conservation of many Vent gene functions during chordate evolution. the ventral mesoderm evidently develops precociously in vertebrates because their relatively large embryos probably require an early and extensive deployment of the mesoderm-derived circulatory system. The vertebrate ventral mesoderm, in spite of its strikingly early advent, still resembles the nascent ventral mesoderm of amphioxus in expressing Vent homologs, This coincidence may indicate that Vent homologs in vertebrates and amphioxus play comparable roles in ventral mesoderm specification. (C) 2001 Wiley-Liss, Inc.

2000
Langlois, MC, Vanacker JM, Holland ND, Escriva H, Queva C, Laudet V, Holland LZ.  2000.  Amphicoup-TF, a nuclear orphan receptor of the lancelet Branchiostoma floridae, is implicated in retinoic acid signalling pathways. Development Genes and Evolution. 210:471-482.   10.1007/s004270000087   AbstractWebsite

In vertebrates, the orphan nuclear receptors of the COUP-TF group function as negative transcriptional regulators that inhibit the hormonal induction of target genes mediated by classical members of the nuclear hormone superfamily, such as the retinoic acid receptors (RARs) or the thyroid hormone receptors (TRs). To investigate the evolutionary conservation of the roles of COUP-TF receptors as negative regulators in the retinoid and thyroid hormone pathways, we have characterized AmphiCOUP-TF, the homologue of COUP-TFI and COUP-TFII, in the chordate amphioxus (Branchiostoma floridae), the closest living invertebrate relative of the vertebrates. Electrophoretic mobility shift assays (EMSA) showed that AmphiCOUP-TF binds to a wide variety of response elements, as do its vertebrate homologues. Furthermore, AmphiCOUP-TF is a transcriptional repressor that strongly inhibits retinoic acid-mediated transactivation. In situ hybridizations revealed expression of AmphiCOUP-TF in the nerve cord of late larvae, in a region corresponding to hindbrain and probably anterior spinal cord. Although the amphioxus nerve cord appears unsegmented at the gross anatomical level, this pattern reflects segmentation at the cellular level with stripes of expressing cells occurring adjacent to the ends and the centers of each myotomal segment, which may include visceral motor neurons and somatic motor neurons respectively, among other cells. A comparison of the expression pattern of AmphiCOUP-TF with those of its vertebrate homologues, suggests that the roles of COUP-TF in patterning of the nerve cord evolved prior to the split between the amphioxus and vertebrate lineages. Furthermore, in vitro data also suggest that AmphiCOUP-TF acts as a negative regulator of signalling by other nuclear receptors such as RAR, TR or ER.

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, Panopoulou GD, Lehrach H, Holland ND.  2000.  Characterization of amphioxus AmphiWnt8: insights into the evolution of patterning of the embryonic dorsoventral axis. Evolution & Development. 2:85-92.   10.1046/j.1525-142x.2000.00047.x   AbstractWebsite

The full-length sequence and developmental expression of an amphioxus Wnt gene (AmphiWnt8 ) are described. In amphioxus embryos, the expression patterns of AmphiWnt8 suggest patterning roles in the forebrain, in the hindgut, and in the paraxial mesoderm that gives rise to the muscular somites. Phylogenetic analysis indicates that a single Wnt8 subfamily gene in an ancestral chordate duplicated early in vertebrate evolution into a Wnt8 clade and a Wnt8b clade. Coincident with this gene duplication, the functions of the ancestral AmphiWnt8-like gene appear to have been divided between vertebrate Wnt8b (exclusively neurogenic, especially in the forebrain) and vertebrate Wnt8 (miscellaneous, especially in early somitogenesis). Amphioxus AmphiWnt8 and its vertebrate Wnt8 homologs probably play comparable roles in the early dorsoventral patterning of the embryonic body axis.

1999
Holland, ND, Holland LZ.  1999.  Amphioxus and the utility of molecular genetic data for hypothesizing body part homologies between distantly related animals. American Zoologist. 39:630-640. AbstractWebsite

Expression domains of developmental genes can indicate body part homologies between distantly related animals and give insights into interesting evolutionary questions. Two of the chief criteria for recognizing homologies are relative position with respect to surrounding body parts and special quality (for instance, a vertebrate testis, regardless of its location, is recognizable by its seminiferous cysts or tubules), When overall body plans of two animals are relatively similar, as for amphioxus versus vertebrates, body part homologies can be supported by developmental gene expression domains, which have properties of special quality and relative position. With expression patterns of AmphiNk2-1 and AmphiPax2/5/8, Re reexamine the proposed homology between the amphioxus endostyle and the vertebrate thyroid gland, and a previously good homology is made better. When body plans of animals are disparate, body part homologies supported by molecular genetic data are less convincing, because the criterion of relative position of gene expression domains becomes uncertain. Thus, when expression of amphioxus AmphiBMP2/4 is used to compare the dorsoventral axis between amphioxus and other animals, a comparison between amphioxus and vertebrates is more convincing than comparison between amphioxus and other invertebrates with disparate body plans. In spite of this difficulty, the use of developmental genetic evidence comparing animals with disparate body plans is currently putting the big picture of evolution into new perspective. For example, some molecular geneticists are non: suggesting that the last common ancestor of all bilaterian animals might have been more annelid-like than flatworm-like.

1998
Panopoulou, GD, Clark MD, Holland LZ, Lehrach H, Holland ND.  1998.  AmphiBMP2/4, an amphioxus bone morphogenetic protein closely related to Drosophila decapentaplegic and vertebrate BMP2 and BMP4: Insights into evolution of dorsoventral axis specification. Developmental Dynamics. 213:130-139.   10.1002/(sici)1097-0177(199809)213:1<130::aid-aja13>3.3.co;2-z   AbstractWebsite

Amphioxus AmphiBMP2/4 appears to be a single gene closely related to vertebrate BMP2 and BMP4. In amphioxus embryos, the expression patterns of AmphiBMP2/4 suggest patterning roles in the ectodermal dorsoventral axis (comparable to dorsoventral axis establishment in the ectoderm by Drosophila decapentaplegic and vertebrate BMP4). In addition AmphiBMP2/4 may be involved in somite evagination, tail bud growth, pharyngeal differentiation (resulting in club-shaped gland morphogenesis), hindgut regionalization, differentiation of olfactory epithelium, patterning of the anterior central nervous system, and establishment of the heart primordium, One difference between the developmental role of amphioxus AmphiBMP2/4 and vertebrate BMP4 is that the former does not appear to be involved in the initial establishment of the dorsoventral polarity of the mesoderm, Dev. Dyn. 1998;213:130-139. (C) 1998 Wiley-Liss, Inc.

1996
Holland, LZ.  1996.  Muscle development in amphioxus: Morphology, biochemistry, and molecular biology. Israel Journal of Zoology. 42:S235-S246. AbstractWebsite

This review concerns the structure and biochemistry of muscle in amphioxus. Most work has focused on the segmented swimming (axial) muscles. These muscles derive from the medial wall of the somites, which arise as evaginations from the gut wall. The myotomal muscle cells of amphioxus, unlike those of vertebrates, never fuse, but remain mononucleate, contain only one myofibril, and span the entire length of the myotome. The muscle cells are very thin and lack a T-tubule system. There are two, maybe three, types of fibers. Innervation is via muscle tails, which contact the basal lamina of the nerve cord. The notochord is also composed of striated muscle cells, which similarly send muscle tails to the nerve cord. Less is known about the biochemistry of muscle. The notochord, like molluskan catch muscle, contains paramyosin. Among the muscle-specific proteins sequenced are alkali myosin light chain, troponin C and sarcoplasmic calcium-binding proteins, calcium-vector protein, and its target protein calcium vector-target protein. The only muscle regulatory factors identified are two MyoD proteins. Almost nothing is known about muscle enzymes in amphioxus.