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Holland, LZ.  2015.  The origin and evolution of chordate nervous systems. Philosophical Transactions of the Royal Society B-Biological Sciences. 370   10.1098/rstb.2015.0048   AbstractWebsite

In the past 40 years, comparisons of developmental gene expression and mechanisms of development (evodevo) joined comparative morphology as tools for reconstructing long-extinct ancestral forms. Unfortunately, both approaches typically give congruent answers only with closely related organisms. Chordate nervous systems are good examples. Classical studies alone left open whether the vertebrate brain was a new structure or evolved from the anterior end of an ancestral nerve cord like that of modern amphioxus. Evodevo plus electron microscopy showed that the amphioxus brain has a diencephalic forebrain, small midbrain, hindbrain and spinal cord with parts of the genetic mechanisms for the midbrain/hindbrain boundary, zona limitans intrathalamica and neural crest. Evodevo also showed how extra genes resulting from whole-genome duplications in vertebrates facilitated evolution of new structures like neural crest. Understanding how the chordate central nervous system (CNS) evolved from that of the ancestral deuterostome has been truly challenging. The majority view is that this ancestor had a CNS with a brain that gave rise to the chordate CNS and, with loss of a discrete brain, to one of the two hemichordate nerve cords. The minority view is that this ancestor had no nerve cord; those in chordates and hemichordates evolved independently. New techniques such as phylostratigraphy may help resolve this conundrum.

Onai, T, Takai A, Setiamarga DHE, Holland LZ.  2012.  Essential role of Dkk3 for head formation by inhibiting Wnt/beta-catenin and Nodal/Vg1 signaling pathways in the basal chordate amphioxus. Evolution & Development. 14:338-350.   10.1111/j.1525-142X.2012.00552.x   AbstractWebsite

To dissect the molecular mechanism of head specification in the basal chordate amphioxus, we investigated the function of Dkk3, a secreted protein in the Dickkopf family, which is expressed anteriorly in early embryos. Amphioxus Dkk3 has three domains characteristic of Dkk3 proteinsan N-terminal serine rich domain and two C-terminal cysteine-rich domains (CRDs). In addition, amphioxus Dkk3 has a TGF beta-receptor 2 domain, which is not present in Dkk3 proteins of other species. As vertebrate Dkk3 proteins have been reported to regulate either Nodal signaling or Wnt/beta-catenin signaling but not both in the same species, we tested the effects of Dkk3 on signaling by these two pathways in amphioxus embryos. Loss of function experiments with an anti-sense morpholino oligonucleotide (MO) against amphioxus Dkk3 resulted in larvae with truncated heads and concomitant loss of expression of anterior gene markers. The resemblance of the headless phenotype to that from upregulation of Wnt/beta-catenin signaling with BIO, a GSK3 beta inhibitor, suggested that Dkk3 might inhibit Wnt/beta-catenin signaling. In addition, the Dkk3 MO rescued dorsal structures in amphioxus embryos treated with SB505124, an inhibitor of Nodal signaling, indicating that amphioxus Dkk3 can also inhibit Nodal signaling. In vitro assays in Xenopus animal caps showed that Nodal inhibition is largely due to domains other than the TGF beta domain. We conclude that amphioxus Dkk3 regulates head formation by modulating both Wnt/beta-catenin and Nodal signaling, and that these functions may have been partitioned among various vertebrate lineages during evolution of Dkk3 proteins.

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, LZ, Albalat R, Azumi K, Benito-Gutierrez E, Blow MJ, Bronner-Fraser M, Brunet F, Butts T, Candiani S, Dishaw LJ, Ferrier DEK, Garcia-Fernandez J, Gibson-Brown JJ, Gissi C, Godzik A, Hallbook F, Hirose D, Hosomichi K, Ikuta T, Inoko H, Kasahara M, Kasamatsu J, Kawashima T, Kimura A, Kobayashi M, Kozmik Z, Kubokawa K, Laudet V, Litman GW, McHardy AC, Meulemans D, Nonaka M, Olinski RP, Pancer Z, Pennacchio LA, Pestarino M, Rast JP, Rigoutsos I, Robinson-Rechavi M, Roch G, Saiga H, Sasakura Y, Satake M, Satou Y, Schubert M, Sherwood N, Shiina T, Takatori N, Tello J, Vopalensky P, Wada S, Xu AL, Ye YZ, Yoshida K, Yoshizaki F, Yu JK, Zhang Q, Zmasek CM, de Jong PJ, Osoegawa K, Putnam NH, Rokhsar DS, Satoh N, Holland PWH.  2008.  The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Research. 18:1100-1111.   10.1101/gr.073676.107   AbstractWebsite

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates-a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

Holland, LZ, Holland ND.  2007.  A revised fate map for amphioxus and the evolution of axial patterning in chordates. Integrative and Comparative Biology. 47:360-372.   10.1093/icb/icm064   AbstractWebsite

The chordates include vertebrates plus two groups of invertebrates (the cephalochordates and tunicates). Previous embryonic fate maps of the cephalochordate amphioxus (Branchiostoma) were influenced by preconceptions that early development in amphioxus and ascidian tunicates should be fundamentally the same and that the early amphioxus embryo, like that of amphibians, should have ventral mesoderm. Although detailed cell lineage tracing in amphioxus has not been done because of limited availability of the embryos and because cleavage is radial and holoblastic with the blastomeres nearly equal in size and not tightly adherent until the mid-blastula stage, a compilation of data from gene expression and function, blastomere isolation and dye labeling allows a more realistic fate map to be drawn. The revised fate map is substantially different from that of ascidians. It shows (1) that the anterior pole of the amphioxus embryo is offset dorsally from the animal pole only by about 20 degrees, (2) that the ectoderm/mesendoderm boundary (the future rim of the blastopore) is at the equator of the blastula, which approximately coincides with the 3rd cleavage plane, and (3) that there is no ventral mesoderm during the gastrula stage. Involution or ingression of cells over the blastopore lip is negligible, and the blastopore, which is posterior, closes centripetally as if by a purse string. During the gastrula stage, the animal pole shifts ventrally, coming to lie about 20 degrees ventral to the anterior tip of the late gastrula/early neurula. Comparisons of the embryos of amphioxus and vertebrates indicate that in spite of large differences in the mechanics of cleavage and gastrulation, anterior/posterior and dorsal/ventral patterning occur by homologous genetic mechanisms. Therefore, the small, nonyolky embryo of amphioxus is probably a reasonable approximation of the basal chordate embryo before the evolution of determinate cleavage in the tunicates and the evolution large amounts of yolk in basal vertebrates.

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.

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.  2004.  Tissue-specific expression of FoxD reporter constructs in amphioxus embryos. Developmental Biology. 274:452-461.   10.1016/j.ydbio.2004.07.010   AbstractWebsite

Cephalochordates (amphioxus), the closest living invertebrate relatives of the vertebrates, are key to understanding the evolution of developmental mechanisms during the invertebrate-to-vertebrate transition. However, a major impediment to amphioxus as a model organism for developmental biology has been the inability to introduce transgenes or other macromolecules into the embryos. Here, we report the development of a reproducible method for microinjection of amphioxus eggs. Specifically, we show that expression of a LacZ reporter construct including 6.3 kb of AmphiFoxD upstream regulatory DNA recapitulates expression of the endogenous gene in the nerve cord, somites, and notochord. We have also identified the 1.6 kb at the 5' end of this region as essential for expression in the first two of these domains and the 4.7 kb at the 3' end as sufficient for expression in the notochord. This study, which is the first report of a method for introduction of large molecules such as DNA into amphioxus embryos, opens the way for studies of gene regulation and function in amphioxus and for comparative studies with vertebrates to understand the relationship between the extensive gene duplications that occurred within the vertebrate lineage and the evolution of vertebrate innovations such as neural crest. (C) 2004 Elsevier Inc. All rights reserved.

Holland, LZ, Yu JK.  2004.  Cephalochordate (amphioxus) embryos: Procurement, culture, and basic methods. Development of Sea Urchins, Ascidians, and Other Invertebrate Deuterostomes: Experimental Approaches. 74:195-215.Website
Schubert, M, Meulemans D, Bronner-Fraser M, Holland LZ, Holland ND.  2003.  Differential mesodermal expression of two amphioxus MyoD family members (AmphiMRF1 and AmphiMRF2). Gene Expression Patterns. 3:199-202.   10.1016/s1567-133x(02)00099-6   AbstractWebsite

To explore the evolution of myogenic regulatory factors in chordates, we isolated two MyoD family genes (AmphiMRF1 and AmphiMRF2) from amphioxus. AmphiMRF1 is first expressed at the late gastrula in the paraxial mesoderm. As the first somites form, expression is restricted to their myotomal region. In the early larva, expression is strongest in the most anterior and most posterior somites. AmphiMRF2 transcription begins at mid/late gastrula in the paraxial mesoderm, but never spreads into its most anterior region. Through much of the neurula stage, AmphiMRF2 expression is strong in the myotomal region of all somites except the most anterior pair; by late neurula expression is downregulated except in the most posterior somites forming just rostral to the tail bud. These two MRF genes of amphioxus have partly overlapping patterns of mesodermal expression and evidently duplicated independent of the diversification of the vertebrate MRF family. (C) 2003 Elsevier Science B.V. All rights reserved.

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.

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, 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.

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>;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.

Holland, LZ, Pace DA, Blink ML, Kene M, Holland ND.  1995.  Sequence and Expression of Amphioxus Alkali Myosin Light-Chain (Amphimlc-Alk) Throughout Development - Implications for Vertebrate Myogenesis. Developmental Biology. 171:665-676.   10.1006/dbio.1995.1313   AbstractWebsite

The lower chordate amphioxus, widely considered the closest living invertebrate relative of the vertebrates, is a key organism for understanding the relationship between gene duplications and evolution of the complex vertebrate body plan. In tetrapod vertebrates, the alkali myosin light chain genes (MLC-alk), which code for proteins associated with the globular head of the myosin heavy chain, constitute a large family with stage-, tissue-, and fiber-type-specific expression of different isoforms thought to have arisen by duplication of a single ancestral gene. In protostome invertebrates, e.g., arthropods, molluscs, and nematodes, only one MLC-alk gene has been found, but the number of such genes in deuterostome invertebrates and lower vertebrates is unknown. The present report, describing the sequence and expression throughout development of the amphioxus gene for alkali myosin light chain (AmphiMLC-alk), thus fills a major gap in understanding the relation between gene duplication and increasing diversity of muscle-cell types. A full-length clone (1 kb) of AmphiMLC-alk was isolated from a larval amphioxus cDNA Library. It coded for a 149-amino-acid protein most closely related to the vertebrate embryonic form of MLC-alk. Southern blot analysis revealed only one copy of AmphiMLC-alk and suggested that it is the only MLC-alk gene in amphioxus. Northern blot analysis indicated that this gene produces only one transcript, which is expressed at all stages of development and in adults. In situ hybridizations showed expression initially in the myotomes of somites 2-5 of neurula embryos and soon thereafter in the myotomes of somite 1 and of newly forming somites progressively added posteriorly. Myotomal expression continues throughout larval development and into the adult stage as the myotomal cells differentiate into striated, mononucleate muscle cells-unlike vertebrate striated muscle cells, those of amphioxus never become multinucleate. In late larvae and adults myotomal expression of AmphiMLC-alk is localized along the medial edge of the myotome and at the ends of the cells. This is the first demonstration of intracellular localization of MLC transcripts in muscle cells of any animal. Expression of AmphiMLC-alk was also detected in smooth muscles as well as in striated muscles not derived from the myotome. These expression data are consistent with the Southern blot analysis in suggesting that there is only one MLC-alk gene in amphioxus. Thus, duplication of an ancestral vertebrate MLC-alk gene probably occurred after the vertebrate and amphioxus lineages split. We conclude that development of a segmented axial musculature preceded the evolution of multiple MLC-alk isoforms, which evidently arose about the time of multinucleation. Since myogenesis in amphioxus is similar to but far simpler than myogenesis in vertebrates at both the structural and gene levels, an understanding of myogenesis in amphioxus can give insights into both the evolutionary history and the detailed mechanisms of vertebrate myogenesis. (C) 1995 Academic Press, Inc.