Publications

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2010
Holland, LZ, Sower SA.  2010.  "Insights of Early Chordate Genomics: Endocrinology and Development in Amphioxus, Tunicates and Lampreys": Introduction to the symposium. Integrative and Comparative Biology. 50:17-21.   10.1093/icb/icq039   AbstractWebsite

This symposium focused on the evolution of chordate genomes, in particular, those events that occurred before the appearance of jawed vertebrates. The aim was to highlight insights that have come from the genome sequences of jawless chordates (lampreys, tunicates, and amphioxus) not only into evolution of chordate genomes, but also into the evolution of the organism. To this end, we brought together researchers whose recent work on these organisms spans the gap from genomics to the evolution of body forms and functions as exemplified by endocrine systems and embryonic development.

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.

Onai, T, Yu JK, Blitz IL, Cho KWY, Holland LZ.  2010.  Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus. Developmental Biology. 344:377-389.   10.1016/j.ydbio.2010.05.016   AbstractWebsite

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity Knockdown and gain-of-function experiments show that these pathways act in opposition to one another Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution (C) 2010 Elsevier Inc. All rights reserved

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.

Boldrin, F, Martinucci G, Holland LZ, Miller RL, Burighel P.  2009.  Internal fertilization in the salp Thalia democratica. Canadian Journal of Zoology-Revue Canadienne De Zoologie. 87:928-940.   10.1139/z09-083   AbstractWebsite

Among tunicates, gamete morphology and sperm-egg interactions have been extensively investigated in ascidians, and to a lesser extent in appendicularians and thaliaceans. Sperm-egg interaction has been studied in only one salp, Pegea socia (Bosc, 1802). To determine if the pattern of internal fertilization of P. socia is generally applicable to salps, we performed an ultrastructural study on blastozooids of Thalia democratica (Forsskal, 1775). The ovary, located in the mantle near the gut, consists of a single oocyte connected to the atrial chamber wall by a "fertilization duct", resembling a stack of single cells without a lumen. The flagellate sperm has a long corkscrew-like head with the single mitochondrion twisted around the nucleus. Fertilization is internal, and sperm actively penetrate the atrial wall and bore through the cells of the fertilization duct. During this process, the fertilization duct shortens as the cells move apart, one to one side and the next to the other, and rejoin to form a central lumen, which contains many sperm. At the same time a few sperm reach the periovular space for fertilizing the oocyte. Comparisons with P. socia indicate that this singular mode of internal fertilization with a complex corkscrew sperm actively penetrating the fertilization duct cells, probably evolved in the salp ancestor and has been modified to some extent in various genera.

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.

Onai, T, Lin HC, Schubert M, Koop D, Osborne PW, Alvarez S, Alvarez R, Holland ND, Holland LZ.  2009.  Retinoic acid and Wnt/beta-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus. Developmental Biology. 332:223-233.   10.1016/j.ydbio.2009.05.571   AbstractWebsite

A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, Upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal. (C) 2009 Elsevier Inc. All rights reserved.

Yu, J-K, Holland LZ.  2009.  Amphioxus (Branchiostoma floridae) spawning and embryo collection . Cold Spring Harbor Protocols. 2009:pdb.prot5285 (9)   10.1101/pdb.prot5285  
Yu, J-K, Holland LZ.  2009.  Amphioxus whole-mount in situ hybridization. Cold Spring Harbor Protocols. 2009:pdb.prot5286(9)   10.1101/pdb.prot5286  
Yu, J-K, Holland LZ.  2009.  Cephalochordates (amphioxus or lancelets): a model for understanding the evolution of chordate characters . Cold Spring Harbor Protocols. 2009:pdb.emo130. doi: (9)   10.1101/pdb.emo130  
Yu, J-K, Holland LZ.  2009.  Extraction of DNA from adult amphioxus tissue. . Cold Spring Harbor Protocols. 2009:pdb.prot5287(9)   10.1101/pdb.prot5287  
Yu, J-K, Holland LZ.  2009.  Extraction of RNA from amphioxus embryos or adult amphioxus tissues. . Cold Spring Harbor Protocols. 2009:pdb.prot5288. (9)   10.1101/pdb.prot5288.  
Yu, J-K, Holland LZ.  2009.  Extraction of RNA from small amounts of amphioxus embryos. Cold Spring Harbor Protocols. 2009:pdb.prot5289.   10.1101/pdb.prot5289  
2008
Holland, LZ, Holland ND, Gilland E.  2008.  Amphioxus and the evolution of head segmentation. Integrative and Comparative Biology. 48:630-646.   10.1093/icb/icn060   AbstractWebsite

Whether or not the vertebrate head is fundamentally segmented has been controversial for over 150 years. Beginning in the late 19th century, segmentalist theories proposed that the vertebrate head evolved from an amphioxus-like ancestor in which mesodermal somites extended the full length of the body with remnants of segmentation persisting as the mesodermal head cavities of sharks and lampreys. Antisegmentalists generally argued either that the vertebrate ancestors never had any mesodermal segmentation anteriorly or that they lost it before the origin of the vertebrates; in either case, the earliest vertebrates had an unsegmented head and the embryonic cranial mesoderm of vertebrates is at best pseudo-segmented, evolving independently of any pre-vertebrate segmental pattern. Recent morphologic studies have generally confirmed the accuracy of the major classical studies of head development in lampreys and sharks, yet disagree with their theoretical conclusions regarding the evolution of head segmentation. Studies of developmental genes in amphioxus and vertebrates, which have demonstrated conservation of the mechanisms of anteriorposterior patterning in the two groups, have shed new light on this controversy. Most pertinently, some homologs of genes expressed in the anterior amphioxus somites, which form as outpocketings of the gut, are also expressed in the walls of the head cavities of lampreys, which form similarly, and in their major derivatives (the velar muscles) as well as in the eye and jaw muscles of bony gnathostomes, which derive from unsegmented head mesoderm. These muscles share gene expression with the corresponding muscles of the shark, which derive from the walls of head cavities that form, not as outpocketings of the gut, but as secondary cavities within solid blocks of tissue. While molecular data that can be compared across all the relevant taxa remain limited, they are consistent with an evolutionary scenario in which the cranial paraxial mesoderm of the lamprey and shark evolved from the anterior somites of an amphioxus-like ancestor. Although, bony vertebrates have lost the mesodermal head segments present in the shark and lamprey, their remnants persist in the muscles of the eye and jaw.

Putnam, NH, Butts T, Ferrier DEK, Furlong RF, Hellsten U, Kawashima T, Robinson-Rechavi M, Shoguchi E, Terry A, Yu JK, Benito-Gutierrez E, Dubchak I, Garcia-Fernandez J, Gibson-Brown JJ, Grigoriev IV, Horton AC, de Jong PJ, Jurka J, Kapitonov VV, Kohara Y, Kuroki Y, Lindquist E, Lucas S, Osoegawa K, Pennacchio LA, Salamov AA, Satou Y, Sauka-Spengler T, Schmutz J, Shin-I T, Toyoda A, Bronner-Fraser M, Fujiyama A, Holland LZ, Holland PWH, Satoh N, Rokhsar DS.  2008.  The amphioxus genome and the evolution of the chordate karyotype. Nature. 453:1064-U3.   10.1038/nature06967   AbstractWebsite

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic similar to 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

Short, S, Holland LZ.  2008.  The evolution of alternative splicing in the Pax family: The view from the basal chordate amphioxus. Journal of Molecular Evolution. 66:605-620.   10.1007/s00239-008-9113-5   AbstractWebsite

Pax genes encode transcription factors critical for metazoan development. Large-scale gene duplication with subsequent gene losses during vertebrate evolution has resulted in two human genes for each of the Pax1/9, Pax3/7, and Pax4/6 subfamilies and three for the Pax2/5/8 subfamily, compared to one each in the cephalochordate amphioxus. In addition, alternative splicing occurs in vertebrate Pax transcripts from all four subfamilies, and many splice forms are known to have functional importance. To better understand the evolution of alternative splicing within the Pax family, we systematically surveyed transcripts of the four amphioxus Pax genes. We have found alternative splicing in every gene. Comparisons with vertebrates suggest that the number of alternative splicing events per gene has not decreased following duplication; there are comparable levels in the four amphioxus Pax genes as in each gene of the equivalent vertebrate families. Thus, the total number of isoforms for the nine vertebrate genes is considerably higher than for the four amphioxus genes. Most alternative splicing events appear to have arisen since the divergence of amphioxus and vertebrate lineages, suggesting that differences in alternative splicing could account for divergent functions of the highly conserved Pax genes in both lineages. However, several events predicted to dramatically alter known functional domains are conserved between amphioxus and vertebrates, suggestive of a common chordate function. Our results, together with previous studies of vertebrate Pax genes, support the theory that alternative splicing impacts functional motifs more than gene duplication followed by divergence.

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.

Yu, JK, Wang MC, Shin T, Kohara Y, Holland LZ, Satoh N, Satou Y.  2008.  A cDNA resource for the cephalochordate amphioxus Branchiostoma floridae. Development Genes and Evolution. 218:723-727.   10.1007/s00427-008-0228-x   AbstractWebsite

Cephalochordates are the basal invertebrate chordates within the phylum Chordata. They are widely used as a model system for research in evolutionary developmental biology (EvoDevo) to understand the basic patterning mechanisms for the chordate body plan and the origin of vertebrates. Recently, the genome of the cephalochordate Branchiostoma floridae was sequenced, which further brings this organism to the front for comparative genomic studies. In this paper, we report the generation of large-scale 5'- and 3'-expressed sequence tags (ESTs) from B. floridae and the complementary deoxyribonucleic acid (cDNA) resource for this species. Both 5'- and 3'-ESTs were sequenced for approximately 140,000 cDNA clones derived from five developmental stages, and the cDNA clones were subsequently grouped into independent clusters using 3'-EST sequences. We identified 21,229 cDNA clusters, and each corresponds to a unique transcript species from B. floridae. We then chose 24,020 cDNA clones representing all of these 21,229 clusters to generate the "Branchiostoma floridae Gene Collection Release 1." We also constructed a database with a searchable interface for this EST dataset and the related information on "Branchiostoma floridae Gene Collection Release 1." This set of cDNA clones along with our cDNA database will serve as an important resource for future research in this basal chordate. This Gene Collection and the original 140,000 individual cDNA clones are available to the research community upon request.

Beaster-Jones, L, Kaltenbach S, Koop D, Yuan SC, Chastain R, Holland LZ.  2008.  Expression of somite segmentation genes in amphioxus: a clock without a wavefront? Development Genes and Evolution. 218:599-611.   10.1007/s00427-008-0257-5   AbstractWebsite

In the basal chordate amphioxus (Branchiostoma), somites extend the full length of the body. The anteriormost somites segment during the gastrula and neurula stages from dorsolateral grooves of the archenteron. The remaining ones pinch off, one at a time, from the tail bud. These posterior somites appear to be homologous to those of vertebrates, even though the latter pinch off from the anterior end of bands of presomitic mesoderm rather than directly from the tail bud. To gain insights into the evolution of mesodermal segmentation in chordates, we determined the expression of ten genes in nascent amphioxus somites. Five (Uncx4.1, NeuroD/atonal-related, IrxA, Pcdh delta 2-17/18, and Hey1) are expressed in stripes in the dorsolateral mesoderm at the gastrula stage and in the tail bud while three (Paraxis, Lcx, and Axin) are expressed in the posterior mesendoderm at the gastrula and neurula stages and in the tail bud at later stages. Expression of two genes (Pbx and OligA) suggests roles in the anterior somites that may be unrelated to initial segmentation. Together with previous data, our results indicate that, with the exception that Engrailed is only segmentally expressed in the anterior somites, the genetic mechanisms controlling formation of both the anterior and posterior somites are probably largely identical. Thus, the fundamental pathways for mesodermal segmentation involving Notch-Delta, Wnt/beta-catenin, and Fgf signaling were already in place in the common ancestor of amphioxus and vertebrates although budding of somites from bands of presomitic mesoderm exhibiting waves of expression of Notch, Wnt, and Fgf target genes was likely a vertebrate novelty. Given the conservation of segmentation gene expression between amphioxus and vertebrate somites, we propose that the clock mechanism may have been established in the basal chordate, while the wavefront evolved later in the vertebrate lineage.

Holland, LZ, Holland ND, Gilland E.  2008.  Amphioxus and the evolution of head segmentation. Integrative and Comparative Biology. 48:630-646.
Koop, D, Holland LZ.  2008.  The basal chordate amphioxus as a simple model for elucidating developmental mechanisms in vertebrates. Birth Defects Research C, Embryo Today. 218:723-727.
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
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.

Holland, LZ.  2007.  Developmental biology - A chordate with a difference. Nature. 447:153-155.   10.1038/447153a   Website