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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 Most of these scenarios are outdated, but several are still seriously considered.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.

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

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

Holland, LZ.  2012.  Amphioxus genomics. Briefings in Functional Genomics. 11:87-88.   10.1093/bfgp/els014   Website
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.

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.

Holland, ND, Holland LZ.  2010.  Laboratory Spawning and Development of the Bahama Lancelet, Asymmetron lucayanum (Cephalochordata): Fertilization Through Feeding Larvae. Biological Bulletin. 219:132-141. AbstractWebsite

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

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.

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.

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, 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

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

Schubert, M, Holland ND, Escriva H, Holland LZ, Laudet V.  2004.  Retinoic acid influences anteroposterior positioning of epidermal sensory neurons and their gene expression in a developing chordate (amphioxus). Proceedings of the National Academy of Sciences of the United States of America. 101:10320-10325.   10.1073/pnas.0403216101   AbstractWebsite

In developing chordates, retinoic acid (RA) signaling patterns the rostrocaudal body axis globally and affects gene expression locally in some differentiating cell populations. Here we focus on development of epidermal sensory neurons in an invertebrate chordate (amphioxus) to determine how RA signaling influences their rostrocaudal distribution and gene expression (for AmphiCoe, a neural precursor gene; for amphioxus islet and AmphiERR, two neural differentiation genes; and for AmphiHox1,-3, -4, and -6). Treatments with RA or an RA antagonist (BMS009) shift the distribution of developing epidermal neurons anteriorly or posteriorly, respectively. These treatments also affect gene expression patterns in the epidermal neurons, suggesting that RA levels may influence specification of neuronal subtypes. Although colinear expression of Hox genes is well known for the amphioxus central nervous system,we find an unexpected comparable colinearity for AmphiHox1, -3, -4, and -6 in the developing epidermis; moreover, RA levels affect the anteroposterior extent of these Hox expression domains, suggesting that RA signaling controls a colinear Hox code for anteroposterior patterning of the amphioxus epidermis. Thus, in amphioxus, the developing peripheral nervous system appears to be structured by mechanisms parallel to those that structure the central nervous system. One can speculate that, during evolution, an ancestral deuterostome that structured its panepidermal nervous system with an RA-influenced Hox code gave rise to chordates in which this patterning mechanism persisted within the epidermal elements of the peripheral nervous system and was transferred to the neuroectoderm as the central nervous system condensed dorsally.

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
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.

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.

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
Yu, JK, Holland LZ, Holland ND.  2002.  An amphioxus nodal gene (AmphiNodal) with early symmetrical expression in the organizer and mesoderm and later asymmetrical expression associated with left-right axis formation. Evolution & Development. 4:418-425.   10.1046/j.1525-142X.2002.02030.x   AbstractWebsite

The full- length sequence and zygotic expression of an amphioxus nodal gene are described. Expression is first detected in the early gastrula just within the dorsal lip of the blastopore in a region of hypoblast that is probably comparable with the vertebrate Spemann's organizer. In the late gastrula and early neurula, expression remains bilaterally symmetrical, limited to paraxial mesoderm and immediately overlying regions of the neural plate. Later in the neurula stage, all neural expression disappears, and mesodermal expression disappears from the right side. All along the left side of the neurula, mesodermal expression spreads into the left side of the gut endoderm. Soon thereafter, all expression is down- regulated except near the anterior and posterior ends of the animal, where transcripts are still found in the mesoderm and endoderm on the left side. At this time, expression also begins in the ectoderm on the left side of the head, in the region where the mouth later forms. These results suggest that amphioxus and vertebrate nodal genes play evolutionarily conserved roles in establishing Spemann's organizer, patterning the mesoderm rostrocaudally and setting up the asymmetrical left - right axis of the body.

Yu, JK, Holland LZ, Jamrich M, Blitz IL, Holland ND.  2002.  AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development. Evolution & Development. 4:9-15.   10.1046/j.1525-142x.2002.01057.x   AbstractWebsite

The full-length sequence and developmental expression of amphioxus AmphiFoxE4 are described. Transcripts of the gene are first detected in the pharyngeal endoderm, where the club-shaped gland is forming and subsequently in the definitive gland itself. AmphiFoxE4 is closely related to vertebrate genes encoding the thyroid-specific transcription factor-2 (TTF2), which plays an early developmental role in the morphogenesis of the thyroid gland and a later role in hormone-mediated control of thyroid function. In amphioxus, AmphiFoxE4 expression is not thyroid specific because the club-shaped gland, the only structure expressing the gene, is not homologous to the vertebrate thyroid; in-stead, the thyroid homologue of amphioxus is a specialized region of the pharyngeal endoderm called the endostyle. We propose that (a) the pharynx of an amphioxus-like ancestor of the vertebrates included a club-shaped gland that expressed FoxE4 as well as an endostyle that did not, and (b) the club-shaped gland soon disappeared in the vertebrate line of descent but (c) not before there was a homeogenetic transfer of FoxE4 expression from the club-shaped gland to the nearby endostyle. Such a transfer could have provided part of the genetic program enabling the endostyle to separate from the pharyngeal endoderm and migrate away as the rudiment of the thyroid gland.

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.

Kusakabe, R, Kusakabe T, Satoh N, Holland ND, Holland LZ.  1997.  Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: Implications for conserved mechanisms of chordate development. Development Genes and Evolution. 207:203-215.   10.1007/s004270050109   AbstractWebsite

The cephalochordate amphioxus is thought to share a common ancestor with vertebrates. To investigate the evolution of developmental mechanisms in chordates, cDNA clones for two amphioxus actin genes, BfCA1 and BfCA1, were isolated. BfCA1 encodes a cytoplasmic actin and is expressed in a variety of tissues during embryogenesis, beginning in the dorsolateral mesendoderm of the mid-gastrula. At the open neural plate stage, BfCA1 transcripts accumulate at the bases of the neuroectodermal cells adjacent the presumptive notochord. The 3' untranslated region of BfCA1 contains a sequence that is similar to the ''zipcode'' sequence of chicken beta-cytoplasmic actin gene, which is thought to direct intracellular mRNA localization. BfCA1 is also expressed in the notochord through the early larval stage, in the pharynx and in the somites at the onset of muscle-cell differentiation. BfMA1 is a vertebrate-type muscle actin gene, although the deduced amino acid sequence is fairly divergent. Transcripts first appear in the early neurula in the somites as they begin to differentiate into axial muscle cells and persist into the adult stage. In young adults, transcripts are localized in the Z-discs of the muscle cells. Smooth muscle cells around the gill slits and striated muscle cells in the pterygeal muscle also express BfMA1; however, there is never any detectable expression in the notochord, which is a modified striated muscle. Together with the alkali myosin light chain gene AmphiMLC-alk, the sequence and muscle-specific expression of BfMA1 implies a conserved mechanism of muscle cell differentiation between amphioxus and vertebrates. Evolution of the chordate actin gene family is discussed based on molecular phylogenetic analysis and expression patterns of amphioxus actin genes.