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

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

Holland, LZ, Gibson-Brown JJ.  2003.  The Ciona intestinalis genome: when the constraints are off. Bioessays. 25:529-532.   10.1002/bies.10302   AbstractWebsite

The recent genome sequencing of a non-vertebrate deuterostome, the ascidian tunicate Ciona intestinalis, makes a substantial contribution to the fields of evolutionary and developmental biology.((1)) Tunicates have some of the smallest bilaterian genomes, embryos with relatively few cells, fixed lineages and early determination of cell fates. Initial analyses of the C. intestinalis genome indicate that it has been evolving rapidly. Comparisons with other bilaterians show that C. intestinalis has lost a number of genes, and that many genes linked together in most other bilaterians have become uncoupled. In addition, a number of independent, lineage-specific gene duplications have been detected. These new results, although interesting in themselves, will take on a deeper significance once the genomes of additional invertebrate deuterostomes (e.g. echinoderms, hemichordates and amphioxus) have been sequenced. With such a broadened database, comparative genomics can begin to ask pointed questions about the relationship between the evolution of genomes and the evolution of bodyplans. BioEssays25:529-532,2003. (C) 2003 Wiley Periodicals, Inc.

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
Holland, LZ, Gorsky G, Fenaux R.  2003.  A diversity of sperm in appendicularians. Are appendicularians monophyletic? pp. 210-239. Response of Marine Ecosystems to Global Change: Ecological Impact of Appendicularians. ( Gorsky G, Youngbluth M, Deibel D, Eds.).: Landes Biosciences
2002
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 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.

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

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

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.

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

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

Holland, LZ.  2002.  Heads or tails? Amphioxus and the evolution of anterior-posterior patterning in deuterostomes Developmental Biology. 241:209-228.   10.1006/dbio.2001.0503   AbstractWebsite

In Xenopus, the canonical Wnt-signaling pathway acting through (beta-catenin functions both in establishing the dorso-ventral axis and in patterning the anterior-posterior axis. This pathway also acts in patterning the animal-vegetal axis in sea urchins. However, because sea urchin development is typically indirect, and adult sea urchins have pentamerous symmetry and lack a longitudinal nerve cord, it has not been clear how the roles of the canonical Wnt-signaling pathway in axial patterning in sea urchins and vertebrates are evolutionarily related. The developmental expression patterns of Notch, brachyury, caudal, and eight Wnt genes have now been determined for the invertebrate chordate amphioxus, which, like sea urchins, has an early embryo that gastrulates by invagination, but like vertebrates, has a later embryo with a dorsal hollow nerve cord that elongates posteriorly from a tail bud. Comparisons of amphioxus with other deuterostomes suggest that patterning of the ancestral deuterostome embryo along its anterior-posterior axis during the late blastula and subsequent stages involved a posterior signaling center including Writs, Notch, and transcription factors such as brachyury and caudal. In tunicate embryos, in which cell numbers are reduced and cell fates largely determined during cleavage stages, only vestiges of this signaling center are still apparent; these include localization of Wnt-5 mRNA to the posterior cytoplasm shortly after fertilization and localization of beta-catenin to vegetal nuclei during cleavage stages. Neither in tunicates nor in amphioxus is there any evidence that the canonical Wnt-signaling pathway functions in establishment of the dorso-ventral axis. Thus, roles for Wnt-signaling in dorso-ventral patterning of embryos may be a vertebrate innovation that arose in connection with the evolution of yolky eggs and gastrulation by extensive involution. (C) 2001 Elsevier Science.

2001
Holland, LZ, Holland ND.  2001.  Evolution of neural crest and placodes: amphioxus as a model for the ancestral vertebrate? Journal of Anatomy. 199:85-98.   10.1046/j.1469-7580.199.parts1-2.8.x   AbstractWebsite

Recent studies of protochordates (ascidian tunicates and amphioxus) have given insights into possible ancestors of 2 of the characteristic features of the vertebrate head: neural crest and placodes. The neural crest probably evolved from cells on either side of the neural plate-epidermis boundary in a protochordate ancestral to the vertebrates. In amphioxus, homologues of several vertebrate neural crest marker genes (BMP2/4, Pax3/7, Msx, Dll and Snail) are expressed at the edges of the neural plate and/or adjacent nonneural ectoderm. Some of these markers are also similarly expressed in tunicates. In protochordates, however, these cells, unlike vertebrate neural crest, neither migrate as individuals through embryonic tissues nor differentiate into a wide spectrum of cell types. Therefore, while the protochordate ancestor of the vertebrates probably had the beginnings of a genetic programme for neural crest formation, this programme was augmented in the earliest vertebrates to attain definitive neural crest. Clear homologues of vertebrate placodes are lacking in protochordates. However, both amphioxus and tunicates have ectodermal sensory cells. In tunicates these are all primary neurons, sending axons to the central nervous system, while in amphioxus, the ectodermal sensory cells include both primary neurons and secondary neurons lacking axons. Comparisons of developmental gene expression suggest that the anterior ectoderm in amphioxus may be homologous to the vertebrate olfactory placode, the only vertebrate placode with primary, not secondary, neurons. Similarly, biochemical, morphological and gene expression data suggest that amphioxus and tunicates also have homologues of the adenohypophysis, one of the few vertebrate structures derived from nonneurogenic placodes. In contrast, the origin of the other vertebrate placodes is very uncertain.

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

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

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

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

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

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

Holland, LZ, Schubert M, Holland LZ, Neuman T.  2001.  Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Developmental Biology. 232:493-508.
Holland, LZ, Holland ND.  2001.  Amphioxus and the evolutionary origin of the vertebrate neural crest and midbrain/hindbrain boundary. Major events in early vertebrate evolution : palaeontology, phylogeny, genetics, and development. ( Ahlberg P, Ed.).:15-32., London ; New York: Taylor & Francis Abstract
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2000
Langlois, MC, Vanacker JM, Holland ND, Escriva H, Queva C, Laudet V, Holland LZ.  2000.  Amphicoup-TF, a nuclear orphan receptor of the lancelet Branchiostoma floridae, is implicated in retinoic acid signalling pathways. Development Genes and Evolution. 210:471-482.   10.1007/s004270000087   AbstractWebsite

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

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

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

Holland, LZ, Schubert M, Holland ND, Neuman T.  2000.  Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Developmental Biology. 226:18-33.   10.1006/dbio.2000.9810   AbstractWebsite

Amphioxus, as the closest living invertebrate relative of the vertebrates, can give insights into the evolutionary origin of the vertebrate body plan. Therefore, to investigate the evolution of genetic mechanisms for establishing and patterning the neuroectoderm, we cloned and determined the embryonic expression of two amphioxus transcription factors, AmphiSox1/2/3 and AmphiNeurogenin. These genes are the earliest known markers for presumptive neuroectoderm in amphioxus. By the early neurula stage, AmphiNeurogenin expression becomes restricted to two bilateral columns of segmentally arranged neural plate cells, which probably include precursors of motor neurons. This is the earliest indication of segmentation in the amphioxus nerve cord, Later, expression extends to dorsal cells in the nerve cord, which may include precursors of sensory neurons. By the midneurula, AmphiSox1/2/3 expression becomes limited to the dorsal part of the forming neural tube. These patterns resemble those of their vertebrate and Drosophila homologs. Taken together with the evolutionarily conserved expression of the dorsoventral patterning genes, BLP2/4 and chordin, in nonneural and neural ectoderm, respectively, of chordates and Drosophila, our results are consistent with the evolution of the chordate dorsal nerve cord and the insect ventral nerve cord from a longitudinal nerve cord in a common bilaterian ancestor. However, AmphiSox1/2/3 differs from its vertebrate homologs in not being expressed outside the CNS, suggesting that additional roles for this gene have evolved in connection with gene duplication in the vertebrate lineage. In contrast, expression in the midgut of AmphiNeurogenin together with the gene encoding the insulin-like peptide suggests that amphioxus may have homologs of vertebrate pancreatic islet cells, which express neurogenin3. In addition, AmphiNeurogenin, like its vertebrate and Drosophila homologs, is expressed in apparent precursors of epidermal chemosensory and possibly mechanosensory cells, suggesting a common origin for protostome and deuterostome epidermal sensory cells in the ancestral bilaterian. (C) 2000 Academic Press.

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

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

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

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

Schubert, M, Holland LZ, Holland ND.  2000.  Characterization of two amphioxus Wnt genes (AmphiWnt4 and AmphiWnt7b) with early expression in the developing central nervous system. Developmental Dynamics. 217:205-215.   10.1002/(sici)1097-0177(200002)217:2<205::aid-dvdy7>3.0.co;2-f   AbstractWebsite

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

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

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