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Holland, LZ.  2000.  Body-plan evolution in the Bilateria: early antero-posterior patterning and the deuterostome-protostome dichotomy. Current Opinion in Genetics & Development. 10:434-442.   10.1016/s0959-437x(00)00109-x   AbstractWebsite

Recent molecular analyses reveal common themes in early antero-posterior patterning in the four major groups of invertebrate deuterostomes and vertebrates in spite of large differences in the mode of gastrulation. Comparisons with Drosophila and Cnidarians suggest a scheme for evolution of the Bilaterian body plan and emphasize the pressing need for similar studies in a wider variety of organisms, especially more basal protostomes.

Holland, LZ, Holland ND.  1999.  Chordate origins of the vertebrate central nervous system. Current Opinion in Neurobiology. 9:596-602.   10.1016/s0959-4388(99)00003-3   AbstractWebsite

Fine structural, computerized three-dimensional (3D) mapping of cell connectivity in the amphioxus nervous system and comparative molecular genetic studies of amphioxus and tunicates have provided recent insights into the phylogenetic origin of the vertebrate nervous system. The results suggest that several of the genetic mechanisms for establishing and patterning the vertebrate nervous system already operated in the ancestral chordate and that the nerve cord of the proximate invertebrate ancestor of the vertebrates included a diencephalon, midbrain, hindbrain, and spinal cord. In contrast, the telencephalon, a midbrain-hindbrain boundary region with organizer properties, and the definitive neural crest appear to be vertebrate innovations.

Holland, LZ, Schubert M, Kozmik Z, Holland ND.  1999.  AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest. Evolution & Development. 1:153-165.   10.1046/j.1525-142x.1999.99019.x   AbstractWebsite

Amphioxus probably has only a single gene (AmphiPax3/7 ) in the Pax3/7 subfamily. Like its vertebrate homologs (Pax3 and Pax7 ), amphioxus AmphiPax3/7 is probably involved in specifying the axial musculature and muscularized notochord. During nervous system development, AmphiPax3/7 is first expressed in bilateral anteroposterior stripes along the edges of the neural plate. This early neural expression may be comparable to the transcription of Pax3 and Pax7 in some of the anterior neural crest cells of vertebrates. Previous studies by others and ourselves have demonstrated that several genes homologous to genetic markers for vertebrate neural crest are expressed along the neural plate-epidermis boundary in embryos of tunicates and amphioxus. Taken together, the early neural expression patterns of AmphiPax3/7 and other neural crest markers of amphioxus and tunicates suggest that cell populations that eventually gave rise to definitive vertebrate neural crest may have been present in ancestral invertebrate chordates. During later neurogenesis in amphioxus, AmphiPax3/7, like its vertebrate homologs, is expressed dorsally and dorsolaterally in the neural tube and may be involved in dorsoventral patterning. However, unlike its vertebrate homologs, AmphiPax3/7 is expressed only at the anterior end of the central nervous system instead of along much of the neuraxis; this amphioxus pattern may represent the loss of a primitive chordate character.

Kozmik, Z, Holland ND, Kalousova A, Paces J, Schubert M, Holland LZ.  1999.  Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development. 126:1295-1304. AbstractWebsite

On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P.W.H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking, We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus, AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region, Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus, The alternative is that the MHB is a vertebrate innovation.

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, Satoh N, Holland LZ, Kusakabe T.  1999.  Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae. Gene. 227:1-10.   10.1016/s0378-1119(98)00608-8   AbstractWebsite

We previously described the cDNA. cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203-215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon-intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in Vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages and that gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates. (C) 1999 Elsevier Science B.V. All rights reserved.

Venkatesh, TV, Holland ND, Holland LZ, Su MT, Bodmer R.  1999.  Sequence and developmental expression of amphioxus AmphiNk2-1: insights into the evolutionary origin of the vertebrate thyroid gland and forebrain. Development Genes and Evolution. 209:254-259. AbstractWebsite

We characterized an amphioxus NK-2 homeobox gene (AmphiNk2-1), a homologue of vertebrate Nkx2-1, which is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2-1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented posteriorly and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2-1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions: this pattern is comparable to Nkx2-1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homologue of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain.

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

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

Holland, LZ, Holland ND.  1998.  Developmental gene expression in amphioxus: New insights into the evolutionary origin of vertebrate brain regions, neural crest, and rostrocaudal segmentation. American Zoologist. 38:647-658. AbstractWebsite

Amphioxus is widely held to be the closest invertebrate relative of the vertebrates and the best available stand-in for the proximate ancestor of the vertebrates. The spatiotemporal expression patterns of developmental genes can help suggest body part homologies between vertebrates and amphioxus, This approach is illustrated using five homeobox genes (AmphiHox1, AmphiHox2, AmphiOtx, AmphiDll, and AmphiEn) to pro,ide insights into the evolutionary origins of three important vertebrate features: the major brain regions, the neural crest, and rostrocaudal segmentation. During amphioxus development, the neural expression patterns of these genes are consistent with the presence of a forebrain (detailed neuroanatomy indicates that the forebrain is all diencephalon without any telencephalon) and an extensive hindbrain; the possible presence of a midbrain requires additional study. Further, during neurulation, the expression pattern of AmphiDll as web as migratory cell behavior suggest that the epidermal cells bordering the neural plate may represent a phylogenetic precursor of the vertebrate neural crest. Finally, when the paraxial mesoderm begins to segment, the earliest expression of AmphiEn is detected in the posterior part of each nascent and newly formed somite, This pattern recalls the expression of the segment-polarity gene engrailed during establishment of the segments of metameric protostomes. Thus, during animal evolution, the role of engrailed in establishing and maintaining metameric body plans may have arisen in a common segmented ancestor of both the protostomes and deuterostomes.

Glardon, S, Holland LZ, Gehring WJ, Holland ND.  1998.  Isolation and developmental expression of the amphioxus Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor evolution. Development. 125:2701-2710. AbstractWebsite

Pax-6 genes have been identified from a broad range of invertebrate and vertebrate animals and shown to be always involved in early eye development. Therefore, it has been proposed that the various types of eyes evolved from a single eye prototype, by a Pax-6-dependent mechanism. Here we describe the characterization of a cephalochordate Pax-6 gene. The single amphioxus Pax-6 gene (AmphiPax-6) can produce several alternatively spliced transcripts, resulting in proteins with markedly different amino and carboxy termini, The amphioxus Pax-6 proteins are 92% identical to mammalian Pax-6 proteins in the paired domain and 100% identical in the homeodomain. Expression of AmphiPax-6 in the anterior epidermis of embryos may be related to development of an olfactory epithelium. Expression is also detectable in Hatschek's left diverticulum as it forms the preoral ciliated pit, part of which gives rise to the homolog of the vertebrate anterior pituitary, A zone of expression in the anterior neural plate of early embryos is carried into the cerebral vesicle (a probable diencephalic homolog) during neurulation, This zone includes cells that will differentiate into the lamellar body, a presumed homolog of the vertebrate pineal eye, In neurulae, AmphiPax-6 is also expressed in ventral cells at the anterior tip of the nerve cord; these cells are precursors of the photoreceptive neurons of the frontal eye, the presumed homolog of the vertebrate paired eyes. However, AmphiPax-6 expression was not detected in two additional types of photoreceptors, the Joseph cells or the organs of Hesse, which are evidently relatively recent adaptations (ganglionic photoreceptors) and appear to be rare exceptions to the general rule that animal photoreceptors develop from a genetic program triggered by Pax-6.

Lacalli, TC, Holland LZ.  1998.  The developing dorsal ganglion of the salp Thalia democratica, and the nature of the ancestral chordate brain. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences. 353:1943-1967. AbstractWebsite

The development of the dorsal ganglion of the salp, Thalia democratica, is described from electron microscope reconstructions up to the stage of central neuropile formation. The central nervous system (CNS) rudiment is initially tubular with an open central canal. Early developmental events include: (i) the formation of a thick dorsal mantle of neuroblasts from which paired dorsal paraxial neuropiles arise; (ii) the differentiation of clusters of primary motor neurons along the ventral margin of the mantle; and (iii) the development from the latter of a series of peripheral nerves. The dorsal paraxial neuropiles ultimately connect to the large central neuropile, which develops later. Direct contact between neuroblasts and muscle appears to be involved in the development of some anterior nerves. The caudal nerves responsible for innervating more distant targets in the posterior part of the body develop without such contacts, which suggests that a different patterning mechanism may be employed in this part of the neuromuscular system. The results are compared with patterns of brain organization in other chordates. Because the salp CNS is symmetrical and generally less reduced than that of ascidian larvae, it is more easily compared with the CNS of amphioxus and vertebrates. The dorsal paraxial centres in the salp resemble the dorsolateral tectal centres in amphioxus in both position and organization; the central neuropile in salps likewise resembles the translumenal system in amphioxus. The neurons themselves are similar in that many of their neurites appear to be derived from the apical surface instead of the basal surface of the cell. Such neurons, with extensively developed apical neurites, may represent a new cell type that evolved in the earliest chordates in conjunction with the formation of translumenal or intralumenal integrative centres. In comparing the salp ganglion with vertebrates, we suggest that the main core of the ganglion is most like the mes-metencephalic region of the vertebrate brain, i.e. the zone occupied by the midbrain, isthmus, and anterior hindbrain. Counterparts of more anterior regions (forebrain) and posterior ones (segmented hindbrain) appear to be absent in salps, but are found in other tunicates, suggesting that evolution has acted quite differently on the main subdivisions of the CNS in different types of tunicates.

Schubert, M, Holland ND, Holland LZ.  1998.  Amphioxus AmphiDRAL encoding a LIM-domain protein: expression in the epidermis but not in the presumptive neuroectoderm. Mechanisms of Development. 76:203-205.   10.1016/s0925-4773(98)00120-8   AbstractWebsite

The sequence and developmental expression have been determined for amphioxus AmphiDRAL, which encodes a homolog of vertebrate DRAL (down-regulated in rhabdomyosarcoma LIM-protein). This is the first clear example of a DRAL homolog in an invertebrate. Detectable developmental expression begins at the gastrula stage in the epidermis, but not in the neuroectoderm; thus the early stages of AmphiDRAL expression indicate the neural/non-neural boundary. During subsequent embryonic stages, expression continues in the epidermis (but not in the developing central nervous system) until it fades during the later larval stages. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Holland, LZ, Venkatesh TV, Gorlin A, Bodmer R, Holland ND.  1998.  Characterization and developmental expression of AmphiNk2-2, an NK2 class homeobox gene from amphioxus (Phylum Chordata; Subphylum Cephalochordata). Development Genes and Evolution. 208:100-105.   10.1007/s004270050159   AbstractWebsite

The genome of amphioxus includes AmphiNk2-2, the first gene of the NK2 homeobox class to be demonstrated in any invertebrate deuterostome. AmphiNk2-2 encodes a protein with a TN domain, homeodomain, and NK2-specific domain; on the basis of amino acid identities in these conserved regions, AmphiNk2-2 is a homolog of Drosophila vnd and vertebrate Nkx2-2. During amphioxus development, expression of AmphiNk2-2 is first detected ventrally in the endoderm of late gastrulae. In neurulae, endodermal expression divides into three domains (the pharynx, midgut, and hindgut), and neural expression commences in two longitudinal bands of cells in the anterior neural tube. These neural tube cells occupy a ventrolateral position on either side of the cerebral vesicle (the probable homolog of the vertebrate diencephalic forebrain). The dynamic expression patterns of AmphiNFkx2-2 suggest successive roles, first in regionalizing the endoderm and nervous system and later during differentiation of specific cell types in the gut (possibly peptide endocrine cells) and brain (possibly including axon outgrowth and guidance).

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.

Holland, LZ, Kene M, Williams NA, Holland ND.  1997.  Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): The metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development. 124:1723-1732. AbstractWebsite

Vertebrate segmentation has been proposed as an evolutionary inheritance either from some metameric protostome or from a more closely related deuterostome, To address this question, we studied the developmental expression of AmphiEn, the engrailed gene of amphioxus, the closest living invertebrate relative of the vertebrates, In neurula embryos of amphioxus, AmphiEn is expressed along the anteroposterior axis as metameric stripes, each located in the posterior part of a nascent or newly formed segment, This pattern resembles the expression stripes of the segment-polarity gene engrailed, which has a key role in establishing and maintaining the metameres in embryos of Drosophila and other metameric protostomes, Later, amphioxus embryos express AmphiEn in non-metameric patterns - transiently in the embryonic ectoderm and dorsal nerve cord. Nerve cord expression occurs in a few cells approximately midway along the rostrocaudal axis and also in a conspicuous group of anterior cells in the cerebral vesicle at a level previously identified as corresponding to the vertebrate diencephalon. Compared to vertebrate engrailed expression at the midbrain/hindbrain boundary, AmphiEn expression in the cerebral vesicle is relatively late, Thus, it is uncertain whether the cerebral vesicle expression marks the rostral end of the amphioxus hindbrain; if it does, then amphioxus may have little or no homolog of the vertebrate midbrain, The segmental expression of AmphiEn in forming somites suggests that the functions of engrailed homologs in establishing and maintaining a metameric body plan may have arisen only once during animal evolution, If so, the protostomes and deuterostomes probably shared a common segmented ancestor.

Holland, LZ, McFallNgai M, Somero GN.  1997.  Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: Differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site. Biochemistry. 36:3207-3215.   10.1021/bi962664k   AbstractWebsite

Orthologous homologs of lactate dehydrogenase-a (LDH-A) (EC; NAD(+):lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent K-m) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in K-m with increasing measurement temperature occur for all homologs, yet K-m at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and K-m. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in K-m and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in K-m between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.

Zhang, SC, Holland ND, Holland LZ.  1997.  Topographic changes in nascent and early mesoderm in amphioxus embryos studied by Dil labeling and by in situ hybridization for a Brachyury gene. Development Genes and Evolution. 206:532-535.   10.1007/s004270050083   AbstractWebsite

In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates. resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently. during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions when segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead. expression is detectable only in the posterior mesoderm and in the notochord, but not in par axial mesoderm where definitive somites have formed.

Holland, ND, Zhang S-C, Clark M, Panopoulou GD, Lehrach H, Holland LZ.  1997.  Sequence and developmental expression of AmphiTob, an amphioxus homolog of vertebrate Tob in the PC3/BTG/Tob family of tumor suppressor genes. Developmental Dynamics. 210:11-18.
Holland, ND, Panganiban G, Henyey EL, Holland LZ.  1996.  Sequence and developmental expression of AmphiDII, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: Insights into evolution of craniate forebrain and neural crest. Development. 122:2911-2920. AbstractWebsite

The dynamic expression patterns of the single amphioxus Distal-less homolog (AmphiDll) during development are consistent with successive roles of this gene in global regionalization of the ectoderm, establishment of the dorsoventral axis, specification of migratory epidermal cells early in neurulation and the specification of forebrain, Such a multiplicity of Distal-less functions probably represents an ancestral chordate condition and, during craniate evolution, when this gene diversified into a family of six or so members, the original functions evidently tended to be parcelled out among the descendant genes, In the amphioxus gastrula, AmphiDll is expressed throughout the animal hemisphere (presumptive ectoderm), but is soon downregulated dorsally (in the presumptive neural plate), During early neurulation, AmphiDll-expressing epidermal cells flanking the neural plate extend lamellipodia, appear to migrate over it and meet mid-dorsally, Midway in neurulation, cells near the anterior end of the neural plate begin expressing AmphiDll and, as neurulation terminates, these cells are incorporated into the dorsal part of the neural tube, which forms by a curling of the neural plate, This group of AmphiDII-expressing neural cells and a second group expressing the gene a little later and even more anteriorly in the neural tube demarcate a region that comprises the anterior three/fourths of the cerebral vesicle; this region of the amphioxus neural tube, as judged by neural expression domains of craniate Distal-less-related genes, is evidently homologous to the craniate forebrain, Our results suggest that craniates evolved from an amphioxus-like creature that had the beginnings of a forebrain and possibly a precursor of neural crest - namely, the cell population leading the epidermal overgrowth of the neural plate during early neurulation.

Holland, LZ, Holland ND.  1996.  Expression of AmphiHox-1 and AmphiPax-1 in amphioxus embryos treated with retinoic acid: Insights into evolution and patterning of the chordate nerve cord and pharynx. Development. 122:1829-1838. AbstractWebsite

Excess all-trans retinoic acid (RA) causes severe craniofacial malformations in vertebrate embryos: pharyngeal arches are fused or absent, and a rostrad expansion of Hoxb-1 expression in the hindbrain shows that anterior rhombomeres are homeotically respecified to a more posterior identity. As a corollary, neural crest migration into the pharyngeal arches is abnormal. We administered excess RA to developing amphioxus, the closest invertebrate relative of the vertebrates and thus a key organism for understanding evolution of the vertebrate body plan. In normal amphioxus, the nerve cord has only a slight anterior swelling, the cerebral vesicle, and apparently lacks migratory neural crest. Nevertheless, excess RA similarly affects amphioxus and vertebrates. The expression domain of AmphiHox-1 (homologous to mouse Hoxb-1) in the amphioxus nerve cord is also extended anteriorly. For both the amphioxus and mouse genes, excess RA causes either (1) continuous expression throughout the preotic hindbrain (mouse) and from the level of somite 7 to the anterior end of the nerve cord (amphioxus) or (2) discontinuous expression with a gap in rhombomere 3 (mouse) and a gap at the posterior end of the cerebral vesicle (amphioxus). A comparison of these expression patterns suggests that amphioxus has a homolog of the vertebrate hindbrain, both preotic and postotic. Although RA alters the expression of AmphiHox-1 expression in the amphioxus nerve cord, it does not alter the expression of AmphiHox-1 in presomitic mesoderm or of alkali myosin light chain (AmphiMlc-alk) in somites, and the axial musculature and notochord develop normally. The most striking morphogenetic effect of RA on amphioxus larvae is the failure of mouth and gill slits to form. In vertebrates effects of excess RA on pharyngeal development have been attributed solely to the abnormal migratory patterns of Hox-expressing cranial neural crest cells. This cannot be true for amphioxus because of the lack of migratory neural crest. Furthermore, expression of Hox genes in pharyngeal tissues of amphioxus has not yet been detected. However, the absence of gill slits in RA-treated amphioxus embryos correlates with an RA-induced failure of AmphiPax-1 to become down-regulated in regions of pharyngeal endoderm that would normally fuse with the overlying ectoderm. In vertebrates, RA might similarly act via Pax-1/9, also expressed in pharyngeal endoderm, to impair pharyngeal patterning.

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

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

Holland, LZ, Holland PWH, Holland ND.  1996.  Revealing homologies between body parts of distantly related animals by in situ hydridization to developmental genes: Amphioxus vs vertebrates. Molecular zoology : advances, strategies, and protocols. ( Ferraris JD, Palumbi SR, Eds.).:267-282,473-483., New York: Wiley-Liss Abstract
Holland, ND, Holland LZ, Kozmik Z.  1995.  An Amphioxus Pax Gene, Amphipax-1, Expressed in Embryonic Endoderm, but Not in Mesoderm - Implications for the Evolution of Class-I Paired Box Genes. Molecular Marine Biology and Biotechnology. 4:206-214. AbstractWebsite

Class I paired box genes are widely distributed through the animal phyla but only fruitfly Pox meso and vertebrate Pax-1 and Pax-9 have been adequately characterized. These vertebrate genes have several developmental functions, but their role in patterning the axial skeleton has received the most attention. Because axial skeletons appear after the origin of the vertebrates, special interest attaches to the possible functions of the precursors of Pax-1 and Pax-g in the invertebrate ancestor of the vertebrates. As a proxy for this ancestor, we studied amphioxus, which is widely thought to be the closest living invertebrate relative of the vertebrates. A cDNA library from developing amphioxus yielded an unequivocal class I paired box gene, AmphiPax-1, that is 2.5 kb long. The gene encodes a 337 amino acid protein that includes a paired domain in which the amino acids are 92% identical to the paired domain amino acids of mouse and human Pax-1 and Pax-g. In situ hybridization detects AmphiPax-1 expression only in the endoderm of the developing pharynx; within this tissue, expression becomes strikingly down-regulated in regions that will fuse with the overlying ectoderm to form gill slits. No transcripts of AmphiPax-1 ever become detectable in any mesodermal structures. We think it likely that, during animal evolution, class I paired box genes originally functioned in endoderm development and were only later co-opted for other roles in mesoderm development; however, other scenarios cannot be ruled out until homologues of these genes are studied in more invertebrate phyla and in the lower vertebrates.

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

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

Holland, PWH, Koschorz B, Holland LZ, Herrmann BG.  1995.  Conservation of Brachyury (T) genes in amphioxus and vertebrates: Developmental and evolutionary implications. Development. 121:4283-4291. AbstractWebsite

Homologues of the murine Brachyury (T) gene have been cloned from several vertebrates, and are implicated in mesoderm formation and in differentiation of the notochord, In contrast, the roles of the ascidian Brachyury gene may be restricted to presumptive notochord, To understand the evolution of Brachyury genes and their developmental roles, we have searched for homologues in amphioxus, representing the third chordate subphylum and the probable closest relative of the vertebrates. We report the isolation of two amphioxus cDNA clones with clear homology to Brachyury genes, and demonstrate that these derive from separate loci resultant from a recent gene duplication. This finding represents an exception to the emerging consensus of an archetypal prevertebrate genome in amphioxus, The spatial and temporal distribution of Brachyury transcripts during amphioxus development is remarkably similar to vertebrate Brachyury, in presumptive mesoderm, posterior mesoderm and the notochord, Gene expression extends throughout the anteroposterior axis of the notochord, despite the most rostral regions being a more recent specialization; it also persists into larval stages, despite differentiation into contractile tissue, We propose that roles of Brachyury in notochord differentiation are more ancient than roles in mesoderm formation, and that the latter are shared by cephalochordates and all vertebrates.