Publications

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2011
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.

2007
Rasmussen, SLK, Holland LZ, Schubert M, Beaster-Jones L, Holland ND.  2007.  Amphioxus AmphiDelta: Evolution of delta protein structure, segmentation, and neurogenesis. Genesis. 45:113-122.   10.1002/dvg.20278   AbstractWebsite

The amphioxus genome has a single Delta gene (AmphiDelta) encoding a protein 766 amino acids long. Comparison of Delta proteins of amphioxus and other animals indicates that AmphiDelta retains features of a basal bilaterian Delta protein-in having nine epidermal growth factor (EGF) repeats and also in having char acteristic numbers of amino acids separating successive cysteines between and within EGF repeats. During development, AmphiDelta is expressed in the forming somites, in some regions of pharyngeal endoderm, and in cells (presumably differentiating neurons) scattered in both the neural plate and ectoderm. Expression is strongly associated with cells initiating movements to separate themselves from parent epithelia, either en masse by evagination (endoderm and mesoderm) or by delamination as isolated cells (ectoderm). The AmphiDelta-expressing cells delaminating from the ectoderm apparently migrate beneath it as they begin differentiating into probable sensory neurons, suggesting a scenario for the evolutionary origin of the placode-derived neurons of vertebrate cranial ganglia. genesis 45:113-122, 2007. Published 2007 Wiley-Liss, lnc.(dagger)

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.

2000
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.

1992
Holland, LZ, Holland ND.  1992.  Early Development in the Lancelet (= Amphioxus) Branchiostoma-Floridae from Sperm Entry through Pronuclear Fusion - Presence of Vegetal Pole Plasm and Lack of Conspicuous Ooplasmic Segregation. Biological Bulletin. 182:77-96.   10.2307/1542182   AbstractWebsite

Lancelet eggs are described from serial fine sections before fertilization and at frequent intervals thereafter until the male and female pronuclei meet at 16 min after insemination. In the unfertilized egg, although mitochondria, as well as yolk granules, are evenly distributed (both are absent only from the egg cortex and meiotic spindle), the mitochondria in the animal third have a more electron-lucent matrix than those elsewhere. The cortex of the unfertilized egg is occupied chiefly by cortical granules, and the subcortical cytoplasm in the vegetal third includes sheets of dense granules interleaved with cisternae of endoplasmic reticulum. By 45 s after insemination, (1) the fertilizing sperm enters (in the animal hemisphere in three out of three observations), (2) yolk granules become patchily distributed around the newly entered sperm, (3) cortical granule exocytosis occurs, and (4) the sheets of dense granules and associated endoplasmic reticulum aggregate with numerous mitochondria into whorls in a yolk-free zone near the vegetal pole. These whorls are the vegetal pole plasm, which is segregated into a single blastomere at each cleavage and might play a role in germ line determination. By 2 min after insemination, the zone of cytoplasm near the animal pole with patchily distributed yolk has enlarged, and the male pronucleus has migrated to the vicinity of the vegetal pole and formed an aster, at the center of which a few mitochondria are aggregated. In lancelets, unlike ascidians, there is no obvious widespread ooplasmic segregation or translocation of cytoplasm from animal to vegetal pole accompanying the movement of the sperm. Between 6 and 16 min, (1) the zone of cytoplasm with patchily distributed yolk enlarges to occupy about the animal third of the egg, (2) the female pronucleus forms by fusion of chromosome-containing vesicles and migrates vegetally, leaving a track of yolk-poor cytoplasm, and (3) the male pronucleus, surrounded by increasing numbers of mitochondria, migrates to meet the female pronucleus just above the equator. In contrast to current opinion, lancelets differ from ascidians both in having a vegetal pole plasm and in lacking marked ooplasmic segregation.