Evolution of the ascidian anural larva: evidence from embryos and molecules. (17/1306)

Most ascidians pass through a tadpole (urodele) larval stage, although some species have derived a tailless (anural) larva. New insights into the evolution of anural larvae in the Roscovita clade of molgulid ascidians were obtained from studing embryonic development of the transitional anural species Molgula bleizi and from phylogenetic analysis based on muscle and cytoskeletal actin gene sequences. By observing in vitro fertilized eggs, we found that M. bleizi, previously described as a typical anural developer, actually forms a short immotile tail during embryogenesis. The short tail contains notochord lineage cells, which undergo abbreviated morphogenetic movements but eventually arrest in development. Molgula bleizi tail muscle lineage cells produce the muscle enzyme acetylcholinesterase (AChE) but do not express muscle actin genes. The presence of a short tail, a vestigial notochord, and AChE-positive muscle cells suggest that M. bleizi is a recently derived anural species. An M. bleizi larval muscle actin gene (MbMA1) was isolated, sequenced, and shown to be a pseudogene based on critical deletions in its coding region that would result in a nonfunctional actin protein. The mutations in MbMA1 are distinct from and have evolved independent of the larval muscle actin pseudogenes MoccMA1a and MoccMA1b in Molgula occulta, another anural developer in the Roscovita clade. Pseudogene formation explains the absence of muscle actin mRNA in M. bleizi embryos. The 3' untranslated region of an M. bleizi cytoskeletal actin gene was also isolated and sequenced. Phylogenetic trees reconstructed using muscle and cytoskeletal actin sequences suggest that the anural developer M. bleizi evolved prior to the divergence of the urodele developer Molgula oculata and the anural developer M. occulta in the Roscovita clade. Since M. bleizi lives attached to hard substrata in the tidal zone, whereas M. oculata and M. occulta live buried in subtidal sand flats, our results suggest that the anural larva evolved at least twice in the Roscovita clade of molgulid ascidians as an adaptation to different habitats.  (+info)

Anteroposterior patterning and organogenesis of Xenopus laevis require a correct dose of germ cell nuclear factor (xGCNF). (18/1306)

The germ cell nuclear factor of Xenopus laevis (xGCNF; NR6A1) is a nuclear orphan receptor that is predominantly expressed during neurula and late tailbud stages. As a strategy to analyze the role of xGCNF in embryogenesis, we have induced a gain of function by overexpression of full-length (fl) GCNF and a functional inhibition by a dominant-negative (dn) GCNF. Early events of embryogenesis including gastrulation and neurulation were not affected and the expression of several early mesodermal markers was normal. Yet specific defects were observed upon organogenesis. Ectopic posterior overexpression of the full-length xGCNF caused posterior defects and disturbed somite formation. In contrast, expression of dnGCNF interfered with differentiation of the neural tube and affected the differentiation of anterior structures, including the cement gland and the eyes. Embryos affected by dnGCNF were rescued by coexpression of flGCNF. After expression of dnGCNF, mRNA encoding the the retinoic acid receptor xRAR gamma 2 was selectively suppressed anteriorly. From the distinct phenotypes obtained, we conclude that GCNF has an essential function in anteroposterior differentiation during organogenesis.  (+info)

Mespo: a novel basic helix-loop-helix gene expressed in the presomitic mesoderm and posterior tailbud of Xenopus embryos. (19/1306)

We have isolated a novel gene from Xenopus, called Mespo, which encodes a protein containing a basic helix-loop-helix (bHLH) motif characteristic of a family of transcriptional activators. Mespo expression begins at the gastrula stage and continues throughout tailbud stages; expression occurs in the presomitic mesoderm and the posterior tailbud. Mespo has high similarity to a subfamily of bHLH transcription factors involved in segmentation of the presomitic paraxial mesoderm.  (+info)

Demodicosis in an American bison. (20/1306)

An 18-month-old, male American bison (Bison bison) was presented with 7- to 9-mm size nodules periorbital, perineal, and on the ventral surface of the tail. Demodex spp. were identified from the exudate by microscopic examination. Examination 6 mo later revealed that the infestation had nearly cleared without treatment.  (+info)

Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF. (21/1306)

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.  (+info)

The role of tolloid/mini fin in dorsoventral pattern formation of the zebrafish embryo. (22/1306)

A highly conserved TGF-&bgr; signaling pathway is involved in the establishment of the dorsoventral axis of the vertebrate embryo. Specifically, Bone Morphogenetic Proteins (Bmps) pattern ventral tissues of the embryo while inhibitors of Bmps, such as Chordin, Noggin and Follistatin, are implicated in dorsal mesodermal and neural development. We investigated the role of Tolloid, a metalloprotease that can cleave Chordin and increase Bmp activity, in patterning the dorsoventral axis of the zebrafish embryo. Injection of tolloid mRNA into six dorsalized mutants rescued only one of these mutants, mini fin. Through chromosomal mapping, linkage and cDNA sequence analysis of several mini fin alleles, we demonstrate that mini fin encodes the tolloid gene. Characterization of the mini fin mutant phenotype reveals that Mini fin/Tolloid activity is required for patterning ventral tissues of the tail: the ventral fin, and the ventroposterior somites and vasculature. Gene expression studies show that mfn mutants exhibit reduced expression of ventrally restricted markers at the end of gastrulation, suggesting that the loss of ventral tail tissues is caused by a dorsalization occurring at the end of gastrulation. Based on the mini fin mutant phenotype and the expression of tolloid, we propose that Mini fin/Tolloid modifes the Bmp activity gradient at the end of gastrulation, when the ventralmost marginal cells of the embryo are in close proximity to the dorsal Chordin-expressing cells. At this time, unimpeded Chordin may diffuse to the most ventral marginal regions and inhibit high Bmp activity levels. In the presence of Mini fin/Tolloid, however, Chordin activity would be negatively modulated through proteolytic cleavage, thereby increasing Bmp signaling activity. This extracellular mechanism is amplified by an autoregulatory loop for bmp gene expression.  (+info)

Topical opioids in mice: analgesia and reversal of tolerance by a topical N-methyl-D-aspartate antagonist. (23/1306)

In addition to its central actions, morphine has important peripheral effects. To examine peripheral analgesic mechanisms, we developed a topical opioid paradigm in which the tail was immersed in a dimethyl sulfoxide (DMSO) solution containing various drugs. Alone, DMSO was inactive in the tail-flick assay in mice. DMSO solutions containing morphine and peptides such as [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO) produced a potent, dose-dependent analgesia with the radiant heat tail-flick assay. The actions of the drugs were local. Analgesia was observed only in regions of the tail exposed to the solution and not in more proximal unexposed portions of the tail. Immersion of the tail in a solution containing either 125I-labeled morphine or 125I-labeled DAMGO revealed no detectable uptake of radioactivity into the brain, spinal cord, or blood. In the tail, radioactivity was limited only to the regions actually immersed in the solutions. The topical drugs potentiated systemic agents, similar to the previously established synergy between peripheral and central sites of action. Local tolerance was rapidly produced by repeated daily exposure of the tail to morphine. Topical morphine tolerance was effectively blocked by the N-methyl-D-aspartate (NMDA) antagonist MK801 given either systemically or topically but not intrathecally. The ability of a topical NMDA antagonist to block local morphine tolerance suggests that peripheral NMDA receptors mediate topical morphine tolerance. Morphine was cross-tolerant to DAMGO, but not to morphine-6beta-glucuronide, implying different mechanisms of action. These observations are significant in the design and use of opioids clinically.  (+info)

Hyperplasia in multiple smooth muscle tissues in transgenic mice expressing a temperature-sensitive SV40 T-antigen under the control of smooth muscle alpha-actin regulatory sequences. (24/1306)

Control of smooth muscle cell (SMC) proliferation is of fundamental importance in the development and pathology of the vasculature. To derive vascular SMC with conditional inactivation of negative cell cycle regulatory proteins in the context of smooth muscle protein expression, a 3.4 kb fragment of the mouse SMC alpha-actin promoter was used to target a temperature-sensitive mutant SV40 T antigen (tsA58) to smooth muscle in transgenic mice. Mice with this genotype display a heritable phenotype of abnormal SMC proliferation in the central tail artery, vasa deferentia, seminal vesicles, prostate, and uterus, with the latter resembling uterine leiomyomatosis and prostatic hypertrophy. Neither the aorta nor other viscera manifested abnormal proliferation. Cultures from aorta, vas deferens, seminal vesicle, and kidney tissue were characterized with regard to protein expression, stability, and matrix remodelling capacity. The alpha-actin content/cell was up to 3-4-fold higher, as well as more stable than in primary SMC cultures, suggesting successful selection for propagation of cells expressing this differentiation marker. All cells displayed enhanced growth at the permissive temperature. As an initial functional assessment, the cells were compared to non-transformed mouse aortic SMC with respect to the ability to remodel collagen gel matrices, and demonstrated conservation of this physiologic function. This in vivo analysis of the SMC alpha-actin promoter supports a broader range of smooth muscle-directed expression activity than previously recognized, and establishes the feasibility of its use to direct transgene expression to vascular as well as genito-urinary smooth muscle. The targeted expression of the tsA58 T antigen has yielded transgenic animals with several manifestations of smooth muscle hyperplasia; these animals have in turn permitted the derivation of several murine SMC lines with phenotypic stability and conditionally-modulated proliferation. These cells will allow expansion of derivative transfected smooth muscle cell lines under permissive conditions, as well as oncogene inactivation at the restrictive temperature when desired for functional studies.  (+info)