RESUMEN
The evolutionary mechanisms responsible for eye degeneration in cave-adapted animals have not been resolved. Opposing hypotheses invoking neural mutation or natural selection, each with certain genetic and developmental expectations, have been advanced to explain eye regression, although little or no experimental evidence has been presented to support or reject either theory. Here we review recent developmental and molecular studies in the teleost Astyanax mexicanus, a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish), which shed new light on this problem. The manner of eye development and degeneration, the ability to experimentally restore eyes, gene expression patterns, and comparisons between different cavefish populations all provide important clues for understanding the evolutionary forces responsible for eye degeneration. A key discovery is that Hedgehog midline signaling is expanded and inhibits eye formation by inducing lens apoptosis in cavefish embryos. Accordingly, eyes could have been lost by default as a consequence of natural selection for constructive traits, such as feeding structures, which are positively regulated by Hh signaling. We conclude from these studies that eye degeneration in cavefish may be caused by adaptive evolution and pleiotropy.
Asunto(s)
Evolución Molecular , Oftalmopatías/genética , Peces/genética , Adaptación Fisiológica/genética , Animales , Ceguera/genética , Peces/embriología , Regulación del Desarrollo de la Expresión Génica , Modelos GenéticosRESUMEN
In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/patología , Cristalino/embriología , Mutación/fisiología , Pez Cebra/embriología , Animales , Muerte Celular , Diferenciación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Cristalino/trasplante , Masculino , Fenotipo , Retina/citología , Retina/embriología , Proteínas Supresoras de TumorRESUMEN
We have compared Pax6 expression during embryonic development in the eyed surface form (surface fish) and several different eyeless cave forms (cavefish) of the teleost Astyanax mexicanus. Despite lacking functional eyes as adults, cavefish embryos form small optic primordia, which later arrest in development and show various degrees of eye degeneration. The pattern of Pax6 mRNA expression was modified early and late during cavefish development. In early surface fish embryos, two bilateral Pax6 expression domains are present in the anterior neural plate, which extend across the midline and fuse to form the forebrain and optic primordia. In cavefish embryos, these Pax6 domains are diminished in size and remain separated, resulting in an anterior gap in Pax6 expression and presumably the formation of smaller optic primordia. The anterior gap in Pax6 expression was confirmed by double staining for Pax6 and distalless-3 mRNA, which marks the anterior margin of the neural plate and is unaltered in cavefish. Similar anterior gaps in Pax6 expression occurred in independently derived cavefish populations, suggesting that they are important in eye degeneration. Later during surface fish development, Pax6 protein is expressed in the cornea, lens, and ganglion and amacrine cells of the neural retina. Pax6 expression was gradually reduced during cavefish lens development, concomitant with lens arrest and degeneration, and was absent in the corneal epithelium, which does not differentiate in cavefish. In contrast, Pax6 expression in the retinal ganglion and amarcine cells is unmodified in cavefish, despite retarded retinal development. The results suggest that changes in Pax6 expression are involved in the evolution of cavefish eye degeneration.
Asunto(s)
Ojo/patología , Peces/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas del Ojo , Peces/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas RepresorasRESUMEN
The Mexican tetra Astyanax mexicanus has many of the favorable attributes that have made the zebrafish a model system in developmental biology. The existence of eyed surface (surface fish) and blind cave (cavefish) dwelling forms in Astyanax also provides an attractive system for studying the evolution of developmental mechanisms. The polarity of evolutionary changes and the environmental conditions leading to the cavefish phenotype are known with certainty, and several different cavefish populations have evolved constructive and regressive changes independently. The constructive changes include enhancement of the feeding apparatus (jaws, taste buds, and teeth) and the mechanosensory system of cranial neuromasts. The homeobox gene Prox 1, which is expressed in the expanded taste buds and cranial neuromasts, is one of the genes involved in the constructive changes in sensory organ development. The regressive changes include loss of pigmentation and eye degeneration. Although adult cavefish lack functional eyes, small eye primordia are formed during embryogenesis, which later arrest in development, degenerate, and sink into the orbit. Apoptosis and lens signaling to other eye parts, such as the cornea, iris, and retina, result in the arrest of eye development and ultimate optic degeneration. Accordingly, an eye with restored cornea, iris, and retinal photoreceptor cells is formed when a surface fish lens is transplanted into a cavefish optic cup, indicating that cavefish optic tissues have conserved the ability to respond to lens signaling. Genetic analysis indicates that multiple genes regulate eye degeneration, and molecular studies suggest that Pax6 may be one of the genes controlling cavefish eye degeneration. Further studies of the Astyanax system will contribute to our understanding of the evolution of developmental mechanisms in vertebrates.
Asunto(s)
Evolución Biológica , Peces/embriología , Animales , Encéfalo/embriología , Ingestión de Alimentos , Ojo/embriología , Peces/fisiología , PigmentaciónRESUMEN
Ascidians have played a major role in studies to understand the function of cytoplasmic determinants in animal development. Special qualities, including eggs with colored cytoplasmic regions, an invariant cleavage pattern and cell lineage, embryos with low cell numbers, larvae with typical chordate features and only six different tissues, rapid development, and a small genome, combine to make these animals a unique system for studying cytoplasmic determinants. There is evidence for determinants that specify the cleavage pattern; the differentiation of epidermal, endodermal, and muscle cells; and cell movements associated with gastrulation. The muscle determinants appear to be modified in concert with tail and muscle regression in species that have evolved an anural, or tailless, larva. Several lines of evidence suggest that determinants may be localized maternal mRNAs, which encode transcription factors or signal transduction components responsible for initiating differential gene activity. Different approaches and strategies are being used to isolate and characterize the function of these localized maternal mRNAs.
Asunto(s)
Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Citoplasma/genética , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Urocordados/embriología , Animales , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Larva/citología , Larva/crecimiento & desarrollo , Larva/metabolismo , Urocordados/citología , Urocordados/metabolismoRESUMEN
Astyanax mexicanus is a teleost with eyed surface-dwelling and eyeless cave-dwelling forms. Eye formation is initiated in cave fish embryos, but the eye subsequently arrests and degenerates. The surface fish lens stimulates growth and development after transplantation into the cave fish optic cup, restoring optic tissues lost during cave fish evolution. Conversely, eye growth and development are retarded following transplantation of a surface fish lens into a cave fish optic cup or lens extirpation. These results show that evolutionary changes in an inductive signal from the lens are involved in cave fish eye degeneration.
Asunto(s)
Ojo/embriología , Peces/embriología , Cristalino/embriología , Cristalino/fisiología , Animales , Apoptosis , Evolución Biológica , Diferenciación Celular , Proteínas de Unión al ADN/biosíntesis , Inducción Embrionaria , Proteínas del Ojo , Proteínas de Homeodominio/biosíntesis , Etiquetado Corte-Fin in Situ , Cristalino/citología , Cristalino/trasplante , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Proteínas Represoras , Retina/embriología , Retina/crecimiento & desarrollo , Retina/metabolismo , Rodopsina/biosíntesis , Proteínas Supresoras de TumorRESUMEN
The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.
Asunto(s)
Proteínas de Unión al ADN/genética , Larva/genética , Proteínas Nucleares/genética , Urocordados/embriología , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Datos de Secuencia Molecular , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Actinas/genética , Filogenia , Urocordados/embriología , Urocordados/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoesqueleto/genética , Embrión no Mamífero , Larva , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Cola (estructura animal)/embriología , Cola (estructura animal)/crecimiento & desarrolloRESUMEN
The Manx gene is required for the development of the tail and other chordate features in the ascidian tadpole larva. To determine the structure of the Manx gene, we isolated and sequenced genomic clones from the tailed ascidian Molgula oculata. The Manx gene contains 9 exons and encodes both major and minor Manx mRNAs, which differ in the length of their 5' untranslated regions. The coding region of the single-copy bobcat gene, which encodes a DEAD-box RNA helicase, is embedded within the first Manx intron. The organization of the bobcat and Manx transcription units was determined by comparing genomic and cDNA clones. The Manx-bobcat gene locus has an unusual organization in which a non-coding first exon is alternatively spliced at the 5' end of two different mRNAs. The bobcat and Manx genes are expressed coordinately during oogenesis and embryogenesis, but not during spermatogenesis, in which bobcat mRNA accumulates independently of Manx mRNA. Similar to Manx, zygotic bobcat transcripts accumulate in the embryonic primordia responsible for generating chordate features, including the dorsal neural tube and notochord, are downregulated during embryogenesis in the tailless species Molgula occulta and are upregulated in M. occulta X M. oculata hybrids, which restore these chordate features. Antisense experiments indicate that zygotic bobcat expression is required for development of the same suite of chordate features as Manx. The results show that the Manx-bobcat gene complex has a role in the development of chordate features in ascidian tadpole larvae.
Asunto(s)
Familia de Multigenes , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , ARN Helicasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Humanos , Larva , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , UrocordadosRESUMEN
We have examined the expression and regulation of cytoskeletal actin genes in ascidians with tailed (Molgula oculata) and tailless larvae (Molgula occulta). Four cDNA clones were isolated representing two pairs of orthologous cytoskeletal actin genes (CA1 and CA2), which encode proteins differing by five amino acids in the tailed and tailless species. The CA1 and CA2 genes are present in one or two copies, although several related genes may also be present in both species. Maternal CA1 and CA2 mRNA is present in small oocytes but transcript levels later decline, suggesting a role in early oogenesis. In the tailed species, embryonic CA1 and CA2 mRNAs first appear in the presumptive mesenchyme and muscle cells during gastrulation, subsequently accumulate in the presumptive notochord cells, and can be detected in these tissues through the tadpole stage. CA1 mRNAs accumulate initially in the same tissues in the tailless species but subsequently disappear, in concert with the arrest of notochord and tail development. In contrast, CA2 mRNAs were not detected in embryos of the tailless species. Fertilization of eggs of the tailless species with sperm of the tailed species, which restores the notochord and the tail, also results in the upregulation of CA1 and CA2 gene expression in hybrid embryos. Antisense oligodeoxynucleotide experiments suggest that CA1 and CA2 expression in the notochord, but not in the muscle cells, is dependent on prior expression of Mocc FHI, an ascidian HNF-3beta-like gene. The expression of the CA1 and CA2 genes in the notochord in the tailed species, downregulation in the tailless species, upregulation in interspecific hybrids, and dependence on HNF-3beta activity is consistent with a role of these genes in development of the ascidian notochord.
Asunto(s)
Actinas/genética , Citoesqueleto/química , Proteínas de Unión al ADN/genética , Notocorda/crecimiento & desarrollo , Proteínas Nucleares/genética , Factores de Transcripción , Urocordados/crecimiento & desarrollo , Actinas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoesqueleto/metabolismo , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Expresión Génica , Gónadas/embriología , Gónadas/metabolismo , Factor Nuclear 3-beta del Hepatocito , Datos de Secuencia Molecular , Urocordados/embriologíaRESUMEN
We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3beta. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are similar to those reported for knockout mutations of HNF-3beta in the mouse, suggesting that HNF-3/forkhead genes have an ancient and fundamental role in organizing the body plan in chordates.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Unión al ADN/genética , Gástrula/fisiología , Proteínas Nucleares/genética , Factores de Transcripción/genética , Urocordados/embriología , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linaje de la Célula , Movimiento Celular , Endodermo/citología , Endodermo/metabolismo , Factores de Transcripción Forkhead , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Larva/crecimiento & desarrollo , Larva/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Datos de Secuencia Molecular , Morfogénesis , Músculos/citología , Músculos/metabolismo , Notocorda/citología , Notocorda/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Oligonucleótidos Antisentido/farmacología , Factores de Transcripción/química , Factores de Transcripción/fisiología , Urocordados/crecimiento & desarrollo , Urocordados/metabolismoRESUMEN
Molgula tectiformis is a new ascidian species recently described by Nishikawa (1991). In Otsuchi Bay, Iwate, Japan, they are easily obtainable from cages for culturing scallops. We report here that M. tectiformis is another example of a direct developer: their embryonic development is lacking the tadpole larva. The fertilized egg is orange and about 150 microns in diameter. At 18 degrees C, the egg cleaves at about 20 min intervals and gastrulation occurs about 5 hr after fertilization. In contrast to conventionally-developing ascidians, M. tectiformis does not form a tadpole larva. Immediately before hatching, three stolons or ampullae begin to extend from the tailless embryo. After hatching the stolons mediate the attachment of the juvenile body to the substratum. Histochemistry for tissue-specific enzyme activity did not detect muscle-specific acetyl-cholinesterase, endoderm-specific alkaline phosphatase, and pigment cell-specific tyrosinase. In addition, in situ hybridization could not prove the presence of muscle actin gene transcripts in the embryo. These results suggest that these larval tissues do not differentiate in M. tectiformis embryos. Because M. tectiformis is common and gravid year-around in Otsuchi Bay, this direct developer provides the opportunity for further analysis of molecular changes during evolution that cause an alternative mode of development.
Asunto(s)
Urocordados/embriología , Acetilcolinesterasa/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular , Histocitoquímica , Larva/citología , Larva/enzimología , Músculos/citología , Músculos/embriología , Músculos/enzimología , Proteínas Tirosina Quinasas/metabolismoRESUMEN
The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon-intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates.
Asunto(s)
Actinas/genética , Cordados no Vertebrados/genética , Evolución Molecular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoplasma/metabolismo , ADN , Exones , Intrones , Datos de Secuencia Molecular , Músculos/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
An evolutionary change in development was studied in two closely related ascidian species, one exhibiting a conventional tadpole larva and the other a modified tailless larva. Interspecific hybridization restores chordate features to the tailless larva. The zinc finger gene Manx is expressed in cells that generate chordate features in the tailed species but is down-regulated in the tailless species. Manx expression is restored in hybrid embryos. Antisense oligodeoxynucleotide treatment inhibited Manx expression and chordate features in hybrid embryos, which suggests that Manx is required for development of the chordate larval phenotype in ascidians.
Asunto(s)
Cordados no Vertebrados/embriología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Urocordados/embriología , Urocordados/genética , Dedos de Zinc/genética , Animales , Cordados no Vertebrados/genética , Hibridación Genética , Hibridación in Situ , Larva/crecimiento & desarrollo , Notocorda/embriología , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cola (estructura animal)/embriología , Tionucleótidos/metabolismo , Tionucleótidos/farmacología , Regulación hacia ArribaRESUMEN
The myoplasm is a localized cytoplasmic region that is involved in axis determination, gastrulation, muscle cell specification, and the pattern of cell divisions during ascidian development. The noncoding yellow crescent (YC) RNA is localized in the myoplasm, but the function of this transcript is unknown. Probes containing the 3' region of YC RNA hybridize to other RNAs in ascidian eggs. A cDNA library from the ascidian Styela clava was screened with a YC probe to identify maternal YC-related RNAs. This screen resulted in isolation of ScYC26b, a cDNA clone encoding the ascidian proliferating cell nuclear antigen (PCNA). The PCNA mRNA has a long 3' untranslated region containing a 521-nucleotide sequence with antisense complementarity to part of the 3' region of YC RNA. The PCNA and YC genes appear to be single copy and may overlap in their 3' regions on opposite DNA strands. The ascidian PCNA protein has 61, 69, and 71% amino acid identity to the Drosophila, Xenopus, and human PCNAs, respectively. S. clava embryos contain maternal and zygotic PCNA mRNAs. Maternal PCNA mRNA is localized in the ectoplasm, a cytoplasmic region that is segregated to cell lineages that proliferate extensively during embryogenesis, and is depleted in the myoplasm, which is segregated to cell lineages that undergo fewer divisions. Zygotic PCNA mRNA is confined to the developing nervous system and is still abundant after the neural cells have ceased to proliferate. PCNA protein, detected with PC10 monoclonal antibody, is also excluded from the myoplasm. These results show that the 3' UTR of PCNA mRNA is antisense and complementary to YC RNA and suggest that differential cell proliferation in the embryo may be limited by localization of maternal PCNA mRNA and protein. Furthermore, zygotic PCNA may have a novel role in neural development in the tadpole larva.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Antígeno Nuclear de Célula en Proliferación/genética , ARN sin Sentido/genética , ARN Mensajero/genética , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Clonación Molecular , Citoplasma/química , ADN Complementario/genética , Embrión no Mamífero/química , Dosificación de Gen , Genes Sobrepuestos/genética , Datos de Secuencia Molecular , Desarrollo de Músculos , Sistema Nervioso/química , Sistema Nervioso/embriología , Oocitos/química , Antígeno Nuclear de Célula en Proliferación/química , ARN/genética , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Urocordados/embriología , Cigoto/químicaRESUMEN
The Msx homeobox genes are expressed in complex patterns during vertebrate development in conjunction with inductive tissue interactions. As a means of understanding the archetypal role of Msx genes in chordates, we have isolated and characterized an Msx gene in ascidians, protochordates with a relatively simple body plan. The Mocu Msx-a and McMsx-a genes, isolated from the ascidians Molgula oculata and Molgula citrina, respectively, have homeodomains that place them in the msh-like subclass of Msx genes. Therefore, the Molgula Msx-a genes are most closely related to the msh genes previously identified in a number of invertebrates. Southern blot analysis suggests that there are one or two copies of the Msx-a gene in the Molgula genome. Northern blot and RNase protection analysis indicate that Msx-a transcripts are restricted to the developmental stages of the life cycle. In situ hybridization showed that Msx-a mRNA first appears just before gastrulation in the mesoderm (presumptive notochord and muscle) and ectoderm (neural plate) cells. Transcript levels decline in mesoderm cells after the completion of gastrulation, but are enhanced in the folding neural plate during neurulation. Later, Msx-a mRNA is also expressed in the posterior ectoderm and in a subset of the tail muscle cells. The ectoderm and mesoderm cells that express Msx-a are undergoing morphogenetic movements during gastrulation, neurulation, and tail formation. Msx-a expression ceases after these cells stop migrating. The ascidian M. citrina, in which adult tissues and organs begin to develop precociously in the larva, was used to study Msx-a expression during adult development. Msx-a transcripts are expressed in the heart primordium and the rudiments of the ampullae, epidermal protrusions with diverse functions in the juvenile. The heart and ampullae develop in regions where mesenchyme cells interact with endodermal or epidermal epithelia. A comparison of the expression patterns of the Molgula genes with those of their vertebrate congeners suggests that the archetypal roles of the Msx genes may be in morphogenetic movements during embryogenesis and in mesenchymal-epithelial interactions during organogenesis.
Asunto(s)
Expresión Génica , Genes Homeobox , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Datos de Secuencia Molecular , Filogenia , ARN Mensajero , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidians Molgula oculata and Molgula occulta are closely related species with different modes of development. M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin gene MocuMA1 was isolated from an M. oculata genomic library. MocuMA1 is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5' upstream region of MocuMA1 is sufficient to drive expression of a lacZ fusion construct in the larval muscle cells, implying that it is a functional gene. MocuMA1 mRNA first appears in the prospective muscle cells of M. oculata embryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA1a and MoccMA1b were isolated from a M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5' upstream regions of the M. occulta muscle actin genes also contain numerous changes, expression of MoccMA1a/lacZ and MoccMA1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMAlb. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.
Asunto(s)
Evolución Biológica , Diferenciación Celular/genética , Músculos/citología , Urocordados/embriología , Actinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Regulación del Desarrollo de la Expresión Génica , Células Híbridas , Datos de Secuencia Molecular , Músculos/embriología , Regiones Promotoras Genéticas , Urocordados/genéticaRESUMEN
We have isolated and characterized the cDNA clone ScYC26a from the ascidian Styela clava based on its relationship to the non-coding yellow crescent (YC) RNA. The ScYC26a mRNA has a long 5' non-coding sequence that is complementary to YC RNA. The deduced amino acid sequence indicates that ScYC26a encodes the ribosomal protein L5. The ScYC26a mRNA is probably encoded by a single copy gene, which shares genomic DNA restriction fragments with the gene encoding YC RNA, suggesting that the ScYC26a and YC genes are closely linked in the S. clava genome. Northern blot hybridization showed that S. clava eggs and embryos contain maternal ScYC26a mRNA and that zygotic ScYC26a transcripts do not accumulate until after metamorphosis. In situ hybridization showed that maternal ScYC26a mRNA is localized in the myoplasm and is segregated primarily to the muscle cell lineages during embryogenesis. The interaction of YC and ScYC26a transcripts may be involved in translational control or localization of L5 mRNA in the myoplasm.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Urocordados/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Femenino , Masculino , Datos de Secuencia Molecular , Proteínas Ribosómicas/metabolismo , Homología de Secuencia de Aminoácido , Urocordados/embriología , Urocordados/genéticaRESUMEN
The ascidian tadpole larva is regarded as a prototype of the ancestral chordate. Here we consider recent studies on the development of the tadpole larva that provide new insights into chordate origins and evolution. The notochord of ascidian larvae and vertebrates appear to be homologous structures based on their induction by endoderm and expression of the Brachyury (T) gene. The muscle cells of ascidian larvae also appear homologous to those of vertebrates based on their expression of bHLH myogenic and muscle-type actin genes, although they are specified by cytoplasmic determinants localized in the egg as well as embryonic induction. Studies of the tailless larvae of anural ascidians have resulted in the identification of Manx, a gene that may control tail development and evolution. These and other results support the ascidian tadpole prototype for the ancestral chordate.