RESUMEN
The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins.
Asunto(s)
Genoma , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/crecimiento & desarrollo , Erizos de Mar/crecimiento & desarrollo , Animales , Axones/fisiología , Conexinas/genética , Electrofisiología , Humanos , Larva/fisiología , Mamíferos , Neuronas/fisiología , Filogenia , Erizos de Mar/clasificación , Erizos de Mar/genética , Sinapsis/fisiología , Factores de Transcripción/genéticaRESUMEN
We have identified a single homolog of goosecoid, SpGsc, that regulates cell fates along both the animal-vegetal and oral-aboral axes of sea urchin embryos. SpGsc mRNA is expressed briefly in presumptive mesenchyme cells of the approximately 200-cell blastula and, beginning at about the same time, accumulates in the presumptive oral ectoderm through pluteus stage. Loss-of-function assays with morpholine-substituted antisense oligonucleotides show that SpGsc is required for endoderm and pigment cell differentiation and for gastrulation. These experiments and gain-of-function tests by mRNA injection show that SpGsc is a repressor that antagonizes aboral ectoderm fate specification and promotes oral ectoderm differentiation. We show that SpGsc competes for binding to specific cis elements with SpOtx, a ubiquitous transcription activator that promotes aboral ectoderm differentiation. Moreover, SpGsc represses transcription in vivo from an artificial promoter driven by SpOtx. As SpOtx appears long before SpGsc transcription is activated, we propose that SpGsc diverts ectoderm towards oral fate by repressing SpOtx target genes. Based on the SpGsc-SpOtx example and other available data, we propose that ectoderm is first specified as aboral by broadly expressed activators, including SpOtx, and that the oral region is subsequently respecified by the action of negative regulators, including SpGsc. Accumulation of SpGsc in oral ectoderm depends on cell-cell interactions initiated by nuclear beta-catenin function, which is known to be required for specification of vegetal tissues, because transcripts are undetectable in dissociated or in cadherin mRNA-injected embryos. This is the first identified molecular mechanism underlying the known dependence of oral-aboral ectoderm polarity on intercellular signaling.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Erizos de Mar/embriología , Transactivadores , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Proteínas del Citoesqueleto/metabolismo , Sistema Digestivo/embriología , Endodermo/citología , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica , Proteína Goosecoide , Proteínas de Homeodominio/genética , Datos de Secuencia Molecular , Boca/embriología , Factores de Transcripción Otx , Pigmentación , Unión Proteica , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción Genética , beta CateninaRESUMEN
Localization of nuclear beta-catenin initiates specification of vegetal fates in sea urchin embryos. We have identified SpKrl, a gene that is activated upon nuclear entry of beta-catenin. SpKrl is upregulated when nuclear beta-catenin activity is increased with LiCl and downregulated in embryos injected with molecules that inhibit beta-catenin nuclear function. LiCl-mediated SpKrl activation is independent of protein synthesis, indicating that SpKrl is a direct target of beat-catenin and TCF. Embryos in which SpKrl translation is inhibited with morpholino antisense oligonucleotides lack endoderm. Conversely, SpKrl mRNA injection rescues some vegetal structures in beta-catenin-deficient embryos. SpKrl negatively regulates expression of the animalizing transcription factor, SpSoxB1. We propose that SpKrl functions in patterning the vegetal domain by suppressing animal regulatory activities.
Asunto(s)
Diferenciación Celular , Proteínas del Citoesqueleto/metabolismo , Endodermo/citología , Erizos de Mar/embriología , Transactivadores , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Blastocisto/citología , Blastocisto/metabolismo , Comunicación Celular , Clonación Molecular , Proteínas del Citoesqueleto/genética , Ectodermo/citología , Ectodermo/metabolismo , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Cloruro de Litio/farmacología , Modelos Biológicos , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción SOXB1 , Erizos de Mar/citología , Erizos de Mar/metabolismo , Alineación de Secuencia , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas Wnt , Dedos de Zinc , beta CateninaRESUMEN
Transcription of SpAN, which encodes a secreted protease related to tolloid and BMP 1, is differentially regulated along the animal-vegetal axis of the sea urchin embryo by a maternally initiated mechanism. Regulatory sites that bind SpSoxB1 and CBF (CCAAT binding factor) are essential for strong transcriptional activity because mutations of these elements reduce promoter activity in vivo 20- and 10-fold, respectively. Here we show that multimerized SpSoxB1 elements cannot activate transcription from the SpAN basal promoter in vivo. However, like other factors containing HMG-class DNA binding domains, SpSoxB1 does induce strong bending of DNA. The CBF binding site lies abnormally far from the transcriptional start site at -200 bp. We show that the SpSoxB1 site is not required if the CCAAT element is moved 100 bp closer to the transcriptional start site, replacing the SpSoxB1 site. This supports a model in which the bending of SpAN promoter DNA by SpSoxB1 facilitates interactions between factors binding to upstream and downstream regulatory elements.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Proteínas de Drosophila , Proteínas de Insectos/genética , Metaloendopeptidasas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 1 , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Factores de Transcripción SOXB1 , Erizos de Mar , TATA Box , Metaloproteinasas Similares a TolloidRESUMEN
To examine whether a BMP signaling pathway functions in specification of cell fates in sea urchin embryos, we have cloned sea urchin BMP2/4, analyzed its expression in time and space in developing embryos and assayed the developmental consequences of changing its concentration through mRNA injection experiments. These studies show that BMP4 mRNAs accumulate transiently during blastula stages, beginning around the 200-cell stage, 14 hours postfertilization. Soon after the hatching blastula stage, BMP2/4 transcripts can be detected in presumptive ectoderm, where they are enriched on the oral side. Injection of BMP2/4 mRNA at the one-cell stage causes a dose-dependent suppression of commitment of cells to vegetal fates and ectoderm differentiates almost exclusively as a squamous epithelial tissue. In contrast, NOGGIN, an antagonist of BMP2/4, enhances differentiation of endoderm, a vegetal tissue, and promotes differentiation of cells characteristic of the ciliated band, which contains neurogenic ectoderm. These findings support a model in which the balance of BMP2/4 signals produced by animal cell progeny and opposing vegetalizing signals sent during cleavage stages regulate the position of the ectoderm/ endoderm boundary. In addition, BMP2/4 levels influence the decision within ectoderm between epidermal and nonepidermal differentiation.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/genética , Ectodermo/fisiología , Endodermo/fisiología , Regulación del Desarrollo de la Expresión Génica , Erizos de Mar/embriología , Factor de Crecimiento Transformador beta , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/fisiología , Clonación Molecular , Embrión no Mamífero/fisiología , Datos de Secuencia Molecular , Oocitos/fisiología , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Erizos de Mar/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Xenopus , Proteínas de XenopusRESUMEN
We discuss recent progress in understanding how cell fates are specified along the animal-vegetal axis of the sea urchin embryo. This process is initiated by cell-autonomous, maternally directed, mechanisms that establish three unique gene-regulatory domains. These domains are defined by distinct sets of vegetalizing (beta-catenin) and animalizing transcription factor (ATF) activities and their region of overlap in the macromeres, which specifies these cells as early mesendoderm. Subsequent signaling among cleavage-stage blastomeres further subdivides fates of macromere progeny to yield major embryonic tissues. Zygotically produced Wnt8 reinforces maternally regulated levels of nuclear beta-catenin in vegetal derivatives to down regulate ATF activity and further promote mesendoderm fates. Signaling through the Notch receptor from the vegetal micromere lineages diverts adjacent mesendoderm to secondary mesenchyme fates. Continued Wnt signaling expands the vegetal domain of beta-catenin's transcriptional regulatory activity and competes with animal signaling factors, including BMP2/4, to specify the endoderm-ectoderm border within veg(1) progeny. This model places new emphasis on the importance of the ratio of maternally regulated vegetal and animal transcription factor activities in initial specification events along the animal-vegetal axis.
Asunto(s)
Tipificación del Cuerpo , Erizos de Mar/embriología , Transactivadores , Animales , Linaje de la Célula , Proteínas del Citoesqueleto/metabolismo , Modelos Biológicos , Proteínas/metabolismo , Transducción de Señal , Proteínas Wnt , Proteínas de Pez Cebra , beta CateninaRESUMEN
We have identified a Sox family transcription factor, SpSoxB1, that is asymmetrically distributed among blastomeres of the sea urchin embryo during cleavage, beginning at 4th cleavage. SpSoxB1 interacts with a cis element that is essential for transcription of SpAN, a gene that is activated cell autonomously and expressed asymmetrically along the animal-vegetal axis. In vitro translated SpSoxB1 forms a specific complex with this cis element whose mobility is identical to that formed by a protein in nuclear extracts. An anti-SpSoxB1 rabbit polyclonal antiserum specifically supershifts this DNA-protein complex and recognizes a single protein on immunoblots of nuclear proteins that comigrates with in vitro translated SpSoxB1. Developmental immunoblots of total proteins at selected early developmental stages, as well as EMSA of egg and 16-cell stage proteins, show that SpSoxB1 is present at low levels in unfertilized eggs and progressively accumulates during cleavage. SpSoxB1 maternal transcripts are uniformly distributed in the unfertilized egg and the protein accumulates to similar, high concentrations in all nuclei of 4- and 8-cell embryos. However, at fourth cleavage, the micromeres, which are partitioned by asymmetric division of the vegetal 4 blastomeres, have reduced nuclear levels of the protein, while high levels persist in their sister macromeres and in the mesomeres. During cleavage, the uniform maternal SpSoxB1 transcript distribution is replaced by a zygotic nonvegetal pattern that reinforces the asymmetric SpSoxB1 protein distribution and reflects the corresponding domain of SpAN mRNA accumulation at early blastula stage ( approximately 150 cells). The vegetal region lacking nuclear SpSoxB1 gradually expands so that, after blastula stage, only cells in differentiating ectoderm accumulate this protein in their nuclei. The results reported here support a model in which SpSoxB1 is a major regulator of the initial phase of asymmetric transcription of SpAN in the nonvegetal domain by virtue of its distribution at 4th cleavage and is subsequently an important spatial determinant of expression in the early blastula. This factor is the earliest known spatially restricted regulator of transcription along the animal-vegetal axis of the sea urchin embryo.
Asunto(s)
Blastómeros/metabolismo , Erizos de Mar/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonación Molecular , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción SOXB1RESUMEN
Differentiation of sea urchin embryo ectoderm, endoderm and mesenchyme cells, whose anlagen are arrayed along the animal-vegetal axis, relies on both maternally regulated localized transcription factor activities and cell-cell signalling. Classic models proposed that fates are determined by opposing animal and vegetal morphogenetic gradients, whereas current models emphasize unidirectional and sequential vegetal-to-animal signalling cascades between adjacent blastomeres. Recent data support aspects of both models: the vegetal micromeres send one or more signals, which depend on a nuclear beta-catenin-dependent pathway, that both activate Notch signalling required for secondary mesenchyme fate and promote endoderm differentiation and gastrulation. This is opposed by an animalizing domain of BMP4 signals that regulates ectodermal cell fates and establishes the ectoderm-endoderm border.
Asunto(s)
Embrión no Mamífero/embriología , Erizos de Mar/embriología , Transducción de Señal/fisiología , Transactivadores , Animales , Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/fisiología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Proteínas de la Membrana/fisiología , Modelos Biológicos , Morfogénesis/fisiología , Receptores Notch , Erizos de Mar/fisiología , Transcripción Genética , beta CateninaRESUMEN
Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined animal-vegetal axis of the very early sea urchin embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity. Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L. M. and Angerer, R. C. (1997) Dev. Biol. 187, 71-78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R. C. and Angerer, L. M. (1999) Mol. Cell. Biol. 19, 1271-1278). Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription. The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the embryo during late cleavage/early blastula stages. Although maternal transcripts are uniformly distributed in the egg and early cleaving embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription. In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a beta-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the animal-vegetal axis of the sea urchin embryo.
Asunto(s)
Metaloendopeptidasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Erizos de Mar/embriología , Factores de Transcripción/metabolismo , Animales , Blastocisto , Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Proteínas Represoras/genética , Factores de Transcripción/genética , Transcripción Genética , CigotoRESUMEN
We have used the Xenopus embryo as a test system for analyzing the activity of SpAN, a sea urchin metalloprotease in the astacin family containing BMP1 and tolloid. Embryos expressing SpAN initiated gastrulation on a time scale indistinguishable from controls, but invagination of the vegetal pole was subsequently delayed by several hours. At tailbud stages the most severely affected embryos were completely ventralized, lacking all dorsal structures. Molecular analysis of injected embryos, using probes for both dorsal (xgsc and xnot) and ventral (xhox3 and xwnt8) mesoderm, indicates that SpAN ventralizes dorsal mesoderm during gastrula stages. These results mirror those previously obtained with BMP4, suggesting that SpAN may enhance the activity of this ventralizing factor. Consistent with this suggestion, we have shown that SpAN blocks the dorsalizing activity of noggin and chordin, two inhibitory binding proteins for BMP4, but not that of a dominant-negative receptor for BMP4. In contrast, a dominant-negative SpAN, in which the metalloprotease domain has been deleted, dorsalizes ventral mesoderm, a phenotype that can be rescued by coexpressing either SpAN or XBMP1. This suggests that SpAN is mimicking a Xenopus metalloprotease responsible for regulating the activity of Xenopus BMPs during gastrulation. Moreover, our results raise the possibility that SpAN may function to facilitate BMP signaling in early sea urchin embryos.
Asunto(s)
Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Xenopus/embriología , Animales , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras , Desarrollo Embrionario , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Microinyecciones , Fenotipo , Proteínas/metabolismo , ARN Mensajero/genética , Erizos de Mar/enzimologíaRESUMEN
We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element. This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output. As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula.
Asunto(s)
Metaloendopeptidasas/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Erizos de Mar/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , ADN Complementario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Oocitos/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Saccharomyces cerevisiae/genética , Erizos de Mar/embriología , Erizos de Mar/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
We have shown previously by in situ hybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoon et al., J. Biol. Chem. 271, 20119-20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find that SpFGFR transcripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence of SpFGFR transcripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive, SpFGFR transcripts in lantern muscle. Together, these results suggest that only a minority of SpFGFR transcripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, IgS ligand binding domain sequences.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Músculos/metabolismo , Procesamiento Postranscripcional del ARN/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Erizos de Mar/crecimiento & desarrollo , Animales , Sitios de Unión/genética , Inmunohistoquímica , Hibridación in Situ , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , ARN Nuclear/genética , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Erizos de Mar/embriología , Transcripción Genética/genéticaRESUMEN
Previous studies of the regulatory region of the SpHE (hatching enzyme) gene of the sea urchin Strongylocentrotus purpuratus (Wei, Z., Angerer, L.M., Gagnon, M.L. and Angerer, R.C. (1995) Characterization of the SpHE promoter that are spatially regulated along the animal-vegetal axis of the sea urchin embryo. Dev. Biol. 171, 195-211) have shown that approximately 330 bp is necessary and sufficient to promote high level expression in embryos of transgenes that reproduce the spatially asymmetric pattern of endogenous gene activity along the maternally determined animal-vegetal embryonic axis. Furthermore, SpHE regulatory elements appear to be redundant since several different combinations are sufficient to elicit strong promoter activity and many subsets function like the endogenous gene only in non-vegetal cells of the blastula (Wei, Z., Angerer, L.M. and Angerer, R.C. (1997) Multiple positive cis-elements regulate the asymmetric expression of the SpHE gene along the sea urchin embryo animal-vegetal axis. Dev. Biol., 187, 71-88). Here we demonstrate by in vivo footprinting that many cis elements on the endogenous promoter are occupied when the gene is active in early blastulae, but the binding of corresponding trans factors is significantly reduced when the gene becomes inactive in late blastulae. In addition, downregulation of the promoter is accompanied by a transition from a non-nucleosomal to a nucleosome-like chromatin structure. Surprisingly, in vitro DNase I footprints of the 300 bp promoter using nuclear protein extracts from early and late blastulae are not detectably different and neither this sequence, nor a longer one extending to -1255, reproduces the loss of endogenous SpHE transcriptional activity after very early blastula stage. These observations imply that temporal repression of SpHE transcription involves a decrease in accessibility of the promoter to activators that are nevertheless present in nuclei and capable of activating transgene promoters. Temporal, but not spatial, downregulation is therefore likely to be regulated by negative activities functioning outside the -1255 promoter region which may serve as direct repressors or mediate an inactive chromatin structure.
Asunto(s)
Blastocisto/enzimología , Regulación hacia Abajo , Metaloendopeptidasas/genética , Regiones Promotoras Genéticas , Erizos de Mar/embriología , Animales , Cromatina/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/metabolismo , TransgenesRESUMEN
Orthodenticle-related (Otx) proteins are a highly conserved class of homeobox-containing transcription factors found in a wide range of organisms. They function in numerous developmental events, most prominently, anterior head patterning in insects and vertebrates. In the sea urchin, Strongylocentrotus purpuratus, an orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specific Spec2a gene and more generally the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from other species, we isolated cDNA and genomic clones containing SpOtx sequences. Here, we report that SpOtx exists in two forms (alpha and beta) that are generated by alternative RNA splicing from a single SpOtx gene. SpOtx(alpha) and SpOtx(beta) had identical C-termini and homeoboxes but were entirely different in their N-terminal domains. SpOtx(alpha) mRNAs were transcribed from a single start site and accumulated in all cells during cleavage, but were gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contrast, three distinct SpOtx(beta) mRNAs resulted from two separate transcriptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later were largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx(beta) appeared later in development than SpOtx(alpha). Overexpression of SpOtx(alpha) and SpOtx(beta) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx(alpha) was able to repress the accumulation of SpOtx(beta) transcripts, whereas SpOtx(beta) had no effect on the accumulation of SpOtx(alpha) transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates.
Asunto(s)
Empalme Alternativo , Proteínas de Homeodominio/genética , Erizos de Mar/genética , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Clonación Molecular , ADN Complementario/genética , Proteínas de Drosophila , Evolución Molecular , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Unión Proteica , Erizos de Mar/embriología , Análisis de Secuencia de ADN , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/metabolismoRESUMEN
The mechanism that establishes the maternally determined animal-vegetal axis of sea urchin embryos is unknown. We have analyzed the cis-regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a beta-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195-211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic beta-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis-acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957-1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo.
Asunto(s)
Tipificación del Cuerpo/genética , Embrión no Mamífero/fisiología , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Erizos de Mar/embriología , Transcripción Genética , Animales , Animales Modificados Genéticamente , Blastocisto/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/biosíntesis , Erizos de Mar/genética , Eliminación de Secuencia , beta-Galactosidasa/biosíntesisRESUMEN
We describe the cloning of a new fibroblast growth factor receptor, SpFGFR1, that is differentially regulated at the level of transcript abundance during sea urchin embryogenesis. Sequence representing the conserved tyrosine kinase domain was obtained by reverse transcription-polymerase chain reaction using degenerate primers, and the entire open reading frame was obtained by standard cDNA library screening methods. SpFGFR contains a series of domains characteristic of FGFRs: three immunoglobulin-like motifs, an acid box, a transmembrane domain, a relatively long juxtamembrane sequence, a split tyrosine kinase domain, and two conserved intracellular tyrosine residues. Alternative splicing of SpFGFR generates two variants (Ig3L and Ig3S), which differ by insertion in the center of the Ig3 domain of 34 extra amino acids, encoded by an additional exon. Transcripts encoding both variants accumulate when morphogenesis begins with mesenchyme cell ingression and gastrulation. SpFGFR transcripts accumulate in all cell types of the embryo, although in situ hybridization shows that they are somewhat enriched in cells of oral ectoderm and endoderm. Transcripts encoding the Ig3S variant, whose structure resembles more closely that of vertebrate receptors, are enriched in endomesoderm, suggesting that the SpFGFR variants could play distinct roles in the sea urchin embryo.
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Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Empalme Alternativo , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/genética , Ectodermo/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Mesodermo/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
In order to investigate how the maternally specified animal-vegetal axis of the sea urchin embryo is established, we have examined the molecular basis of regulation of several genes transcribed differentially in nonvegetal and vegetal domains of the very early blastula. Here we present an initial characterization of the regulatory region of one of these, SpAN, which encodes a protease in the astacin family related to Drosophila tolloid and vertebrate BMP-1 (Reynolds et al., Development 114, 769-786). Tests of SpAN promoter function in vivo show that high-level activity and correct not-vegetal expression are mediated by sequences within 300 bp upstream of the basal promoter. In vitro studies have identified six protein binding sites serviced by at least five different proteins. Comparison of the structure of the SpAN promoter to that of SpHE, whose expression pattern is identical, shows that both promoters contain multiple positively acting upstream elements close to the basal promoter. We show that two elements are critical for high-level transcription of SpAN, since exact replacement of either results in 10- to 20-fold reduction in promoter strength. These shared elements are, however, not essential for spatially correct SpHE gene transcription. We conclude that the coordinate strong activities of the SpAN and SpHE promoters in the nonvegetal domain of the embryo rely primarily on different transcription factor activities.
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Blastocisto/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Metaloendopeptidasas/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Sitios de Unión , Blastocisto/química , ADN/metabolismo , Huella de ADN , Sondas de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Microinyecciones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Erizos de Mar , Análisis de Secuencia , Transcripción Genética/genética , TransgenesRESUMEN
To understand how the maternally determined animal-vegetal polarity of the sea urchin embryo is established, we have begun to examine the regulatory apparatus of the gene encoding the Strongylocentrotus purpuratus hatching enzyme (SpHE). Previous studies have shown that the pattern of SpHE mRNA accumulation reflects the animal-vegetal developmental axis in that transcription is strongly upregulated during early cleavage in more animal blastomeres, but not in those around the maternally specified vegetal pole of the 16-cell embryo [Reynolds et al., Development 114, 769-786 (1992)]. Tests of SpHE promoter function in vivo using chloramphenicol acetyltransferase and beta-galactosidase enzymatic reporters define a regulatory region within several hundred nucleotides of the transcription initiation site. This region is sufficient to mediate both strong expression in the early blastula and spatially correct transcription. However, neither this region nor longer upstream sequences are sufficient to reproduce the transcriptional downregulation after very early blastula stage that is observed for endogenous genes. Biochemical assays of protein-DNA interactions within the regulatory region identify at least nine sites binding at least six different factors. These cis elements include Otx (an orthodenticle homologue), CCAAT, ets-related, and three unidentified motifs. Deletions and/or replacements of these cis-elements, alone and in combination, indicate that no single factor is essential for SpHE promoter activity, but instead that various combinations of subsets of these elements are capable of eliciting levels of transcription similar to those of the unaltered regulatory region. This density of regulatory elements is consistent with the intense transcription of endogenous SpHE genes during cleavage.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Metaloendopeptidasas/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Erizos de Mar , Transcripción GenéticaRESUMEN
Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus. The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.