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E Amaya

Publications and source records attributed to E Amaya.

At least 19 recordsLinked to original sources
PubMed2001

Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.

Animals↗
PubMed2001

Xenopus Sprouty2 inhibits FGF-mediated gastrulation movements but does not affect mesoderm induction and patterning.

Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior-posterior axis. The Drosophila gene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. We have cloned Xenopus sprouty2 and show that it is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Finally, using Xenopus oocytes we show that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis.

Amino Acid Sequence↗
PubMed2001

A role for BMP signalling in heart looping morphogenesis in Xenopus.

The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.

Animals↗
PubMed2001

Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis.

Morpholino (MO) based inhibition of translational initiation represents an attractive methodology to eliminate gene function during Xenopus development (Heasman et al., 2000). However, the degree to which a given target protein can be eliminated and the longevity of this effect during embryogenesis has not been documented. To examine the efficacy of MOs, we have used transgenic Xenopus lines that harbour known numbers of integrations of a GFP reporter under the control of the ubiquitous and highly expressed CMV promoter (Fig. 1a). In addition we have investigated the longevity of the inhibitory effect by using transgenic lines expressing GFP specifically in the lens of tadpoles. These transgenic lines represent the ideal control for the technique as the promoters are highly expressed and GFP can be easily detected by fluorescence and immunoblotting. Moreover, as GFP has no function in development, the levels of inhibition can be tested in an otherwise normal individual. Here we report that MOs are able to efficiently and specifically inhibit the translation of GFP in transgenic lines from Xenopus laevis and Xenopus tropicalis and the inhibitory effect is long-lived, lasting into the tadpole stages. genesis 30:110--113, 2001.

Animals↗
PubMed2001

Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides.

Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma factors. Of these, sigma(F) is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma(F) (E-sigma(F)), and only two genes of known function are exclusively under the control of E-sigma(F), spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma(F), we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma(F) in vivo. The results indicate a weak sigma(F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma factors, as well as to sigma(A). There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma factors. The looseness of the sigma(F) promoter requirement contrasts with the strict requirement for sigma(A)-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma(F) in vivo.

Amino Acid Sequence↗
PubMed2000

Analysis of vertebrate SCL loci identifies conserved enhancers.

The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.

Amino Acid Sequence↗
PubMed1999

A gene trap approach in Xenopus.

The frog transgenesis technique ultimately promises to make mutagenesis possible through random insertion of plasmid DNA into the genome. This study was undertaken to evaluate whether a gene trap approach combined with transgenesis would be appropriate for performing insertional mutagenesis in Xenopus embryos. Firstly, we confirmed that the transgenic technique results in stable integration into the genome and that transmission through the germline occurs in the expected Mendelian fashion. Secondly, we developed several gene trap vectors, using the green fluorescent protein (GFP) as a marker. Using these vectors, we trapped several genes in Xenopus laevis that are expressed in a spatially restricted manner, including expression in the epiphysis, the olfactory bulb and placodes, the eyes, ear, brain, muscles, tail and intestine. Finally, we cloned one of the trapped genes using 5' rapid amplification of cDNA ends polymerase chain reaction (RACE PCR). These results suggest that the transgenic technique combined with a gene trap approach might provide a powerful method for generating mutations in endogenous genes in Xenopus.

Amino Acid Sequence↗
PubMed1996

Inhibition of FGF receptor activity in retinal ganglion cell axons causes errors in target recognition.

Native fibroblast growth factor receptor (FGFR) function was inhibited in developing Xenopus retinal ganglion cells (RGCs) by in vivo transfection of a dominant negative FGFR. Axons expressing the dominant negative protein advanced at 60% of the normal speed, but nevertheless navigated appropriately in the embryonic optic pathway. When they neared the optic tectum, however, many axons made erroneous turns, causing them to bypass rather than enter their target. By contrast, RGC axons expressing nonfunctional FGFR mutants entered the tectum correctly. These findings demonstrate a role for FGFR signaling in the extension and targeting of RGC axons and suggest that receptor tyrosine kinase/growth factor interactions play a critical function in establishing initial connectivity in the vertebrate visual system.

Animals↗
PubMed1996

Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.

We have developed a simple approach for large-scale transgenesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Transgenic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.

Animals↗
PubMed1995

Temporal regulation of the Xenopus FGF receptor in development: a translation inhibitory element in the 3' untranslated region.

Early frog embryogenesis depends on a maternal pool of mRNA to execute critical intercellular signalling events. FGF receptor-1, which is required for normal development, is stored as a stable, untranslated maternal mRNA transcript in the fully grown immature oocyte, but is translationally activated at meiotic maturation. We have identified a short cis-acting element in the FGF receptor 3' untranslated region that inhibits translation of synthetic mRNA. This inhibitory element is sufficient to inhibit translation of heterologous reporter mRNA in the immature oocyte without changing RNA stability. Deletion of the poly(A) tract or polyadenylation signal sequences does not affect translational inhibition by this element. At meiotic maturation, we observe the reversal of translational repression mediated by the inhibitory element, mimicking that seen with endogenous maternal FGF receptor mRNA at meiosis. In addition, the activation of synthetic transcripts at maturation does not appear to require poly(A) lengthening. We also show that an oocyte cytoplasmic protein specifically binds the 3' inhibitory element, suggesting that translational repression of Xenopus FGF receptor-1 maternal mRNA in the oocytes is mediated by RNA-protein interactions. These data describe a mechanism of translational control that appears to be independent of poly(A) changes.

Animals↗
PubMed1993

FGF signalling in the early specification of mesoderm in Xenopus.

We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateral mesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.

Animals↗
PubMed1992

Fibroblast growth factor receptors contain a conserved HAV region common to cadherins and influenza strain A hemagglutinins: a role in protein-protein interactions?

The first extracellular domain of the cadherins has been shown to exhibit extensive sequence homology with the amino termini of the HA1 chains of influenza strain A hemagglutinins. These regions of homology are known to be functionally important in both the cadherins and the hemagglutinins. The homologous regions harbor the tripeptide HAV, which has been identified as being the cadherin cell adhesion recognition sequence. Here we report that members of the rapidly expanding family of fibroblast growth factor receptors also possess HAV-containing regions. These regions are homologous to the HAV-containing regions present within both the hemagglutinins and the cadherins and appear to be involved in regulating the function of the fibroblast growth factor receptors. We speculate that the HAV motif may represent an evolutionarily conserved amino acid sequence that will prove to be functionally important in a wide variety of proteins.

Amino Acid Sequence↗
PubMed1991

Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.

Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.

Actins↗
PubMed1990

Regulation of the fibroblast growth factor receptor in early Xenopus embryos.

Recent evidence suggests that fibroblast growth factor (FGF) is a primary mesoderm inducer in Xenopus development. We have isolated a full-length cDNA clone for the Xenopus FGF receptor. Like other FGF receptors, the Xenopus homolog is a membrane-spanning protein with a split intracellular tyrosine kinase domain. The Xenopus FGF receptor mRNA is present as a maternal message whose levels are constant through early development. There is no specific regional localization of the transcript by analysis of FGF receptor mRNA levels in microdissected embryonic tissue. In isolated animal-pole blastomeres, FGF receptor mRNA declines over 16 hr in culture and this loss can be prevented by incubation with FGF or activin. Despite the presence of the FGF receptor mRNA in the oocyte, oocytes in culture do not respond to added FGF. However, injection of exogenous Xenopus FGF receptor transcripts into oocytes does generate a functional response to FGF. Our data suggest that posttranscriptional response to FGF. Our data suggest that posttranscriptional mechanisms regulate the FGF receptor in the oocyte and early embryo and further suggest that mesoderm-inducing factors influence receptor mRNA levels during the time of early tissue formation.

Amino Acid Sequence↗
PubMed1988

Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae.

We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases. CEN3 DNA is organized in chromatin such that a 220-250-bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encoding the Bam HI-recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984). Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete structural and functional centromeric unit. A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI. The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.

Cell Nucleus↗
PubMed1987

UV-induced damage and repair in centromere DNA of yeast.

The centromere is the region within a chromosome that is required for proper segregation during mitosis and meiosis. Lesions in this sequence represent a unique type of damage, as loss of function could result in catastrophic loss of the genetic material of an entire chromosome. We have measured the induction by ultraviolet (UV) light of pyrimidine dimers in a 2550-bp restriction fragment that includes the centromere region of chromosome III in Saccharomyces cerevisiae. Yeast cells were exposed to ultraviolet light, cellular DNA was gently extracted, and subsequently treated with a UV-specific endonuclease to cleave all pyrimidine dimers. The sites of UV-specific nuclease scission within the centromere were determined by separating the DNA according to molecular weight, transferring the fragments to nitrocellulose, and hybridizing to a radiolabeled 624-bp fragment homologous to the centromere DNA from chromosome III. Several hotspots were identified in chromatin DNA from cells, as well as in irradiated deproteinized DNA. Double strand damage due to closely opposed pyrimidine dimers was also observed. At biological doses (35% survival) there are approximately 0.1 to 0.2 pyrimidine dimers per centromere. These dimers are efficiently repaired in the centromere and surrounding region.

Base Sequence↗