pinecone/core-2020-05-10-deduplication huggingface dataset:
Two Otx Proteins Generated from Multiple Transcripts of a Single Gene inStrongylocentrotus purpuratus

AbstractOrthodenticle-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-specificSpec2agene 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 containingSpOtxsequences. Here, we report that SpOtx exists in two forms (α and β) that are generated by alternative RNA splicing from a singleSpOtxgene. SpOtx(α) and SpOtx(β) had identical C-termini and homeoboxes but were entirely different in their N-terminal domains.SpOtx(α)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 distinctSpOtx(β)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(β) appeared later in development than SpOtx(α). Overexpression of SpOtx(α) and SpOtx(β) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx(α) was able to repress the accumulation ofSpOtx(β)transcripts, whereas SpOtx(β) had no effect on the accumulation ofSpOtx(α)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
Characterization of the Binding of Recombinant Mouse Sperm Fertilin β Subunit to Mouse Eggs: Evidence for Adhesive Activity via an Egg β1Integrin-Mediated Interaction

AbstractThe sperm protein fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes. Fertilin is a heterodimer. The β subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface. To investigate this hypothesis and determine what role fertilin β plays in fertilization, we have expressed the putative extracellular domain of mouse fertilin β in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant fertilin β-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant fertilin β-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant fertilin β-EC to single eggs. We find that recombinant fertilin β-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that fertilin β has adhesive properties. Peptides corresponding to the disintegrin domain of fertilin β reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of fertilin β by binding sites on the egg. Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant fertilin β-EC, implicating an egg surface protein as a binding site for recombinant fertilin β-EC. Binding of recombinant fertilin β-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mMCa2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant fertilin β-EC prior toin vitrofertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for fertilin β, since an anti-α6integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeidaet al.(1995)Cell81, 1095–1104). We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant fertilin β-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant fertilin β-EC binding; and (c) recombinant fertilin β-EC binding is reduced in the presence of anti-β1integrin antibodies. These results suggest that a β1-containing integrin participates in the binding of recombinant fertilin β-EC to mouse eggs
Characterization of the Binding of Recombinant Mouse Sperm Fertilin α Subunit to Mouse Eggs: Evidence for Function as a Cell Adhesion Molecule in Sperm-Egg Binding

AbstractFertilin (previously known as PH-30) is a sperm protein that is a candidate molecule for mediating the binding and fusion of the sperm and egg plasma membranes. Fertilin is a heterodimer, with a β subunit that has a region of homology to the disintegrin family of integrin ligands and an α subunit that has a region of homology to viral fusion peptides. It has been hypothesized that fertilin β and α subunits mediate the interactions between sperm and egg plasma membranes, namely, binding and fusion, respectively. To address this hypothesis and to examine specifically the role of fertilin α in fertilization, we have expressed the predicted extracellular domain of mouse fertilin α as a bacterial fusion protein with maltose-binding protein. This fusion protein (hereafter referred to as recombinant fertilin α-EC) binds to the microvillar region ofzona pellucida(ZP)-free eggs, the region of the membrane to which sperm bind. This binding is reduced in the absence of divalent cations and is supported by Ca2+, Mg2+, or Mn2+. Eggs that have been treated with chymotrypsin bind less recombinant fertilin α-EC than do untreated eggs, suggesting that a chymotrypsin-sensitive binding site for recombinant fertilin α-EC is present on egg surfaces. Binding to eggs is also affected by the method used to remove the ZP. Finally, recombinant fertilin α-EC inhibits the binding of sperm to eggs duringin vitrofertilization of ZP-free eggs. These data are the first evidence to suggest that fertilin α can function as a cell adhesion molecule during fertilization, mediating the binding of sperm and egg plasma membranes
Twist Protein in Mouse Embryogenesis

AbstractThe genes in thetwistfamily of bHLH transcription factors are essential for embryogenesis of both vertebrates and invertebrates, as demonstrated by the embryonic lethal phenotypes of theDrosophilaand mousetwist-null mutants. Presented here is the embryonic distribution of murine Twist protein (MTwist). Its appearance and presence in the course of early embryogenesis was followed with a monoclonal antibody, αTwiMab-1, the first antibody generated against any of the vertebrate Twist proteins. The specificity for MTwist was demonstrated in comparative Western blot experiments, reticulocyte lysate assays, and immunohistochemistry of embryonic mouse samples. Consistent with its probable role as a transcription factor, MTwist localized to the nuclei. MTwist signal first appeared in 8- to 10-somite embryos at 8.25 dpc in the cranial neural crest cells and branchial arches, in the limb-bud mesenchyme and the somatic lateral plate, and in the sclerotome and the dermatome of the somites. The presence of MTwist protein in these tissues corroborates the reportedMTwistRNA distribution and the phenotype of theMTwist-null mutants. There also emerged, however, an unexpected difference betweenMTwistRNA and protein expression. No protein could be detected prior to 8.25 dpc, despite the reported high levels of transcripts as early as 7.0 dpc. Also, the presomitic mesoderm, epithelial somites, and anterior mesoderm expressed abundantMTwistRNA, but no protein. The results suggest posttranscriptional downregulation ofMTwistin these regions. A proposed role of MTwist in somite formation and maturation is inhibition of myogenic bHLH and MEF2 genes and thus prevention of premature and/or ectopic differentiation in the presomitic mesoderm and epithelial somites. The absence of MTwist protein from these areas indicates that its role in somitogenesis must be reevaluated
A Role forIndian hedgehogin Extraembryonic Endoderm Differentiation in F9 Cells and the Early Mouse Embryo

AbstractHedgehoggenes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression ofIndian hedgehogmRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulatesIndian hedgehogexpression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or noIndian hedgehogmessage is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells.In situhybridization analysis forIndian hedgehogmRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mountin situhybridization analysis of Day 7.5 and 8.5 embryos indicates thatIndian hedgehogexpression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full lengthIndian hedgehogcDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest thatIndian hedgehogis involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis
Chicken Transcription Factor AP-2: Cloning, Expression and Its Role in Outgrowth of Facial Prominences and Limb Buds

AbstractEmbryonic facial development in chick embryos involves a sequential activation of genes that control differential growth and patterning of the beak. In the present study we isolate one such gene, the transcription factor, AP-2, that is known to be expressed in the face of mouse embryos. The protein sequence of chick AP-2α is 94% homologous to human and mouse AP-2. Wholemountin situhybridization with a probe for chick AP-2 identifies expression from primitive streak stages up to stage 28. The most striking expression patterns in the head are during neural crest cell migration when AP-2 transcripts follow closely the tracts previously mapped for neural crest cells. Later, expression in the facial mesenchyme is strongest in the frontonasal mass and lateral nasal prominences and is downregulated in the maxillary and mandibular prominences. Once limb buds are visible, high expression is seen in the distal mesenchyme but not in the apical ectodermal ridge. The expression patterns of AP-2 in stage 20 embryos suggested that the gene may be important in “budding out” of facial prominences and limb buds. We implanted beads soaked in retinoic acid in the right nasal pit of stage 20 embryos resulting in a specific inhibition of outgrowth of the frontonasal mass and lateral nasal prominences. AP-2 expression was completely down-regulated in the lateral nasal within 8 hr of bead application. In addition, the normal up-regulation of AP-2 in the frontonasal mass did not occur following retinoic-acid treatment. There was an increase in programmed cell death around the right nasal pit that accompanied the down-regulation of AP-2. Prominences whose morphogenesis were not affected by retinoic acid did not have altered expression patterns. We removed the apical ectodermal ridge in stage 20 limb buds and found that AP-2 expression was partially downregulated 4 hr following ridge removal and completely downregulated 8 hr following stripping. Application of an FGF-4 soaked bead to the apex of the limb bud maintained AP-2 expression. Thus AP-2 is involved in outgrowth and could be regulated by factors such as FGFs that are present in the ectoderm of both the face and limb
The Removal of the Sperm Perinuclear Theca and Its Association with the Bovine Oocyte Surface during Fertilization

AbstractThe perinuclear theca (PT) is a unique cytoskeletal structure whose anterior part is intercalated between the inner acrosomal membrane and the nuclear envelope of the mammalian sperm head and is important for spermiogenesis and stabilization of sperm structures (Oko and Maravei,Biol. Reprod.50, 1000–1014, 1994; Oko and Maravei,Microsc. Res. Tech.32, 520–532, 1995). Using immunofluorescence labeling of inseminated bovine oocytes and serial sectioning–ultrastructural analysis, we demonstrate that the PT is removed from the sperm nucleus following the loss of the sperm plasma membrane and the interaction of oocyte cortex with the PT. These events precede the development of the male pronucleus. The removal of the PT involves the elongated oocyte microvilli, rich in actin microfilaments, since it can be blocked by the microfilament-disrupting drug cytochalasin B. Reduction of disulfide bonds, which is a major factor supporting the disassembly of the sperm nucleus and accessory structures during mammalian fertilization, seems to exert little effect on the PTin vitro,as evidenced by the treatment of isolated bull sperm with the disulfide bond-reducing agent dithiothreitol.In vivo,intact bull sperm microinjected into mature oocytes do not undergo disassembly of the PT. Consequently, the decondensation of the sperm nucleus does not occur. These data suggest that the binding of the PT to the oocyte microvillar region and its removal from the sperm nucleus constitute an early step in mammalian fertilization, which is required for the conversion of the sperm nucleus into a male pronucleus
Both Myoblast Lineage and Innervation Determine Fiber Type and Are Required for Expression of the Slow Myosin Heavy Chain 2 Gene

AbstractSkeletal muscle fibers express members of the myosin heavy chain (MyHC) gene family in a fiber-type-specific manner. In avian skeletal muscle it is the expression of the slow MyHC isoforms that most clearly distinguishes slow- from fast-contracting fiber types. Two hypotheses have been proposed to explain fiber-type-specific expression of distinct MyHC genes during development—an intrinsic mechanism based on the formation of different myogenic lineage(s) and an extrinsic, innervation-dependent mechanism. We developed a cell culture model system in which both mechanisms were evaluated during fetal muscle development. Myoblasts isolated from prospective fast (pectoralis major) or slow (medial adductor) fetal chick muscles formed muscle fibers in cell culture, none of which expressed slow MyHC genes. By contrast, when muscle fibers formed from myoblasts derived from the slow muscle were cocultured with neural tube, the muscle fibers expressed a slow MyHC gene, while muscle fibers formed from myoblasts of fast muscle origin continued to express only fast MyHC. Motor endplates formed on the fibers derived from myoblasts of both fast and slow muscle origin in cocultures, and slow MyHC gene expression did not occur when neuromuscular transmission or depolarization was blocked. We have cloned the slow MyHC gene that is expressed in response to innervation and identified it as the slow MyHC 2 gene, the predominant adult slow isoform. cDNAs encoding portions of the three slow myosin heavy chain genes (MyHC1, slow MyHC 2, and slow MyHC 3) were isolated. Only slow MyHC 2 mRNA was demonstrated to be abundant in the cocultures of neural tube and muscle fibers derived from myoblasts of slow muscle origin. Thus, expression of the slow MyHC 2 gene in thisin vitrosystem indicates that formation of slow muscle fiber types is dependent on both myoblast lineage (intrinsic mechanisms) and innervation (extrinsic mechanisms), and suggests neither mechanism alone is sufficient to explain formation of muscle fibers of different types during fetal development
Relationships between Protein Isoforms and Genetic Functions Demonstrate Functional Redundancy at theBroad-ComplexduringDrosophilaMetamorphosis

AbstractMetamorphosis in holometabolous insects is an ecdysone-dependent process by which the larval form is replaced by a reproductive, adult form. At the onset of metamorphosis ecdysone induces a set of early genes which coordinate tissue-specific responses to hormone. TheBroad-Complex(BR-C) early gene, which acts as a global regulator of tissue-specific responses to ecdysone, encodes a family of zinc-finger DNA binding proteins known as Z1, Z2, Z3, and Z4. Genetically theBR-Cencodes three complementing functions,br, rbp,and2Bc,and a class ofnpr1alleles that fail to complement any of the other genetic functions. The effects ofBR-Cmutations on metamorphic development are highly pleiotropic, yet little is known about the roles of individualBR-Cproteins in directing the required responses to ecdysone. Because theBR-Cis a vital regulator of metamorphosis it is essential to establish the relationships betweenBR-Cgenetic functions and protein products. We present here the first general and definitive study of these relationships. Using heat-inducible transgenes we have rescued lethality associated with each of the complementing genetic functions and have restored transcriptional activity of tissue-specificBR-C+-dependent target genes. Our data lead us to conclude thatbr+function is only provided by the Z2 isoform. We find that Z1 transgenes provide fullrbp+function, while Z4 provides partial function. Likewise, while Z3 provides full2Bc+function, Z2 also provides partial function. These results indicate possible functional redundancy or regulatory dependence (via autoregulation) associated with therbp+and2Bc+functions. The establishment of these relationships betweenBR-Cgenetic functions and protein isoforms is an important step toward understanding the roles ofBR-Cproteins in directing metamorphic responses to ecdysone
Evidence for Serum Response Factor-Mediated Regulatory Networks GoverningSM22αTranscription in Smooth, Skeletal, and Cardiac Muscle Cells

AbstractSM22α is an adult smooth muscle-specific protein that is expressed in the smooth, cardiac, and skeletal muscle lineages during early embryogenesis before becoming restricted specifically to all vascular and visceral smooth muscle cells (SMC) in late fetal development and adulthood. We have used theSM22αgene as a marker to define the regulatory mechanisms that control muscle-specific gene expression in SMCs. Previously, we reported that the 445-base-pair promoter ofSM22αwas sufficient to direct transcription of a lacZ reporter gene in early cardiac and skeletal muscle cell lineages and in a subset of arterial SMCs, but not in venous nor visceral SMCs in transgenic mice. Here we describe two evolutionarily conserved CArG (CC(A/T)6GG) boxes in theSM22αpromoter, both of which are essential for full promoter activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, skeletal, and cardiac muscle lineages in transgenic mice. Both CArG boxes bind serum response factor (SRF), but SRF binding is not sufficient forSM22αpromoter activity, since overexpression of SRF in the embryonal teratocarcinoma cell line F9, which normally expresses low levels of SRF, fails to activate the promoter. However, a chimeric protein in which SRF was fused to the transcription activation domain of the viral coactivator VP16 is able to activate theSM22αpromoter in F9 cells. These results demonstrate the SM22α promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to theSM22αpromoter and they suggest that SRF activatesSM22αtranscription in conjunction with additional regulatory factors that are cell type-restricted
Regionalized Transcriptional Domains of Myosin Light Chain 3f Transgenes in the Embryonic Mouse Heart: Morphogenetic Implications

AbstractWithin the embryonic heart, five segments can be distinguished: two fast-conducting atrial and ventricular compartments flanked by slow-conducting segments, the inflow tract, the atrioventricular canal, and the outflow tract. These compartments assume morphological identity as a result of looping of the linear heart tube. Subsequently, the formation of interatrial, interventricular, and outflow tract septa generates a four-chambered heart. The lack of markers that distinguish right and left compartments within the heart has prevented a precise understanding of these processes. Transgenic mice carrying annlacZreporter gene under transcriptional control of regulatory sequences from the MLC1F/3F gene provide specific markers to investigate such regionalization. Our results show that transgene expression is restricted to distinct regions of the myocardium: β-galactosidase activity in 3F-nlacZ-2E mice is confined predominantly to the embryonic right atrium, atrioventricular canal, and left ventricle, whereas, in 3F-nlacZ-9 mice, the transgene is expressed in both atrial and ventricular segments (right/left) and in the atrioventricular canal, but not in the inflow and outflow tracts. These lines of mice illustrate that distinct embryonic cardiac regions have different transcriptional specificities and provide early markers of myocardial subdivisions. Regional differences in transgene expression are not detected in the linear heart tube but become apparent as the heart begins to loop. Subsequent regionalization of transgene expression provides new insights into later morphogenetic events, including the development of the atrioventricular canal and the fate of the outflow tract
Retinoic Acid Can Block Differentiation of the Myocardium after Heart Specification

AbstractWhile a number of transcription factors that are likely to play a role in cardiac differentiation have recently been described, the signals that lead to the expression of these factors remains poorly understood. Here we report that exposure ofXenopusembryos to continuous low levels of all-transretinoic acid (RA), starting at the time of neural fold closure, blocks expression of myocardial differentiation markers. The development of the remainder of the embryo is relatively normal, suggesting that retinoic acid can act rather specifically on myocardial precursors. Indeed, the pattern of endocardial gene expression appears to remain unaffected by RA treatment. Although RA blocks myocardial gene expression, a superficially normal heart tube forms. The heart tube, however, fails to loop during subsequent development and never forms beating tissue. The effect of RA treatment on expression of myocardial genes is developmental stage dependent, since no influence is observed after myocardial differentiation has commenced. These data indicate that a vital component of the myocardial determination pathway is sensitive to retinoid signaling