pinecone/core-2020-05-10-deduplication huggingface dataset:
The Effect of Pertussis Toxin on Zebrafish Development: A Possible Role for Inhibitory G-Proteins in Hedgehog Signaling

AbstractRecent results have indicated that cAMP-dependent protein kinase (PKA) acts as a negative regulator of Hedgehog signaling in target cells of the vertebrate embryo. Consequently, suppression of PKA activity is sufficient to mimic the effect of receiving a Hedgehog signal. We have explored whether PKA-inhibiting Gi-proteins (GiPs) may also be involved in the regulation of Hedgehog signaling. Zebrafish embryos were injected with RNA encoding pertussis toxin (Ptx), a specific inhibitor of GiPs. These embryos developed phenotypic traits opposite to embryos expressing a dominant negative form of the PKA regulatory subunit (dnPKA), including a fusion of the eyes, a lack of ventral specification in the forebrain, and an expansion of the sclerotome at the expense of adaxial fates in the posterior somites. These effects can be partially rescued by coexpression of dnPKA, but not by coexpression of Indian Hedgehog, suggesting that GiPs act upstream of PKA and downstream of Hedgehogs. Other Hedgehog- and PKA-dependent processes, sclerotomal specification and adaxial specification in the first five somites, are not negatively affected by Ptx. Thus, GiPs may be involved in Hedgehog signaling in some, but not all target cells
LEF-1/TCF Proteins Mediate Wnt-Inducible Transcription from the Xenopus Nodal-Related 3 Promoter

AbstractThe Xenopus nodal-related 3 gene (Xnr3) is expressed in the Spemann organizer of the embryo and encodes a member of the transforming growth factor β family that mediates some activities of the organizer. Xnr3 is transcriptionally activated by wnt signaling during gastrulation in the Xenopus embryo. Here we show that a small region of the Xnr3 promoter is sufficient to confer wnt-inducible transcription. By mutational analysis of the promoter, we have identified two distinct sequence elements required for the response to wnt signals. One regulatory sequence interacts with a factor which accumulates in Xenopus gastrulae independent of wnt signaling. The other functionally important site can bind mammalian LEF-1 protein, a member of the LEF-1/TCF family of transcription factors. In addition, misexpression of LEF-1 in embryo explants induces transcription of the endogenous Xnr3 gene. Taken together, these data provide further evidence for a role of LEF-1/TCF proteins in wnt signaling and identify the Spemann organizer-specific gene Xnr3 as a direct target of these transcription factors in vertebrates
Regulation of the Transcription Factor E75 by 20-Hydroxyecdysone and Juvenile Hormone in the Epidermis of the Tobacco Hornworm,Manduca sexta,during Larval Molting and Metamorphosis

AbstractThe homolog of the ecdysteroid-induced transcription factor E75A inDrosophila melanogasterwas cloned from the tobacco hornworm,Manduca sexta,and its developmental expression and hormonal regulation were analyzed. Both E75A and E75B mRNAs were found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20Ein vitrocaused the rapid, transient induction of E75A RNA with a peak at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) showed a 10-fold higher sensitivity to 20E (EC50= 2 × 10−8M for fourth instar and 2 × 10−7M for fifth instar epidermis). The presence of the protein synthesis inhibitor anisomycin had no effect on the induction but prevented the decline, indicating that E75A RNA was directly induced by 20E, but its down-regulation depended on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, caused an increased accumulation of E75A RNA throughout the culture period although the temporal pattern was unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis
Depletion of Intracellular Ca2+Stores, Mediated by Mg2+-Stimulated InsP3Liberation or Thapsigargin, Induces a Capacitative Ca2+Influx in Prawn Oocytes

AbstractBy voltage clamp technique and intracellular calcium measurements, we recorded in prawn oocytes simultaneous [Ca2+]iand ionic current changes stimulated by external Mg2+. The [Ca2+]iresponse consists of an oscillation period followed by a second state of sustained [Ca2+]ilevel. The oscillation period successively comprises a first [Ca2+]ipeak, a series of [Ca2+]itransients, and a [Ca2+]ioscillatory plateau respectively concurrent with an initial transient outward K+Cacurrent, an inward Na+Cacurrent, and a final K+Caoutward current. By using inhibitor (heparin) or sensitizers (thimerosal or caffeine) of calcium release ER channels, and caged InsP3, we established that InsP3is the sole second messenger releasing Ca2+from intracellular stores. By sequential substitutions and reapplications of external Ca2+, and using econazole (50 μM), a Ca2+influx inhibitor, we documented Ca2+influx during the [Ca2+]ioscillatory plateau. The intracellular Ca2+store was depleted with thapsigargin (75–350 nM) in Ca2+-free ASW. Reapplication of external Ca2+evoked a rise in [Ca2+]i, indicating a store-dependent capacitative Ca2+influx, correlated with a K+Caoutward current increase. No measurable Ca2+release-activated Ca2+current (Icrac) could be detected, but was indirectly demonstrated using the sensitivity of the K+Cachannels to [Ca2+]i. We propose that the involvement of external Ca2+, in the physiological [Ca2+]iresponse of prawn oocytes to external Mg2+, consists of a store-dependent capacitative Ca2+influx
Role of Mitf in Differentiation and Transdifferentiation of Chicken Pigmented Epithelial Cell

AbstractMitfencodes a basic helix-loop-helix-leucine-zipper (bHLHzip) protein that is known to function in the development of melanocytes, pigmented epithelial cells (PECs), osteoclasts, and mast cells. In this paper, we report on the isolation, expression, and overexpression of the chickenMitfand discuss the role of its protein product in the differentiation and transdifferentiation of PECs. Northern blotting showed that chickenMitfis predominantly expressed in embryonic retinal pigmented epithelium (PE), but is expressed at low levels in other tissues. A 5′ RACE analysis revealed differences in the 5′ region ofMitfmRNA in PE and other tissues. Immunological analysis revealed that Mitf, the protein encoded byMitf,is first detected in the nuclei of the optic vesicle cells at embryonic stage 13 in a restricted region covered with mesenchymal cells. From stage 14 to 24, the specific staining is observable in the PE and precursor of the PE, the outer layer of the optic cup. In embryos at stages later than stage 29 the signals for Mitf in the future iris, ciliary body, and posterior retinal regions become faint. These results show that expression of Mitf starts at the optic vesicle stage at which no other marker genes for PECs such asmmp115andtyrosinaseare expressed. Dedifferentiation of cultured retinal PECs (rPECs) was induced by phenylthiourea and testicular hyaluronidase, bFGF, or TGF-β.Mitfexpression was inhibited by these factors and reactivated during redifferentiation of the dedifferentiated cells into rPECs, showing the correlation betweenMitfexpression and rPEC differentiation. Retrovirus-mediated overexpression ofMitfinhibited bFGF-induced dedifferentiation and transdifferentiation of rPECs to both lens and neural cells. These findings showed that downregulation ofMitfexpression is essential for the transdifferentiation of rPEC.Mitfoverexpression caused hyperpigmentation in cultured rPECs and suppressed the changes in gene expression induced by bFGF.Mitfoverexpression promoted expression ofmmp115andtyrosinasein bFGF-treated rPECs suggesting a critical role for Mitf in rPEC differentiation.Mitfoverexpression, however, did not promote expression of another rPEC-specific gene,pP344,in bFGF-treated rPECs. This result suggests the presence of other regulatory genes promoting rPEC differentiation. The expression patterns ofpax6andMitfare complementary bothin vivoandin vitro.Overexpression ofMitfinhibited expression ofpax6in cultured rPECs. These observations suggest that Mitf regulatespax6expression negatively
Common Epicardial Origin of Coronary Vascular Smooth Muscle, Perivascular Fibroblasts, and Intermyocardial Fibroblasts in the Avian Heart

AbstractPrevious studies have shown that during avian heart development, epicardial and coronary vascular smooth muscle precursors are derived from the proepicardium, a derivative of the developing liver. This finding led to a model of coronary vascular development in which epicardial cells migrate over the postlooped heart, followed by migration of committed endothelial and smooth muscle precursors from the proepicardium through the subepicardial matrix where the coronary arteries develop. Here we show that epicardial cells undergo epithelial–mesenchymal transformation to become coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts. We began by establishing primary cultures of quail epicardial cells that retain morphologic and antigenic identity to epicardial cellsin vivo.Quail epicardial monolayers stimulated with serum or vascular growth factors produced invasive mesenchyme in collagen gels. Chick epicardial cells labeledin ovowith DiI invaded the subepicardial extracellular matrix, demonstrating that mesenchymal transformation of epicardium occursin vivo.To determine the fates of epicardially derived mesenchymal cells, quail epicardial cells labeledin vitrowithLacZwere grafted into the pericardial space of E2 chicks. These cells attached to the heart, formed a chimeric epicardium, invaded the subepicardial matrix and myocardial wall, and became coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts, demonstrating the common epicardial origin of these cell types. A general model of coronary vascular development should now include epicardial–mesenchymal transformation and direct participation of mesenchyme derived from the epicardium in coronary morphogenesis
Structure and Function of a Sea Urchin Orthodenticle-Related Gene (HpOtx)

AbstractTwo distinct types of orthodenticle-related proteins (HpOtxE/L) have been implicated as transcription activators of the aboral ectoderm-specific arylsulfatase (Ars) gene. Here, we describe the structure of HpOtx gene and present evidence that mRNAs ofHpOtxE/Lare transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing. By transactivation experiments, we have also demonstrated that HpOtxLactivates theArspromoter in the gastrula-stage embryo
HGF/SF Is Present in Normal Adult Skeletal Muscle and Is Capable of Activating Satellite Cells

AbstractWe have shown that hepatocyte growth factor/scatter factor can stimulate activation and early division of adult satellite cells in culture, and that the action of hepatocyte growth factor/scatter factor is similar to the action of the unidentified satellite cell activator found in extracts of crushed muscle. We now provide new evidence that hepatocyte growth factor/scatter factor is present in uninjured adult rat skeletal muscle and that the activating factor in crushed muscle extract is hepatocyte growth factor/scatter factor. Immunoblots of crushed muscle extract demonstrate the presence of hepatocyte growth factor/scatter factor. Furthermore, crushed muscle extract stimulates the scattering of cultured MDCK cells. Immunolocalization studies with adult rat skeletal muscle show the presence of hepatocyte growth factor/scatter factor in the extracellular matrix surrounding muscle fibers; in addition, the receptor for hepatocyte growth factor/scatter factor, c-met, is localized to putative satellite cells. In muscle from mdx mice, hepatocyte growth factor/scatter factor and c-met are colocalized in activated satellite cells in regions of muscle repair. Moreover, the satellite cell-activating activity of crushed muscle extract is abolished by preincubation with anti-hepatocyte growth factor antibodies. Finally, direct injection of hepatocyte growth factor/scatter factor into uninjured tibialis anterior muscle of 12-month-old rats stimulated satellite cell activation. These experiments demonstrate that hepatocyte growth factor/scatter factor is present in muscle, can be released upon injury, and has the ability to activate quiescent satellite cellsin vivo
Signal Transduction Pathways Leading to Spore Differentiation inDictyostelium discoideum

AbstractCells that overexpress PKA as a consequence of carrying multiple copies of the gene encoding the catalytic subunit can be induced to sporulate when developing as single cells. A peptide phosphorylated by PKA, termed SDF-1, has recently been shown to stimulate this process (Anjardet al.,1997). Several genes have been implicated in a signal transduction pathway by which prestalk cells induce encapsulation of prespore cells during terminal differentiation including a prestalk-specific putative membrane protease (TagC) and a two-component system consisting of a receptor-histidine kinase (DhkA) and a response regulator with cAMP phosphodiesterase activity (RegA). To determine whether SDF-1 uses this pathway, strains carrying null mutations in the pertinent genes were transformed with apkaCplasmid such that they can overexpress PKA. Since these mutant strains all sporulated efficiently when SDF-1 was added, it appears that other gene products mediate the response. However, we found thatregA−mutant cells release a distinct factor, SDF-2, that rapidly induces encapsulation of test cells overexpressingpkaC.Since cells in whichtagCis disrupted do not form SDF-2 and cells in whichdhkAis disrupted do not respond to SDF-2, this peptide appears to use the two-component system that regulates PKA activity. SDF-2 is a small peptide released by prestalk cells in a manner dependent on TagC. It appears to act on prespore cells through the DhkA receptor to inhibit the cAMP phosphodiesterase of RegA, thereby activating PKA via cAMP. The process of induction by SDF-2 can be shown to be distinct from that by SDF-1. SDF-2 appears to stimulate prestalk cells to release additional SDF-2 by acting through a signal transduction pathway that also involves DhkA, RegA, and PKA. Based on these results we present a model for the signal transduction cascade regulating spore differentiation
Stability of Frog Motor Nerve Terminals in the Absence of Target Muscle Fibers

AbstractUsing repeatedin vivoimaging, we addressed the role of target muscle fibers in the maintenance of frog motor nerve terminals at synaptic sites. Target-deprived nerve terminals were generated by selective and permanent removal of muscle fibers without damage to the innervation. Individual nerve terminals, stained with the dye FM1-43, were imaged before and again during the subsequent 1–9 months of target deprivation and the stability of the nerve terminal arbors over time was determined. Repeated observation of motor nerve terminals showed that nerve terminals were well maintained at synaptic sites during the first 1–2 months after target loss; the original number of nerve terminal segments was retained at 85% of the synaptic sites after muscle damage. After long periods of target deprivation, 6–9 months, loss or retraction of nerve terminal segments resulted in a reduction in the arbor of most but not all nerve terminals. This apparent nerve terminal destabilization was not a result of illumination or irradiation because a similar decrease in the extent of nerve terminal arbors was not observed at control irradiated neuromuscular junctions. The persistence of many complete target-deprived nerve terminal arbors at synaptic sites long after target degeneration suggested that the cues that confer stability to frog motor nerve terminals likely reside external to muscle fibers and may be associated with the synaptic basal lamina or the terminal Schwann cell. Since the arbors of many target-deprived nerve terminals were eventually reduced, the nonmuscle stabilization cues may not persist indefinitely at target-deprived synaptic sites
An Autocrine Function for Transforming Growth Factor (TGF)-β3 in the Transformation of Atrioventricular Canal Endocardium into Mesenchyme during Chick Heart Development

AbstractTransformation of atrioventricular canal endocardium into invasive mesenchyme is a critical antecedent of cardiac septation and valvulogenesis. Previous studies by Pottset al.(Proc. Natl. Acad. Sci. USA88, 1510–1520, 1991) showed that treatment of atrioventricular canal endocardial and myocardial cocultures with TGFβ3 antisense oligodeoxynucleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGFβ3 antisense oligos in this transformation bioassay, and (ii) more clearly define the mechanism of TGFβ3 function in atrioventricular canal mesenchyme formation.In situhybridization and immunohistochemistry showed little or no TGFβ3 mRNA or protein in the atrioventricular canal myocardium or endocardium prior to mesenchyme formation (stage 14; paraformaldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were positive for both TGFβ3 mRNA and protein. In culture bioassays, atrioventricular canal endocardial monolayers pretreated with antisense phosphorothioate oligodeoxynucleotides to TGFβ3 did not transform into invasive mesenchyme in response to cardiocyte conditioned medium: the subsequent addition of exogenous TGFβ3 protein relieved this inhibition. Control cultures without pretreatment or those receiving missense oligos generated similar numbers of invasive mesenchyme in response to cardiocyte conditioned medium. Direct addition of TGFβ3 protein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associations and stimulated cellular hypertrophy, but did not engender invasive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGFβ2 protein, either alone or in combination with TGFβ3. The results of this study indicate that: (i) atrioventricular canal endocardium expresses TGFβ3 in response to a myocardially derived signal other than TGFβ3, (ii) atrioventricular canal endocardial TGFβ3 functions in an autocrine fashion to elicit selected characteristics necessary for cushion tissue formation, and (iii) TGFβ3 alone or in combination with TGFβ2 is insufficient to transform atrioventricular canal endocardium into invasive mesenchyme in culture
Involvement of Mash1 in EGF-Mediated Regulation of Differentiation in the Vertebrate Retina

AbstractIt is believed that signaling through the epidermal growth factor (EGF) receptor plays a critical role in the development ofDrosophilaeyes. In the present study we have analyzed the role that EGF-mediated signaling plays in vertebrate retinal development. We have observed that during late retinal neurogenesis EGF delays rod photoreceptor differentiation and that this effect of EGF involves the modulation of expression of a homologue ofDrosophilaproneural genes,Mash1.EGF causes a significant decrease in Mash1 expression and an increase in the proportion of proliferating cells in the retinain vitro.The decrease in Mash1 expression is accompanied by a concomitant decrease in opsin expression, a marker for overt rod photoreceptor differentiation. Withdrawal of EGF leads to an increase in both Mash1 and opsin expression; however, the onset of expression of Mash1 precedes that of opsin. Our study identifies a proliferative intermediate precursor, characterized by Mash1 expression, that is the target of EGF-mediated suppression of rod photoreceptor differentiation. Based on the evolutionarily conserved roles of EGF- and Notch-mediated signaling in the delay of differentiation in proliferating precursors we propose that these distinct signaling mechanisms act in concert to ensure the fidelity of the strict temporal and spatial nature of cell fate determination in the retina
Lineage-Specific Regulation of theCiona snailGene in the Embryonic Mesoderm and Neuroectoderm

AbstractThesnailgene encodes a highly conserved, zinc-finger transcription factor that has been implicated in the specification of mesodermal and neuronal tissues in a variety of organisms. In the ascidian,Ciona intestinalis, snail(Ci-sna) is expressed at the 32-cell stage in derivatives of the B4.1 blastomere, including B6.2, B6.4, and B7.5, which give rise to the primary-lineage tail muscles of the tadpole. At later stages,Ci-snais expressed in the lineages that will form the secondary tail muscle, the lateral ependymal cells of the spinal cord, and the dorsal cells of the cerebral vesicle. A minimal, 504-bpcis-regulatory sequence from theCi-snapromoter region, the B4.1 enhancer, is shown to direct the expression of heterologous promoters in primary-lineage muscles. Furthermore, evidence is presented thatcis-regulatory elements necessary for expression in both the secondary muscle and neuronal lineages are separate from the B4.1 enhancer. We discuss the possibility that the classical muscle determinant present in ascidian eggs may correspond to bHLH activators, which bind to specific E-box sequences contained in the B4.1 enhancer
Murine Cerberus Homologue mCer-1: A Candidate Anterior Patterning Molecule

AbstractXenopuscerberus (Xcer) is a cytokine expressed in anterior mesendoderm overlapping and surrounding Spemann's gastrula organiser. When misexpressed in blastomeres, Xcer can induce ectopic heads with well-defined brain, cement gland, olfactory placodes, cyclopic eye, and occasionally liver and heart. We report here the identification ofmCer-1,a murine gene related tocerberus.Both mCer-1 and Xcer appear to belong to the cystine knot superfamily, which includes TGFβs and BMPs. InXenopusanimal cap assays, mCer-1 and Xcer induced cement glands and markers of anterior neural tissue and endoderm, characteristic of BMP inhibition. Furthermore, both antagonised the ventrolateral mesoderm-inducing activity of coexpressed BMP4. In mouse embryos,mCer-1was expressed at early gastrulation in a stripe of primitive endoderm along the future anterior side of the egg cylinder, a region essential for anterior patterning. A second phase of expression was detected in anterior embryonic mesendoderm, and by late-streak stages most of the anterior half of the embryo was positive, except for the node and cardiac progenitors. Expression was later seen in the cranial portion of the two most-recently formed somites and in two stripes within presomitic mesoderm. In embryos lackingOtx2,a homeogene with a demonstrated role in anterior patterning,mCer-1was still expressed in an anterior zone, although often abnormally. The data suggest that mCer-1 shares structural, functional, and expression characteristics with Xcer and may participate in patterning the anterior of the embryo and nascent somite region, in part, through a BMP-inhibitory mechanism