pinecone/core-2020-05-10-deduplication huggingface dataset:
Vaccinia as a Tool for Functional Analysis in Regenerating Limbs: Ectopic Expression of Shh

AbstractAxolotls, with their extensive abilities to regenerate as adults, provide a useful model in which to study the mechanisms of regeneration in a vertebrate, in hopes of understanding why other vertebrates cannot regenerate. Although the expression of many genes has been described in regeneration, techniques for functional analysis have so far been limited. In this paper we demonstrate a new method for efficient overexpression of foreign genes in axolotls. Using vaccinia virus expressing β-galactosidase microinjected into regenerating limbs, we show that vaccinia can infect both dividing and nondividing limb cells. The site of infection remains discrete and there is no secondary spread of infection to nearby cells. β-Gal is expressed at high levels in blastema cells for about a week and in differentiated cells for longer. Blastemas that have been injected with vaccinia at different stages regenerate normally. As a test of the utility of vaccinia for functional analysis in regeneration, we constructed a virus expressing Shh and injected it into the anterior of regenerating limbs. Ectopic Shh expression caused extra digits, carpals, and tarsals in the hands and feet of regenerating limbs, suggesting that despite differences in the timing of expression and the eventual pattern, the function of Shh appears to be similar to that in the developing limbs of other vertebrates. Our results demonstrate that vaccinia virus is an excellent vector for ectopically expressing genes for secreted proteins and is a useful tool to study the function of signaling molecules during the process of regeneration in urodeles
Disruption of Lens Fiber Cell Differentiation and Survival at Multiple Stages by Region-Specific Expression of Truncated FGF Receptors

AbstractTo determine if fibroblast growth factor signaling mechanisms are required for terminal differentiation and survival of lens fiber cells, we evaluated the effects of expressing truncated fibroblast growth factor receptors (tFGFRs) in different regions of the developing lens. Two sets of transgenic mice were generated, one expressing tFGFRs from the αA-crystallin promoter (αA-tFGFR), which expresses linked genes in fiber cells throughout their differentiation program, and the other expressing tFGFRs from the γF-crystallin promoter (γF-tFGFR), which expresses linked genes beginning later during their differentiation. Histological and TUNEL analyses of lenses from αA-tFGFR and γF-tFGFR transgenic mice suggest that FGFR signaling is required for both early and late fiber cell differentiation and/or survival of the terminally differentiated cells. Additionally, multilayering and increased levels of apoptosis were observed in the anterior epithelium after the onset of fiber cell abnormalities. In situ hybridizations suggest that tFGFR transgenes were not expressed at significant levels in the epithelium. Combined with TUNEL and X-gal analyses on the lens epithelium from γF-tFGFR/Rosaβ-geo26 and nontransgenic/Rosaβ-geo26 chimeras, these results suggest that the organization and survival of the epithelial cells depend on appropriate structure and/or function of the differentiated fiber cells
The Morphology of Heart Development in Xenopus laevis

AbstractWe have used serial histological sections to document heart formation in Xenopus laevis, from the formation of a linear heart tube to the appearance of morphologically distinct atrial and ventricular chambers. 3D reconstruction techniques have been used to derive accurate models from digital images, revealing the morphological changes that accompany heart differentiation. To demonstrate the utility of this approach in analysing cardiac gene expression, we have reexamined the distribution of Hand1 transcripts in the linear and looped heart tube. Our results demonstrate that prior to looping, an initial asymmetric, left-sided pattern is replaced by more symmetrical localisation of transcripts to the ventral portion of the myocardium. After the onset of looping, Hand1 expression is restricted to the ventral ventricular myocardium and extends along the entire length of the single ventricle
An Nkx-Dependent Enhancer Regulates cGATA-6 Gene Expression during Early Stages of Heart Development

AbstractThe evolutionarily conserved GATA-6 transcription factor is an early and persistent marker of heart development in diverse vertebrate species. We previously found evidence for a functionally conserved heart-specific enhancer upstream of the chicken GATA-6 (cGATA-6) gene and in the present study we used transgenic mouse assays to further characterize this regulatory module. We show that this enhancer is activated in committed precursor cells within the cardiac crescent and that it remains active in essentially all cardiogenic cells through the linear heart stage. Although this enhancer can account for cGATA-6 gene expression early in the cardiogenic program, it is not able to maintain expression throughout the heart later in development. In particular, the enhancer is sequentially downregulated along the posterior to anterior axis, with activity becoming confined to outflow tract myocardium. Enhancers with similar properties have been shown to regulate the early heart-restricted expression of the mouse Nkx2.5 transcription factor gene. Whereas these Nkx2.5 enhancers are GATA-dependent, we show that the cGATA-6 enhancer is Nkx-dependent. We speculate that these enhancers are silenced to allow GATA-6 and Nkx2.5 gene expression to be governed by region-specific enhancers in the multichambered heart
The Nudel Protease of Drosophila Is Required for Eggshell Biogenesis in Addition to Embryonic Patterning

AbstractThe dorsoventral axis of the Drosophila embryo is defined by a ventral signal that arises within the perivitelline space, an extracellular compartment between the embryo plasma membrane and the vitelline membrane layer of the eggshell. Production of the ventral signal requires four members of the serine protease family, including a large modular protein with a protease domain encoded by the nudel gene. Here we provide evidence that the Nudel protease has an integral role in eggshell biogenesis. Mutations in nudel that disrupt Nudel protease function produce eggs having vitelline membranes that are abnormally permeable to the dye neutral red. Permeability varies among mutant nudel alleles but correlates with levels of Nudel protease catalytic activity and function in embryonic dorsoventral patterning. These mutations also block cross-linking of vitelline membrane proteins that normally occurs upon egg activation, just prior to fertilization. In addition, Nudel protease autoactivation temporally coincides with vitelline membrane cross-linking and can be triggered in mature eggs in vitro by conditions that lead to egg activation. We discuss how the Nudel protease might be involved in both eggshell biogenesis and embryonic patterning
Modulation of Mouse Preimplantation Embryo Development by Acrogranin (Epithelin/Granulin Precursor)

AbstractPreimplantation mammalian embryos in culture secrete autocrine growth factors into the surrounding medium that, in turn, stimulate the development of the embryos. The full complement of these factors is unknown. Since one hallmark of embryo development is the formation of an epithelium, the trophectoderm, we tested the hypothesis that one such embryo-derived growth factor is acrogranin (epithelin/granulin precursor), a factor that possesses growth-regulatory activities principally toward epithelial cells. We found that acrogranin mRNA was expressed in preimplantation mouse embryos with the transcript levels rising to their highest point in blastocysts, coincident with the appearance of the trophectoderm. Indirect immunofluorescence confocal microscopy of preimplantation mouse embryos at different developmental stages revealed that acrogranin immunostaining was most concentrated in the trophectoderm of blastocysts. Immunoblotting and immunoprecipitation experiments demonstrated that the embryos secreted acrogranin into the surrounding medium. To determine how altering the levels of acrogranin in the culture medium surrounding the embryos might affect embryonic growth and development, acrogranin protein levels in the culture medium were decreased with a function-blocking antibody or increased by adding the purified acrogranin to the medium. In both a concentration-dependent and a reversible manner, affinity-purified anti-acrogranin antibody significantly inhibited the development of eight-cell embryos to the blastocyst stage compared to controls (no added immunoglobulin or nonspecific IgG). Furthermore, embryo cell numbers were significantly decreased in the presence of the highest concentrations of acrogranin antibody compared to control embryos. Exogenous acrogranin added to cultures of eight-cell embryos accelerated the time for the onset of cavitation, as well as stimulating the rate of blastocoel expansion and increasing the number of trophectoderm cells compared to controls. These results indicate that acrogranin can regulate the appearance of the epithelium in the developing mouse blastocyst, the growth of the trophectoderm, and/or the function of the embryonic epithelium
A New Look at the Origin, Function, and “Stem-Cell” Status of Muscle Satellite Cells

AbstractMuscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair, and maintenance of skeletal muscle. Recent studies in mice, however, have revealed the potential for highly purified hematopoietic stem cells from bone marrow to participate in muscle regeneration. Perhaps more significantly, a population of putative stem cells isolated directly from skeletal muscle efficiently reconstitutes the hematopoietic compartment and participates in muscle regeneration following intravenous injection in mice. The plasticity of muscle stem cells has raised important questions regarding the relationship between the muscle-derived stem cells and the skeletal muscle satellite cells. Furthermore, the ability of hematopoietic cells to undergo myogenesis has prompted new investigations into the embryonic origin of satellite cells. Recent developmental studies suggest that a population of satellite cells is derived from progenitors in the embryonic vasculature. Taken together, these studies provide the first evidence that pluripotential stem cells are present within adult skeletal muscle. Tissue-specific stem cells, including satellite cells, may share a common embryonic origin and possess the capacity to activate diverse genetic programs in response to environmental stimuli. Manipulation of such tissue-specific stem cells may eventually revolutionize therapies for degenerative diseases, including muscular dystrophy
Hydra Metalloproteinase 1: A Secreted Astacin Metalloproteinase Whose Apical Axis Expression Is Differentially Regulated during Head Regeneration

AbstractThe newly emerging astacin metalloproteinase family comprises multiple members with diverse functions. Most recently, the development-related functions have been attributed to both (1) proteolytic cleavage and subsequent release of active TGF-β-like growth factors from latent inhibitory complexes and (2) modification of extracellular matrix (ECM) assembly and composition. We previously identified and purified hydra metalloproteinase 1 (HMP-1), a developmentally important astacin proteinase that functions in head regeneration and transdifferentiation of tentacle battery cells (L. Yan et al., 1995, Development 121, 1591–1602). In the present study, further cloning revealed that HMP-1 is produced as a secreted zymogen with a conserved hydrophobic signal sequence and a putative propeptide. The processed HMP-1 is composed of a characteristic astacin proteinase domain and a unique Cys-rich C-terminus. With this simple domain structure, HMP-1 represents an ancestral astacin proteinase. Consistent with its role in head regeneration, HMP-1 mRNA is expressed at highest levels by endodermal cells at the apical pole of the body column just inferior to the base of tentacles, the region of active cell differentiation or transdifferentiation. A modified immunocytochemical procedure demonstrated that HMP-1 protein can be localized not only to ECM of tentacles as we previously reported, but also to endodermal cells of the body column in a pattern similar to its mRNA distribution. The localization of HMP-1 protein in tentacles was confirmed using an enzymatic approach. A translocation of HMP-1 protein from cells in the body column to the extracellular milieu in tentacles further suggests that HMP-1 is a secreted protein. HMP-1 expression undergoes extensive regulation at the transcriptional level both temporally and spatially during head regeneration. The involvement of HMP-1 in this morphogenetic process is further supported by the blockage of head regeneration with localized antisense treatment. Taken together, these results suggest that HMP-1 is a secreted astacin metalloproteinase that has an important role in regulating hydra head morphogenesis potentially through its differential expression along the body axis
Plasticity of Retrovirus-Labelled Myotubes in the Newt Limb Regeneration Blastema

AbstractTwo important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question
A Conserved Role for H15-Related T-Box Transcription Factors in Zebrafish and Drosophila Heart Formation

AbstractT-box transcription factors are critical regulators of early embryonic development. We have characterized a novel zebrafish T-box transcription factor, hrT (H15-related T box) that is a close relative of Drosophila H15 and a recently identified human gene. We show that Drosophila H15 and zebrafish hrT are both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrT expression in anterior lateral plate mesoderm suggest a very early role for hrT in the differentiation of the cardiac precursors. hrT is coexpressed with gata4 and nkx2.5 not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, we analyzed hrT expression in pandora mutant embryos, since these have defects in many of the tissues that express hrT, including the heart. hrT expression is much reduced in the early heart fields of pandora mutants, whereas it is ectopically expressed subsequently. Using hrT expression as a marker, we describe a midline patterning defect in pandora affecting the anterior hindbrain and associated midline mesendodermal derivatives. We discuss the possibility that the cardiac ventricular defect previously described in pandora and the midline defects described here are related
Spatiotemporal Analysis of Ca2+ Waves in Relation to the Sperm Entry Site and Animal–Vegetal Axis during Ca2+ Oscillations in Fertilized Mouse Eggs

AbstractFertilized mouse eggs exhibit repetitive rises in intracellular Ca2+ concentration ([Ca2+]i) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca2+]i rise were investigated by high-speed Ca2+ imaging during early development of monospermic eggs. Every [Ca2+]i rise involved a Ca2+ wave. In the first Ca2+ transient, [Ca2+]i increased in two steps separated by a “shoulder” point, suggesting two distinct Ca2+ release mechanisms. The first step was a Ca2+ wave that propagated from the sperm-fusion site to its antipode in 4–5 s (velocity, ∼20 μm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca2+]i rise of 12–15 s. A slight cytoplasmic movement followed the Ca2+ wave in the same direction and recovered in 25–35 s. These characteristics changed as follows, as Ca2+ oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation (∼3 h after fertilization). (1) The duration of Ca2+ transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca2+]i rise became greater and wave speeds increased up to 80–100 μm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca2+ wave, although its displacement became smaller. (5) The Ca2+ wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature
Purification and Analysis of in Vivo-Differentiated Oligodendrocytes Expressing the Green Fluorescent Protein

AbstractA complete understanding of the molecular mechanisms involved in the formation and repair of the central nervous system myelin sheath requires an unambiguous identification and isolation of in vivo-differentiated myelin-forming cells. In order to develop a novel tool for the analysis of in vivo-differentiated oligodendrocytes, we generated transgenic mice expressing a red-shifted variant of the green fluorescent protein under the control of the proteolipid protein promoter. We demonstrate here that green fluorescent protein-derived fluorescence in the central nervous system of 9-day- to 7-week-old mice is restricted to mature oligodendrocytes, as determined by its spatiotemporal appearance and by both immunocytochemical and electrophysiological criteria. Green fluorescent protein-positive oligodendrocytes could easily be visualized in live and fixed tissue. Furthermore, we show that this convenient and reliable identification now allows detailed physiological analyses of differentiated oligodendrocytes in situ. In addition, we developed a novel tissue culture system for in vivo-differentiated oligodendrocytes. Initial data using this system indicate that, for oligodendrocytes isolated after differentiation in vivo, as yet unidentified factors secreted by astrocytes are necessary for survival and/or reappearance of a mature phenotype in culture
Differential Expression and Regulation by 20-Hydroxyecdysone of Mosquito Ultraspiracle Isoforms

AbstractUltraspiracle (USP), the insect homologue of the vertebrate retinoid X receptor, is an obligatory dimerization partner for the ecdysteroid receptor (EcR). Two USP isoforms, USP-A and USP-B, with distinct N-termini, occur in the mosquito Aedes aegypti. In the fat body and ovary, USP-A mRNA is highly expressed during the pre- and late vitellogenic stages, corresponding to a period of low ecdysteroid titer, while USP-B mRNA exhibits its highest levels during the vitellogenic period, correlating with a high ecdysteroid titer. Remarkably, 20-hydroxyecdysone (20E) has opposite effects on USP isoform transcripts in in vitro fat body culture. This steroid hormone upregulates USP-B transcription and its presence is required to sustain a high level of USP-B expression. In contrast, 20E inhibits activation of USP-A transcription. Although EcR·USP-A recognizes the same ecdysteroid-responsive elements, EcR·USP-B binds them with an affinity twofold higher than that of EcR·USP-A. Likewise, EcR·USP-B transactivates a reporter gene in CV-1 cells twofold more strongly than EcR·USP-A. These results suggest that USP-B functions as a major heterodimerization partner for EcR during the vitellogenic response to 20E in the mosquito
Functional Analysis of Repressor Binding Sites in the iab-2 Regulatory Region of the abdominal-A Homeotic Gene

AbstractSpatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. Previously, the Hunchback (HB) protein has been implicated in setting the anterior expression limit of the UBX homeotic protein in parasegment 6. Here we investigate DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that in contrast to Ubx enhancer elements, both HB and Krüppel (KR) are required to set the ABD-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that HB and KR are direct regulators of this iab-2 enhancer. The single KR site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that KR repression occurs through a local quenching mechanism. We also show that the gap repressor Giant (GT) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. Finally, we show that this iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (PHO). These iab-2 PHO sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the PHO sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer
Premeiotic Aster as a Device to Anchor the Germinal Vesicle to the Cell Surface of the Presumptive Animal Pole in Starfish Oocytes

AbstractThe germinal vesicle (GV) of starfish oocytes stays just beneath the oocyte cortex at the presumptive animal pole during the long period of oogenesis. We subjected oocytes to a centrifugal force field to detach the GV from the cortex. The association between the cortex and the GV persisted and withstood a small amount of centrifugal acceleration at 200g. The GV was eventually separated from the cortex at 700g. The amount of acceleration sufficient for the GV separation was lowered when the oocytes were pretreated with Nocodazole and was increased by Taxol pretreatment. Observation of microtubular structures with an anti-α-tubulin antibody revealed the presence of a complex of spots and radiating arrays as was described by J. J. Otto and T. E. Schroeder (1984, Dev. Biol. 101, 274–281) and called the premeiotic aster. Nocodazole shortened the astral arrays, and Taxol enhanced them. These observations indicate that the premeiotic aster works as a device to hold the GV in an eccentric position just beneath the oocyte cortex