pinecone/core-2020-05-10-deduplication huggingface dataset:
Zygotic Expression of the caudal Homolog pal-1 Is Required for Posterior Patterning in Caenorhabditis elegans Embryogenesis

AbstractPrevious work has shown that the Caenorhabditis elegans gene pal-1, a homolog of Drosophila caudal, is required maternally for blastomere specification in the early embryo and postembryonically for tail development in males. We show here that embryonic (zygotic) transcription of pal-1 is also required for posterior patterning during later embryogenesis. Embryos homozygous for strong loss-of-function mutations arrest as nonviable L1 larvae with gross posterior defects. PAL-1 protein produced from zygotic transcripts is expressed dynamically during gastrulation and morphogenesis in specific cells of all major lineages except the germ line. Most expressing cells are undergoing cell movements or forming midline structures or both. Mutant embryos exhibit defects involving most of the expressing cells. Aberrant early cell positions are observed in posterior hypodermis, both in the C-lineage cells that express pal-1 and in the neighboring hypodermal seam cell precursors, which do not, as well as in posterior muscle derived from the C and D lineages. Defects in late gastrulation, ventral hypodermal enclosure, and formation of the rectum result from failures of cell movements of ABp and MS descendants. Limited mosaic analysis supports the view that most of the required pal-1 functions are cell autonomous
Retinoic Acid-Mediated Gene Expression in Transgenic Reporter Zebrafish

AbstractRetinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis
Gdf11 Is a Negative Regulator of Chondrogenesis and Myogenesis in the Developing Chick Limb

AbstractGDF11, a new member of the TGF-β gene superfamily, regulates anterior/posterior patterning in the axial skeleton during mouse embryogenesis. Gdf11 null mice display skeletal abnormalities that appear to represent anterior homeotic transformations of vertebrae consistent with high levels of Gdf11 expression in the primitive streak, presomitic mesoderm, and tail bud. However, despite strong Gdf11 expression in the limb throughout development, this structure does not appear to be affected in the knockout mice. In order to understand this dichotomy of Gdf11 expression versus Gdf11 function, we identified the chicken Gdf11 gene and studied its role during limb formation. In the early limb bud, Gdf11 transcripts are detected in the subectodermal mesoderm at the distal tip, in a region overlapping the progress zone. At these stages, Gdf11 is excluded from the central core mesenchyme where precartilaginous condensations will form. Later in development, Gdf11 continues to be expressed in the distal most mesenchyme and can also be detected more proximally, in between the forming skeletal elements. When beads incubated in GDF11 protein were implanted into the early wing bud, GDF11 caused severe truncations of the limb that affected both the cartilage elements and the muscle. Limb shortening appeared to be the result of an inhibition of chondrogenesis and myogenesis and using an in vitro micromass assay, we confirmed the negative effects of GDF11 on both myogenic and chondrogenic cell differentiation. Analysis of molecular markers of skeletal patterning revealed that GDF11 induced ectopic expression of Hoxd-11 and Hoxd-13, but not of Hoxa-11, Hoxa-13, or the Msx genes. These data suggest that GDF11 may be involved in controlling the late distal expression of the Hoxd genes during limb development and that misregulation of these Hox genes by excess GDF11 may cause some of the observed alterations in skeletal element shape. In addition, GDF11 induced the expression of its own antagonist follistatin, indicating that the activity of GFD11 may be limited by a negative feedback mechanism. The data from our studies in the chick suggest that Gdf11 plays a role in the formation and development of the avian limb skeleton
Double-Stranded RNA Injection Produces Nonspecific Defects in Zebrafish

AbstractWe have investigated the ability of dsRNA to inhibit gene functions in zebrafish using sequences targeted to the maternal gene pouII-1, the transgene GFP, and an intron of the zebrafish gene terra. We found that embryos injected with all of these dsRNAs at approximately 7.5 pg/embryo or higher had general growth arrest during gastrulation and displayed various nonspecific defects at 24 h postfertilization, although embryonic development was unaffected before the midblastula stage. Reducing dsRNA concentration could alleviate the global defects. Injection of GFP dsRNA (7.5–30 pg/embryo) did not inhibit GFP expression in transgenic fish, although abnormal embryos were induced. Co-injection of GFP mRNA with either GFP or non-GFP dsRNA caused reduction of GFP expression. Whole-mount in situ hybridization clearly showed that embryos injected with dsRNA degraded co-injected and endogenous mRNA without sequence specificity, indicating that dsRNA has a nonspecific effect at the posttranscriptional level. It appears that RNAi is not a viable technique for studying gene function in zebrafish embryos
Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses

AbstractThe neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution
Neurogenin2 Expression in Ventral and Dorsal Spinal Neural Tube Progenitor Cells Is Regulated by Distinct Enhancers

AbstractThe basic helix-loop-helix transcription factor Neurogenin2 (NGN2) is expressed in distinct populations of neural progenitor cells within the developing central and peripheral nervous systems. Transgenic mice containing ngn2/lacZ reporter constructs were used to study the regulation of ngn2 in the developing spinal cord. ngn2/lacZ transgenic embryos containing sequence found 5′ or 3′ to the ngn2 coding region express lacZ in domains that reflect the spatial and temporal expression profile of endogenous ngn2. A 4.4-kb fragment 5′ of ngn2 was sufficient to drive lacZ expression in the ventral neural tube, whereas a 1.0-kb fragment located 3′ of ngn2 directed expression to both dorsal and ventral domains. Persistent β-gal activity revealed that the NGN2 progenitor cells in the dorsal domain give rise to a subset of interneurons that send their axons to the floor plate, and the NGN2 progenitors in the ventral domain give rise to a subset of motor neurons. We identified a discrete element that is required for the activity of the ngn2 enhancer specifically in the ventral neural tube. Thus, separable regulatory elements that direct ngn2 expression to distinct neural progenitor populations have been defined
MyoD Cannot Compensate for the Absence of Myogenin during Skeletal Muscle Differentiation in Murine Embryonic Stem Cells

Abstractmyogenin (−/−) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (−/−) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (−/−) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (−/−) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (−/−) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (−/−) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties
Functional Domains of the LIM Homeodomain Protein Xlim-1 Involved in Negative Regulation, Transactivation, and Axis Formation in Xenopus Embryos

AbstractThe Xenopus LIM homeodomain protein Xlim-1 is specifically expressed in the Spemann organizer region and assumed to play a role in the establishment of the body axis as a transcriptional activator. To further elucidate the mechanism underlying the regulation of its transcriptional activity, we focused on the region C-terminal to the homeodomain of Xlim-1 (CT239-403) and divided it into five regions, CCR1–5 (C-terminal conserved regions), based on similarity between Xlim-1 and its paralog, Xlim-5. The role of Xlim-1 CT239-403 in the Spemann organizer was analyzed by assaying the axis-forming ability of a series of CCR-mutated constructs in Xenopus embryos. We show that high doses of Xlim-1 constructs deleted of CCR1 or CCR2 initiate secondary axis formation in the absence of its coactivator Ldb1 (LIM-domain-binding protein 1), suggesting that CCR1 and CCR2 are involved in negative regulation of Xlim-1. In contrast, while Xlim-1 is capable of initiating secondary axis formation at low doses in the presence of Ldb1, deletion of CCR2 (aa 275–295) or substitution of five conserved tyrosines in CCR2 with alanines (CCR2-5YA) abolished the activity. In addition, UAS-GAL4 one-hybrid reporter assays in Xenopus showed that CCR2, but not CCR2-5YA, with its flanking regions (aa 261–315) functions as a transactivation domain when fused to the GAL4 DNA-binding domain. Finally, we show that none of the known transcriptional coactivators tested (CBP, SRC-1, and TIF2) interacts with the Xlim-1 transactivation domain (aa 261–315). Thus, Xlim-1 not only contains a unique tyrosine-rich activation domain but also contains a negative regulatory domain in CT239-403, suggesting a complex regulatory mechanism underlying the transcriptional activity of Xlim-1 in the organizer
Role of Caspases and Mitochondria in the Steroid-Induced Programmed Cell Death of a Motoneuron during Metamorphosis

AbstractAccessory planta retractor (APR) motoneurons of the hawk moth, Manduca sexta, undergo a segment-specific pattern of programmed cell death (PCD) 24 to 48 h after pupal ecdysis (PE). Cell culture experiments show that the PCD of APRs in abdominal segment 6 [APR(6)s] is a cell-autonomous response to the steroid hormone 20-hydroxyecdysone (20E) and involves mitochondrial demise and cell shrinkage. Twenty-four hours before PE, at stage W3-noon, APR(6)s require further 20E exposure and protein synthesis (as tested with cycloheximide) to undergo PCD, and death can be blocked by a broad-spectrum caspase inhibitor. By PE, death is 20E- and protein synthesis-independent and the caspase inhibitor blocks cell shrinkage but not loss of mitochondrial function. Thus, the commitment to mitochondrial demise precedes the commitment to execution events. The phenotype of necrotic cell death induced by a mitochondrial electron transfer inhibitor differs unambiguously from 20E-induced PCD. By inducing PCD pharmacologically, the readiness of APR(6)s to execute PCD was found to increase during the final larval instar. These data suggest that the 20E-induced PCD of APR(6)s includes a premitochondrial phase which includes 20E-induced synthetic events and apical caspase activity, a mitochondrial phase which culminates in loss of mitochondrial function, and a postmitochondrial phase during which effector caspases are activated and APR(6) is destroyed
The LAX1 and FRIZZY PANICLE 2 Genes Determine the Inflorescence Architecture of Rice by Controlling Rachis-Branch and Spikelet Development

AbstractWe have analyzed two mutants that exhibit altered panicle architecture in rice (Oryza sativa L.). In lax1-2, which is a new and stronger allele of the previously reported lax mutant, initiation and/or maintenance of rachis-branches, lateral spikelets, and terminal spikelets was severely prevented. In situ hybridization analysis using OSH1, a rice knotted1 (kn1) ortholog, confirmed the absence of lateral meristems in lax1-2 panicles. These defects indicate that the LAX1 gene is required for the initiation/maintenance of axillary meristems in the rice panicle. In addition to its role in forming lateral meristems, the wild-type LAX1 gene acts as a floral meristem identity gene which specifies the terminal spikelet meristem. A comparison of the defects in lax1-1 and lax1-2 plants suggested that the sensitivities to reduced LAX1 activity were not uniform among different types of meristems. In the fzp2 mutant panicle, the basic branching pattern of the panicle was indistinguishable from that of the wild type; however, specification of both terminal and lateral spikelet meristems was blocked, and sequential rounds of branching occurred at the point where the spikelet meristems are initiated in the wild-type panicle. This resulted in the generation of a panicle composed of excessive ramification of rachis-branches. The lax1-1 fzp2 double mutants exhibited a novel, basically additive, phenotype, which suggests that LAX1 and FZP2 function in genetically independent pathways
Autonomous Transition into Meiosis of Mouse Fetal Germ Cells in Vitro and Its Inhibition by gp130-Mediated Signaling

AbstractMouse primordial germ cells (PGCs) arrive at the urogenital ridge (UGR) at around 10.5 days postcoitum (dpc). They proliferate until around 13.5 dpc, then enter into meiosis in the female or become mitotically arrested in the male gonads. In this study, meiotic transition of mouse PGCs was examined in vitro. Female PGCs obtained from UGRs or genital ridges at 10.5–11.5 dpc began to express meiosis-specific genes, Scp3 and Dmc1, after dissociation and cultivation on feeder cells for several days. Meiotic transition into the leptotene stage was confirmed by the formation of axial cores. Male PGCs at 10.5–11.5 dpc and migratory PGCs obtained from mesenteries at 10.5 dpc also expressed Scp3 and formed axial cores after several days of culture, supporting the hypothesis that PGCs are capable of entering meiosis before arriving at the UGR. gp130-mediated signaling, known to promote survival/growth of PGCs and also to inhibit the differentiation of embryonic stem cells, suppressed the expression of Scp3 in PGCs and inhibited the following formation of axial cores in vitro. This novel activity of gp130-mediated signaling may provide some clues for the understanding of pluripotency of mammalian germ-line cells and/or the sex differentiation of fetal germ cells
Dispersion of Cyclin B mRNA Aggregation Is Coupled with Translational Activation of the mRNA during Zebrafish Oocyte Maturation

AbstractCyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), an event prerequisite for initiating oocyte maturation in this species. We investigated localization of cyclin B mRNA in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes were treated with 17α,20β-DP, a meshwork of microfilaments in the oocyte cortex disappeared and the aggregation of cyclin B mRNA dispersed just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deformed the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17α,20β-DP, cytochalasin B (10 μg/ml) induced both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17α,20β-DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration (1 μg/ml) of cytochalasin B inhibited 17α,20β-DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation
Expression and Function of a Stem Cell Promoter for the Murine CBFα2 Gene: Distinct Roles and Regulation in Natural Killer and T Cell Development

AbstractThe Runt family transcription factor CBFα2 (AML1, PEBP2αB, or Runx1) is required by hematopoietic stem cells and expressed at high levels in T-lineage cells. In human T cells CBFα2 is usually transcribed from a different promoter (distal promoter) than in myeloid cells (proximal promoter), but the developmental and functional significance of this promoter switch has not been known. Here, we report that both coding and noncoding sequences of the distal 5′ end are highly conserved between the human and the murine genes, and the distal promoter is responsible for the overwhelming majority of CBFα2 expression in murine hematopoietic stem cells as well as in T cells. Distal promoter activity is maintained throughout T cell development and at lower levels in B cell development, but downregulated in natural killer cell development. The distal N-terminal isoform binds to functionally important regulatory sites from known target genes with two- to threefold higher affinity than the proximal N-terminal isoform. Neither full-length isoform alters growth of a myeloid cell line under nondifferentiating conditions, but the proximal isoform selectively delays mitotic arrest of the cell line under differentiating conditions, resulting in the generation of greater numbers of neutrophils
Ligand-Induced Signaling in the Absence of Furin Processing of Notch1

AbstractNotch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor which is both present on the cell surface and able to generate an intracellular signal in response to ligand. Consistent with previous reports, we found that disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbed ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL (CBF1, Su(H), and LAG-1). Yet contrary to these reports, we could detect the full-length p300 Notch1 product on the cell surface. Moreover, this uncleaved form of Notch1 could suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support our previous studies characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. Our results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor could mediate two distinct signaling pathways in response to ligand
Regulation of Cell Fate in Caenorhabditis elegans by a Novel Cytoplasmic Polyadenylation Element Binding Protein

AbstractThe fog-1 gene of Caenorhabditis elegans specifies that germ cells differentiate as sperm rather than as oocytes. We cloned fog-1 through a combination of transformation rescue experiments, RNA-mediated inactivation, and mutant analyses. Our results show that fog-1 produces two transcripts, both of which are found in germ cells but not in the soma. Furthermore, two deletion mutants alter these transcripts and are likely to eliminate fog-1 activity. The larger transcript is expressed under the control of sex-determination genes, is necessary for fog-1 activity, and is sufficient to rescue a fog-1 mutant. This transcript encodes a novel member of the CPEB family of RNA-binding proteins. Because CPEB proteins in Xenopus and Drosophila regulate gene expression at the level of translation, we propose that FOG-1 controls germ cell fates by regulating the translation of specific messenger RNAs