pinecone/core-2020-05-10-deduplication huggingface dataset:
The Casein Kinase I Family in Wnt Signaling

AbstractThe canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Recently, sufficiency tests identified casein kinase Iϵ (CKIϵ) as a positive component of the canonical Wnt/β-catenin pathway, and necessity tests showed that CKIϵ is required in vertebrates to transduce Wnt signals. In addition to CKIϵ, the CKI family includes several other isoforms (α, β, γ, and δ) and their role in Wnt sufficiency tests had not yet been clarified. However, in Caenorhabditis elegans studies, loss-of-function of a CKI isoform most similar to α produced the mom phenotype, indicative of loss-of-Wnt signaling. In this report, we examine the ability of the various CKI isoforms to activate Wnt signaling and find that all the wild-type CKI isoforms do so. Dishevelled (Dsh), another positive component of the Wnt pathway, becomes phosphorylated in response to Wnt signals. All the CKI isoforms, with the exception of γ, increase the phosphorylation of Dsh in vivo. In addition, CKI directly phosphorylates Dsh in vitro. Finally, we find that CKI is required in vivo for the Wnt-dependent phosphorylation of Dsh. These studies advance our understanding of the mechanism of Wnt action and suggest that more than one CKI isoform is capable of transducing Wnt signals in vivo
The Spatial and Temporal Expression of Ch-en, the engrailed Gene in the Polychaete Chaetopterus, Does Not Support a Role in Body Axis Segmentation

AbstractWe are interested in understanding whether the annelids and arthropods shared a common segmented ancestor and have approached this question by characterizing the expression pattern of the segment polarity gene engrailed (en) in a basal annelid, the polychaete Chaetopterus. We have isolated an en gene, Ch-en, from a Chaetopterus cDNA library. Genomic Southern blotting suggests that this is the only en class gene in this animal. The predicted protein sequence of the 1.2-kb cDNA clone contains all five domains characteristic of en proteins in other taxa, including the en class homeobox. Whole-mount in situ hybridization reveals that Ch-en is expressed throughout larval life in a complex spatial and temporal pattern. The Ch-en transcript is initially detected in a small number of neurons associated with the apical organ and in the posterior portion of the prototrochophore. At later stages, Ch-en is expressed in distinct patterns in the three segmented body regions (A, B, and C) of Chaetopterus. In all segments, Ch-en is expressed in a small set of segmentally iterated cells in the CNS. In the A region, Ch-en is also expressed in a small group of mesodermal cells at the base of the chaetal sacs. In the B region, Ch-en is initially expressed broadly in the mesoderm that then resolves into one band/segment coincident with morphological segmentation. The mesodermal expression in the B region is located in the anterior region of each segment, as defined by the position of ganglia in the ventral nerve cord, and is involved in the morphogenesis of segment-specific feeding structures late in larval life. We observe banded mesodermal and ectodermal staining in an anterior–posterior sequence in the C region. We do not observe a segment polarity pattern of expression of Ch-en in the ectoderm, as is observed in arthropods
Mesoderm Formation in Eleutherodactylus coqui: Body Patterning in a Frog with a Large Egg

AbstractThe direct developing frog, Eleutherodactylus coqui, develops from a large egg (diameter 3.5 mm). To investigate the effect of egg size on germ-layer formation, we studied mesoderm formation in E. coqui and compared it to that of Xenopus laevis (diameter 1.3 mm). First, we identified the position of prospective mesoderm in the 16-cell E. coqui embryo by cell-lineage tracing. Although the animal blastomeres are small, they form most of the blastocoel roof and make extensive contributions to some mesodermal tissues. Second, we performed recombinant analysis with X. laevis animal caps to define the distribution of mesoderm-inducing activity. Mesoderm-inducing activity in E. coqui was restricted around the marginal zone with strong activity in the superficial cells. Neither the vegetal pole nor the blastocoel floor had activity, although these same regions from X. laevis induced mesoderm. Third, we cloned Ecbra, a homologue of Xbra, an early mesoderm marker in X. laevis. Ecbra was expressed in the marginal ring close to the surface, similar to X. laevis, but E. coqui had weaker expression on the dorsal side. Our results suggest that mesoderm formation is shifted more animally and superficially in E. coqui compared to X. laevis
Evidence That Protein Kinase C (PKC) Participates in the Meiosis I to Meiosis II Transition in Mouse Oocytes

AbstractOocytes from LTXBO mice exhibit a delayed entry into anaphase I and frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity was detected in LTXBO oocytes at prophase I and increased with meiotic maturation, with the highest (P < 0.05) activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduced (P < 0.05) M-phase-promoting factor (MPF) activity and promoted (P < 0.05) progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remained elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-δ associated with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevented untimely entry into interphase, but only when PKC activity was reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, as suppression of PKC activity failed to prevent activation by Mostm1Ev/ Mostm1Ev LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis
In Vivo Imaging Indicates Muscle Fiber Dedifferentiation Is a Major Contributor to the Regenerating Tail Blastema

AbstractDuring tail regeneration in urodele amphibians such as axolotls, all of the tissue types, including muscle, dermis, spinal cord, and cartilage, are regenerated. It is not known how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. Using Nomarski imaging, we followed the process of regeneration in the larval axolotl tail. Combining this with in vivo fluorescent labeling of single muscle fibers, we show that mature muscle dedifferentiates. Muscle dedifferentiation occurs by the synchronous fragmentation of the multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation and the extension of cell processes. We further show that direct clipping of the muscle fiber and severe tissue damage around the fiber are both required to initiate dedifferentiation. Our observations also make it possible to estimate for the first time how many of the blastema cells arise specifically from muscle dedifferentiation. Calculations based on our data suggest that up to 29% of nondermal-derived cells in the blastema come from dedifferentiation of mature muscle fibers. Overall, these results show that endogenous multinucleate muscle fibers can dedifferentiate into mononucleate cells and contribute significantly to the blastema
Novel Cell Lines Promote the Discovery of Genes Involved in Early Heart Development

AbstractClonal cell lines representing early cardiomyocytes would provide valuable reagents for the dissection of the genetic program of early cardiogenesis. Here we describe the establishment and characterization of cell lines from the hearts of transgenic mice and embryos with SV40 large T antigen expressed in the heart-forming region. Ultrastructure analysis by transmission electron microscopy showed the primitive, precontractile nature of the resulting cells, with the absence of myofilaments, Z lines, and intercalated disks. Immunohistochemistry, RT-PCR, Northern blots, and oligonucleotide microarrays were used to determine the expression levels of thousands of genes in the 1H and ECL-2 cell lines. The resulting gene-expression profiles showed the transcription of early cardiomyocyte genes such as Nkx2.5, GATA4, Tbx5, dHAND, cardiac troponin C, and SM22-α. Furthermore, many genes not previously implicated in early cardiac development were expressed. Two of these genes, Hic-5, a possible negative regulator of muscle differentiation, and the transcription enhancing factor TEF-5 were selected and shown by in situ hybridizations to be expressed in the early developing heart. The results show that the 1H and ECL-2 cell lines can be used to discover novel genes expressed in the early cardiomyocyte
The Zinc Finger Protein DIE-1 Is Required for Late Events during Epithelial Cell Rearrangement in C. elegans

AbstractThe mechanism by which epithelial cells undergo directed rearrangement is central to morphogenesis, yet the regulation of these movements remains poorly understood. We have investigated epithelial cell rearrangement (intercalation) in the dorsal hypodermis, or embryonic epidermis, of the C. elegans embryo by analyzing the die-1(w34) mutant, which fails to undergo normal intercalation. Dorsal hypodermal cells of die-1(w34) homozygous embryos initiate but fail to complete the process of intercalation. Multiphoton microscopy reveals that intercalating cells extend monopolar, basolateral protrusions in their direction of migration; posterior dorsal hypodermal cells in die-1(w34) mutants appear to extend protrusions normally, but fail to translocate their cell bodies to complete rearrangement. Despite abnormal intercalation, the subsequent morphogenetic movements that enclose the embryo with epithelial cells and the process of dorsal cell fusion still occur. However, elongation of the embryo into a wormlike shape is disrupted in die-1(w34) embryos, suggesting that intercalation may be necessary for subsequent elongation of the embryo. Actin filaments are not properly organized within the dorsal hypodermis of die-1(w34) embryos, consistent with intercalation's being a necessary prerequisite for elongation. The die-1 gene encodes a C2H2 zinc finger protein containing four fingers, which likely acts as a transcriptional regulator. DIE-1 is present in the nuclei of hypodermal, muscle, gut, and pharyngeal cells; its distribution suggests that DIE-1 acts in each of these tissues to regulate morphogenetic movements. die-1(w34) mutants display morphogenetic defects in the pharynx, gut, and muscle quadrants, in addition to the defects in the dorsal hypodermis, consistent with the DIE-1 expression pattern. Mosaic analysis indicates that DIE-1 is autonomously required in the posterior dorsal hypodermis for intercalation. Our analysis documents for the first time the dynamics of protrusive activity during epithelial cell rearrangement. Moreover, our analysis of die-1 shows that the events of epithelial cell rearrangement are under transcriptional control, and that early and later phases of epithelial cell rearrangement are genetically distinguishable
Sox3 Expression Defines a Common Primordium for the Epibranchial Placodes in Chick

AbstractThe epibranchial placodes are ectodermal thickenings that generate sensory neurons of the distal ganglia of the branchial nerves. Although significant advances in our understanding of neurogenesis from the placodes have recently been made, the events prior to the onset of neurogenesis remain unclear. We found that chick Sox3 (cSox3) shows a highly dynamic pattern of expression before becoming confined to the final placodes: one pre-otic (geniculate) and three post-otic (one petrosal and two nodose) placodes. A fate-mapping study using lipophilic dyes revealed that all post-otic placodes arise within a single broad cSox3-positive domain, where cSox3 expression and epithelial thickness will be retained only in much smaller final neurogenic placodes. The data presented here suggest that post-otic placodes are remnants of a common primordium defined as a discrete domain of cSox3 expression
Wingless Capture by Frizzled and Frizzled2 in Drosophila Embryos

AbstractA variety of factors could influence how far developmental signals spread. For example, the Patched receptor limits the range of its ligand Hedgehog. Somehow, the Frizzled2 receptor has the opposite effect on its ligand. Increasing the level of Frizzled2 stabilizes Wingless and thus extends the Wingless gradient in Drosophila wing imaginal disks. Here we ask whether Frizzled or Frizzled2 affects the spread of Wingless in Drosophila embryos. We show that in the embryonic epidermis, the combined expression of both receptors is lowest in the engrailed domain. This is because expression of Frizzled is repressed by the Engrailed transcription factor, whereas that of Frizzled2 is repressed by Wingless signaling. Receptor downregulation correlates with an early asymmetry in Wingless distribution, characterized by the loss of Wingless staining in the engrailed domain. Raising the expression of either Frizzled or Frizzled2 in this domain prevents the early disappearance of Wingless-containing vesicles. Apparently, Wingless is captured, stabilized, and quickly internalized by either receptor. As far as we can tell, captured Wingless is not passed on to further cells and does not contribute to the spread of Wingless. Receptor downregulation in the posterior compartment may contribute to dampening the signal at the time when cuticular fates are specified
Defects in Pulmonary Vasculature and Perinatal Lung Hemorrhage in Mice Heterozygous Null for the Forkhead Box f1 Transcription Factor

AbstractDecreased pulmonary expression of Forkhead Box f1 (Foxf1) transcription factor was associated with lethal alveolar hemorrhage in 55% of the Foxf1 +/− newborn mice. The severity of the pulmonary abnormalities correlates with the levels of Foxf1 mRNA. Defects in alveolarization and vasculogenesis were observed in subsets of the Foxf1 +/− mice with relatively low levels of expression from the normal Foxf1 allele. Lung hemorrhage was coincident with disruption of the mesenchymal–epithelial cell interfaces in the alveolar and bronchiolar regions of the lung parenchyma and was associated with increased apoptosis and reduced surfactant protein B (SP-B) expression. Finally, the lung defect associated with the Foxf1 +/− mutation was accompanied by reduced expression of vascular endothelial growth factor (VEGF), the VEGF receptor 2 (Flk-1), bone morphogenetic protein 4 (Bmp-4), and the transcription factors of the Brachyury T-Box family (Tbx2–Tbx5) and Lung Kruppel-like Factor. Reduction in the level of Foxf1 caused neonatal pulmonary hemorrhage and abnormalities in alveologenesis, implicating this transcription factor in the regulation of mesenchyme–epithelial interaction critical for lung morphogenesis
A Sustained Increase in Intracellular Ca2+ Is Required for the Acrosome Reaction in Sea Urchin Sperm

AbstractThe acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca2+ ([Ca2+]i). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca2+]i elevation lasts minutes and involves two Ca2+ permeable channels. Both the opening of the second channel and the onset of the AR occur ∼5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca2+ from sea water or addition of Ca2+ channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a “rapid” (t1/2 = 3–15 s) decrease in [Ca2+]i and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH4Cl and either EGTA, Co2+, or Ni2+ 5 s after egg factor prevents the partial inhibition of the evoked pHi change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca2+]i caused by the second Ca2+ channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca2+ uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca2+ channel
Regulation of the mRNAs Encoding Proteins of the BMP Signaling Pathway during the Maternal Stages of Xenopus Development

AbstractActivation of the Xenopus bone morphogenetic protein (BMP) pathway is coincident with the onset of zygotic transcription but requires maternal signaling proteins. The mechanisms controlling the translation of mRNAs that encode proteins of the BMP pathway were investigated by using polysome association as an assay for translational activity. Our results indicate that five different mRNAs encoding proteins of the BMP pathway were translationally regulated during Xenopus development. These mRNAs were either not associated or inefficiently associated with polysomes in oocytes, and each was recruited to polysomes at a different developmental stage. The Smad1 and ALK-2 mRNAs were recruited to polysomes during oocyte maturation, whereas the BMP-7 and XSTK9 mRNAs were recruited during the early stages of embryogenesis. The ALK-3 mRNA was not efficiently associated with polysomes during any maternal stage of development and was efficiently recruited to polysomes only after the onset of zygotic transcription. In general, for all stages except oocytes, polysome recruitment was associated with the presence of a 3′ poly(A) tail. However, there was not an obvious correlation between the absolute length of poly(A) and the efficiency of polysome recruitment, indicating that the relationship between poly(A) tail length and translation during early frog embryogenesis is complex. We further focused on the BMP-7 mRNA and demonstrated that sequence elements within the 3′UTR were necessary for recruitment of the BMP-7 mRNA to polysomes and sufficient to direct the addition of poly(A) and activate translation of a reporter during embryogenesis. Interestingly, the BMP-7 mRNA lacks the previously defined eCPE sequences proposed to direct poly(A) addition and translational activation during embryogenesis. The implications of our findings for translational regulation of maternal mRNAs during embryogenesis and for the activation of the BMP pathway are discussed
The Caenorhabditis elegans Six/sine oculis Class Homeobox Gene ceh-32 Is Required for Head Morphogenesis

AbstractCaenorhabditis elegans has four members of the Six/sine oculis class of homeobox genes, ceh-32, ceh-33, ceh-34, and ceh-35. Proteins encoded by this gene family are transcription factors sharing two conserved domains, the homeodomain and the Six/sine oculis domain, both involved in DNA binding. ceh-32 expression was detected during embryogenesis in hypodermal and neuronal precursor cells and later in descendants of these cells as well as in gonadal sheath cells. RNAi inactivation studies suggest that ceh-32 plays a role in head morphogenesis, like vab-3, the C. elegans Pax-6 orthologue. ceh-32 and vab-3 are coexpressed in head hypodermal cells and ceh-32 mRNA levels are reduced in vab-3 mutants. Moreover, ectopic expression of VAB-3 in transgenic worms is able to induce ceh-32 ectopically. In addition, we demonstrate that VAB-3 is able to bind directly to the ceh-32 upstream regulatory region in vitro and to activate reporter gene transcription in a yeast one-hybrid system. Our results suggest that VAB-3 acts upstream of ceh-32 during head morphogenesis and directly induces ceh-32. Thus, ceh-32 appears to be the first target gene of VAB-3 identified so far
Developmental Clustering of Ion Channels at and near the Node of Ranvier

AbstractVoltage-gated Na+ and K+ channels are localized to distinct subcellular domains in mammalian myelinated nerve fibers. Specifically, Na+ channels are clustered in high densities at nodes of Ranvier, while K+ channels are found in juxtaparanodal zones just beyond regions of axoglial contact where sequential layers of the myelin sheath terminate. Specific targeting, clustering, and maintenance of these channels in their respective domains are essential to achieve high conduction velocities of action potential propagation. The cellular, molecular, and developmental mechanisms that exist to achieve this neuronal specialization are discussed and reviewed. Current evidence points to a prominent role in channel clustering played by myelinating glial cells, and sites of axoglial contact in particular
Imprinted Expression of Neuronatin from Modified BAC Transgenes Reveals Regulation by Distinct and Distant Enhancers

AbstractNeuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression
Calcium-Mediated Inactivation of the MAP Kinase Pathway in Sea Urchin Eggs at Fertilization

AbstractWe have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 μM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca2+ ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca2+ was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca2+