pinecone/core-2020-05-10-deduplication huggingface dataset:
Spermiogenesis Is Impaired in Mice Bearing a Targeted Mutation in the Protein Phosphatase 1cγ Gene

AbstractType 1 protein phosphatases (PP1) are involved in diverse cellular activities, ranging from glycogen metabolism to chromatin structure modification, mitosis, and meiosis. The holoenzymes are composed of two or more subunits, including a catalytic subunit (PP1c) and one or more regulatory subunits. Many eukaryotes possess several catalytic subunit genes which encode highly conserved isoforms. In rodents, one of these isoforms, PP1cγ2, appears to be expressed predominantly in testes. Whether PP1cγ2 performs a testis-specific function is unclear. To address this and other questions, the PP1cγ gene was disrupted by targeted insertion in murine embryonic stem cells. Mice derived from these cells were viable, and homozygous females were fertile. However, males homozygous for the targeted insertion were infertile. Histological examination revealed severe impairment of spermiogenesis beginning at the round spermatid stage. In addition, defects in meiosis were inferred from the presence of polyploid spermatids. Immunohistochemistry revealed the presence of PP1cα protein on condensing spermatids in both wild-type and mutant testes, suggesting that this closely related isoform is unable to compensate for the loss of PP1cγ. These defects are discussed in the light of known functions of protein phosphatase 1
Acquisition of Competence to Condense Metaphase I Chromosomes during Spermatogenesis

AbstractLittle is known about the timing of meiotic prophase events during spermatogenesis in the mouse or how these events are related to cell-cycle progression. This work was designed to test hypotheses about the timing and biochemical correlates of developmental acquisition of competence to condense bivalent pairs of homologous chromosomes held together by chiasmata. The experimental approach takes advantage of the fact that okadaic acid (OA) treatment of pachytene spermatocytes causes precocious entry into metaphase I (MI) of meiosis. Leptotene and zygotene (L/Z) spermatocytes are not competent to respond to OA with condensation of chiasmate bivalent chromosomes. Competence for MI condensation of chiasmate bivalents is acquired by the middle of the pachytene stage of meiotic prophase, several days after homologous chromosomes become fully synapsed. The acquisition of MI competence is paralleled by the accumulation of histone H1t in the nuclei of mid-pachytene spermatocytes. Biochemical differences also exist between the incompetent L/Z spermatocytes and the competent pachytene spermatocytes. Both have the molecular components of metaphase promoting factor, CDC2 and CYCLIN B1; however, the histone H1 kinase activity of metaphase promoting factor of incompetent L/Z spermatocytes is not activated by OA, as it is in competent pachytene spermatocytes. Additionally, the CDC25C protein phosphatase is present in competent pachytene spermatocytes, but not in incompetent L/Z or early pachytene spermatocytes. Both incompetent and competent spermatocytes accumulate MPM-2 phosphoepitopes and phosphorylated histone H3 in response to OA treatment, indicating that presence of these antigens is not sufficient to promote condensation of meiotic chromosomes. These data demonstrate that meiotic competence of spermatocytes is acquired after homologous chromosome pairing is established and is coincident with first appearance of histone H1t and CDC25C protein phosphatase in spermatocytes
Quantitative Variations in the Level of MAPK Activity Control Patterning of the Embryonic Termini inDrosophila

AbstractWe have examined the role in patterning of quantitative variations of MAPK activity in signaling from theDrosophilaTorso (Tor) receptor tyrosine kinase (RTK). Activation of Tor at the embryonic termini leads to differential expression of the genestaillessandhuckebein.We demonstrate, using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. We also demonstrate that two chimeric receptors, Torextracellular-Egfrcytoplasmicand Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. Altogether, our results illustrate how a gradient of MAPK activity controls differential gene expression and, thus, the establishment of various cell fates. We discuss the roles of quantitative mechanisms in defining RTK specificity
Essential Role forSonic hedgehogduring Hair Follicle Morphogenesis

AbstractThe hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control andShh−/− embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression ofGli1andPtc1was reduced inShh−/− dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected inShh−/− skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development
Overexpression of Agrin Isoforms inXenopusEmbryos Alters the Distribution of Synaptic Acetylcholine Receptors during Development of the Neuromuscular Junction

AbstractSynapse formation involves a large number of macromolecules found in both presynaptic nerve terminals and postsynaptic cells. Many of the molecules involved in synaptogenesis of the neuromuscular junction have been discovered through morphological localization to the synapse and functional cell culture assays, but their role in embryonic development has been more difficult to study. One of the best understood of these molecules is agrin, a synaptic extracellular matrix protein secreted by both motor neurons and muscle cells, that organizes the postsynaptic apparatus, including high-density aggregates of acetylcholine receptors (AChRs), at the neuromuscular junction. We tested the specific hypothesis that different agrin isoforms made by neurons and muscle cells contribute to agrin's synapse organizing activity in the embryo. Agrin isoforms were overexpressed by injecting synthetic RNA intoXenopus laevisembryos at the one- or two-cell stage. To mark cells containing agrin RNA, green fluorescent protein (GFP) RNA was coinjected. The relative area of muscle AChR aggregates was measured by confocal microscopy and image analysis in GFP-positive segments of injected embryos. Innervated regions of myotomal muscles were compared in animals injected with a mixture of agrin and GFP RNAs or with GFP RNA alone. Overexpression of COOH-terminal 95-kDa fragments of a rat agrin isoform made only by neurons (4,8) and the major isoform (0,0) made by muscle cells both increased AChR cluster area by 100–200%. Rat agrin protein was colocalized with AChR aggregates in innervated regions of muscles in injected embryos. These results show that agrin derived from both the nerve terminal and the muscle cell could contribute to synaptic differentiation at the embryonic neuromuscular junction. They further demonstrate the usefulness of overexpression by RNA injection as an assay for molecular function in embryonic synapse formation
A Role for DNA Methylation in Gastrulation and Somite Patterning

AbstractDNA methylation constitutes an important epigenetic factor in the control of genetic information. In this study, we analyzed expression of the DNA methyltransferase gene and examined DNA methylation patterns during early development of the zebrafish. Maternal transcripts of the zebrafish DNA methyltransferase gene (MTase) are ubiquitously present at high levels in early embryos with overall levels decreasing after the blastula stage. At 24 h, methyltransferase mRNA is predominantly found in the brain, neural tube, eyes, and differentiating somites. Expression of MTase in the somites is highest in the anterior cells of the somites. Despite the high levels of MTase mRNA in blastula-stage embryos, we observe DNA hypomethylation at the blastula and gastrula stages compared to sperm or older embryos. Zebrafish embryos treated with 5-azacytidine (5-azaC) and 5-aza-2-deoxycytidine (5-azadC), nucleotide analogs known to induce cellular differentiation and DNA hypomethylation in mammalian cells, exhibit DNA hypomethylation and developmental perturbations. These defects are specifically observed in embryos treated at the beginning of the blastula period, just prior to midblastula transition. The most common phenotype is the loss of tail and abnormal patterning of somites. Head development is also affected in some embryos. Histological andin situhybridization analyses reveal whole or partial loss of a differentiated notochord and midline muscle in treated embryos. When examined during gastrulation, 5-azaC-treated embryos have a shortened and thickened axial mesoderm. We propose that DNA methylation is required for normal gastrulation and subsequent patterning of the dorsal mesoderm
Expression of Matrix Metalloproteinases 2 and 9 in Regenerating Skeletal Muscle: A Study in Experimentally Injured andmdxMuscles

AbstractMatrix metalloproteinases (MMPs) cooperatively degrade all components of the extracellular matrix (ECM). Remodeling of ECM during skeletal muscle degeneration and regeneration suggests a tight regulation of matrix-degrading activity during muscle regeneration. In this study, we investigated the expression of MMP-2 and MMP-9, in normal muscles and their regulation during regeneration process. We further investigated their secretion by C2C12 myogenic cell line. Two models of muscle degeneration–regeneration were used: (1) normal muscles in which necrosis was experimentally induced by cardiotoxin injection; (2)mdxmuscles which exhibit recurrent signs of focal myofiber necrosis followed by successful regeneration. MMPs were studied by zymography; their free activity was quantified using3H-labeled gelatin substrate and mRNA expression was followed by Northern hybridization. Muscle degeneration–regeneration was analyzed by conventional morphological methods andin situhybridization was performed on muscle sections to identify the cells expressing these MMPs. Results show that MMP-2, but not MMP-9 expression, is constitutive in normal muscles. Upon injury, the active form of MMP-2 is transiently increased, whereas MMP-9 is induced within 24 h and remains present for several days. Quantitative assays of free gelatinolytic activity show a progressive and steady increase that culminates at 7 days postinjury and slowly returns to normal levels. In adultmdxmice, both pro and active forms of MMP-2 and MMP-9 are expressed. Northern blot results support these findings. Zymography of C2C12-conditioned medium shows that myogenic cells produce MMP-2. Byin situhybridization we localized MMP-9 mRNA in inflammatory cells and putative activated satellite cells in injured muscles. Our data allow the correlation of the differential expression of pro and/or active forms of MMP-2 and MMP-9 with different stages of the degeneration–regeneration process: MMP-9 expression is related to the inflammatory response and probably to the activation of satellite cells, whereas MMP-2 activation is concomitant with the regeneration of new myofibers
FGF Is Required for Posterior Neural Patterning but Not for Neural Induction

AbstractFibroblast growth factor (FGF) has been implicated in a variety of developmental processes including posterior mesoderm and neural patterning. Previous work has led to contradictory roles for FGF in neural induction and anteroposterior neural patterning. Launayet al.(Development122, 869–880, 1996) suggested a requirement for FGF in anterior neural induction. In contrast, Kroll and Amaya (Development122, 3173–3183, 1996) and Banget al.(Development124, 2075–2085, 1997) proposed that FGF is not required for early neural patterning. Here we use a loss-of-function assay to examine whether FGF is required for neural patterning in three experimental situations: (i) inXenopusearly embryos, (ii) in embryonic explants consisting of presumptive dorsal mesoderm and neurectoderm (Keller explants), and (iii) in explants of dorsal ectoderm and posterior mesoderm in which FGF signaling is specifically blocked in the ectoderm. When cultured until tailbud stages, Keller explants develop neural tissue with normal anteroposterior pattern. Overexpression of the dominant-negative FGF receptor (XFD) in Keller explants inhibited the posterior neural markers En-2, Krox-20, and HoxB9, but not the panneural marker nrp-1 and the anterior neurectodermal markers XAG-1 and Xotx-2. Similar results were seen in whole embryos, but only when XFD RNA was targeted to both the dorsal and lateral regions. In contrast, addition of FGF to Keller explants resulted in a shift of the midbrain–hindbrain boundary marker En-2 to a more anterior position normally fated to become cement gland. We also determined whether FGF is required specifically by the neurectoderm for anteroposterior neural patterning. Recombinants of dorsal ectoderm and posterior mesoderm were made in which FGF was specifically blocked in the ectoderm. Spinal cord and hindbrain markers were inhibited in these recombinants, whereas anterior markers and cement gland development were enhanced. Our results demonstrate that FGF is important for posterior development in both mesoderm and neurectoderm and that neural induction and posteriorization represent separable developmental events
On the Control of Oocyte Meiotic Maturation and Ovulation inCaenorhabditis elegans

AbstractPrior to fertilization, oocytes undergo meiotic maturation (cell cycle progression) and ovulation (expulsion from the ovary). To begin the study of these processes inCaenorhabditis elegans,we have defined a time line of germline and somatic events by video microscopy. As the oocyte matures, its nuclear envelope breaks down and its cell cortex rearranges. Immediately thereafter, the oocyte is ovulated by increasing contraction of the myoepithelial gonadal sheath and relaxation of the distal spermatheca. By systematically altering the germ cell contents of the hermaphrodite using mutant strains, we have uncovered evidence of four cell–cell interactions that regulate maturation and ovulation. (1) Both spermatids and spermatozoa induce oocyte maturation. In animals with a feminized germline, maturation is inhibited and oocytes arrest in diakinesis. The introduction of sperm by mating restores maturation. (2) Sperm also directly promote sheath contraction. In animals with a feminized or tumorous germline, contractions are infrequent, whereas in animals with a masculinized germline or with sperm introduced by mating, contractions are frequent. (3 and 4) The maturing oocyte both induces spermathecal dilation and modulates sheath contractions at ovulation; dilation of the distal spermatheca and sharp increases in sheath contraction rates are only observed in the presence of a maturing oocyte
Venation Pattern Formation inArabidopsis thalianaVegetative Leaves

AbstractBranching net-like structures are a trait common to most multicellular organisms. However, our knowledge is still poor when it comes to the genetic operations at work in pattern formation of complex network structures such as the vasculature of plants and animals. In order to initiate a causal analysis of venation pattern formation in dicotyledonous plant leaves, we have first studied its developmental profile in vegetative leaves of a wild-type strain of the model organismArabidopsis thaliana.As landmarks of the complexity of the venation pattern, we have defined three main developmental parameters, which have been quantitatively followed in time: the ratios of (a) the length and (b) the number of branchpoints of the vein network with the surface of the lamina, which decrease in parallel as the leaf grows, only small differences existing between successive leaves, and (c) the number of hydathodes per leaf, which increases both during leaf expansion and from juvenile to adult rosette leaves. We next searched for natural variations in the first vegetative leaves of 266 ecotypes, finding only 2 which showed a venation pattern unequivocally different from that of the rest, Ba-1 and Ei-5, the latter displaying an extremely simple pattern that we have called Hemivenata. This phenotype, which is inherited as a monogenic recessive trait, is visible both in leaves and in cotyledons and seems to arise from a perturbation in an early acting patterning mechanism. Finally, we have screened for mutants with abnormal venation pattern but normally shaped leaves, concluding that such a phenotype is rare, since only one recessive mutation was obtained,extrahydathodes,characterized by the presence of an increased number of hydathodes per leaf
Synapse Formation by Hippocampal Neurons from Agrin-Deficient Mice

AbstractAgrin, a proteoglycan secreted by motoneurons, is a critical organizer of synaptic differentiation at skeletal neuromuscular junctions. Agrin is widely expressed in the nervous system so other functions seem likely, but none have been demonstrated. To test roles for agrin in interneuronal synapse formation, we studied hippocampi from mutant mice that completely lack the z+splice form of agrin essential for neuromuscular differentiation and also exhibit severely (∼90%) reduced levels of all agrin isoforms (M. Gautamet al.,1996,Cell85, 525–535). The brains of neonatal homozygous agrin mutants were often smaller than those of heterozygous and wild-type littermates, but were morphologically and histologically indistinguishable. In particular, antibodies to pre- and postsynaptic components of glutamatergic synapses were similarly coaggregated at synaptic sites in both mutants and controls. Because mutants die at birth due to neuromuscular defects, we cultured neurons to assess later stages of synaptic maturation. In primary cultures, the agrin-deficient neurons formed MAP2-positive dendrites and tau-1-positive axons. Synaptic vesicle proteins, AMPA- and NMDA-type glutamate receptors, GABAAreceptors, and the putative synapse-organizing proteins PSD-95, GKAP, and gephyrin formed numerous clusters at synaptic sites. Quantitatively, the number of SV2-labeled contacts per neuron at day 5 and the number of PSD-95 clusters per dendrite length at day 18 in culture showed no significant differences between genotypes. Furthermore, exogenous z+agrin was unable to induce ectopic accumulation of components of central glutamatergic or GABAergic synapses as it does for neuromuscular cholinergic synapses. These results indicate that the z+forms of agrin are dispensable for glutamatergic and GABAergic synaptic differentiation in the central nervous system
Msx2Gene Dosage Influences the Number of Proliferative Osteogenic Cells in Growth Centers of the Developing Murine Skull: A Possible Mechanism forMSX2-Mediated Craniosynostosis in Humans

AbstractThroughout its complex morphogenesis, the vertebrate skull must at once protect the brain and expand to accommodate its growth. A key structural adaptation that allows this dual role is the separation of the bony plates of the skull with sutures, fibrous joints that serve as growth centers and allow the calvarial bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of one or more calvarial bones with consequent abnormalities in skull shape, is a common developmental anomaly that disrupts this process. We found previously that a single amino acid substitution in the homeodomain of the humanMSX2gene is associated with the autosomal dominant disorder craniosynostosis, Boston type. This mutation enhances the affinity ofMsx2for its target sequence, suggesting that the mutation acts by a dominant positive mechanism. Consistent with this prediction, we showed that general overexpression ofMsx2under the control of the broadly expressed CMV promoter causes the calvarial bones to invade the sagittal suture. Here we use tissue-specific overexpression ofMsx2within the calvarial sutures to address the developmental mechanisms of craniosynostosis and skull morphogenesis. We demonstrate that a segment of theMsx2promoter directs reporter gene expression to subsets of cells within the sutures. In late embryonic and neonatal stages, this promoter is expressed in undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter activity is reduced in the suture, exhibiting a punctate pattern in undifferentiated osteoblastic cells in the outer margin of the osteogenic front. Overexpression ofMsx2under the control of this promoter is sufficient to enhance parietal bone growth into the sagittal suture by P6. This phenotype is preceded by an increase in both the number and the BrdU labeling of osteoblastic cells in the osteogenic fronts of the calvarial bones. These findings suggest that an important early event inMSX2-mediated craniosynostosis in humans is a transient retardation of osteogenic cell differentiation in the suture and a consequent increase in the pool of osteogenic cells
Paired-Related Homeobox Genes Cooperate in Handplate and Hindlimb Zeugopod Morphogenesis

AbstractThe closely related homeobox genesprx-1andprx-2are expressed in lateral plate and limb bud mesoderm, but targeted inactivation of these genes failed to demonstrate a limb phenotype. Here we report that mice carrying compound mutations inprx-1andprx-2have severe limb deformities. In the forelimb autopod, pre- and postaxial polydactyly were found most commonly, but also syndactyly, oligodactyly, and abnormal digit placement affecting posterior elements were observed. In the hindlimb, preaxial polydactyly with variable expressivity was seen in all cases. Extreme distal digit duplications were seen in both the fore- and hindlimbs.prx-1; prx-2double-mutant mice also displayed extreme shortening and impaired ossification of the hindlimb zeugopods. Integrity of the forelimb apical ectodermal ridge was abnormal as determined by expression ofFGF8andBMP4.Expression ofmsx-1andmsx-2,markers forBMPsignaling pathways, was absent in regions of the posterior handplates, while expression ofShhandpatchedwas unaffected. The mutant phenotypes were dosage dependent, sinceprx-1 −/−; prx-2 +/−mice also displayed severe limb abnormalities. These data suggest thatprx-1andprx-2cooperatively regulate handplate and hindlimb zeugopod morphogenesis throughBMP-mediated signaling pathways