pinecone/core-2020-05-10-deduplication huggingface dataset:
Twist Is Required for Muscle Template Splitting during AdultDrosophilaMyogenesis

AbstractThe basic helix–loop–helix transcription factor Twist is required for normal development of larval and adult somatic muscles inDrosophila.Adult flies normally have six pairs of dorsal longitudinal indirect flight muscles (DLMs), whereas when Twist function is reduced, only three pairs of DLMs are formed. Althoughtwistis expressed in precursors of adult muscles throughout the larval and early pupal stages, we demonstrate that Twist function is required only during the late larval stage for DLM patterning. In wild-type flies, this is just prior to the time when three pairs of persistent larval muscle fibers split longitudinally to form templates for the six pairs of DLMs. By examining sections at various times during pupal development, we found that splitting of the larval muscles does not occur intwistmutants, indicating that Twist function is required to induce major changes in the larval templates prior to differentiation. The function of Twist in larval muscle splitting is likely mediated by myocyte enhancer factor-2 (MEF2) since inMef2hypomorphic mutants splitting is also reduced andMef2expression is dependent upon Twist. Our findings define specific roles for Twist and MEF2 during pupal myogenesis and demonstrate that these transcription factors function in adult muscle precursor cells to regulate downstream factors controlling muscle cell splitting and morphogenesis
A Crucial Role forPax3in the Development of the Hypaxial Musculature and the Long-Range Migration of Muscle Precursors

AbstractActivated by dorsalizing and lateralizing signals, thePax3gene is an early marker for the entire paraxial mesoderm and its dorsal derivative, the dermomyotome. Later, its expression becomes restricted to the lateral dermomyotome and to the migratory muscle precursors giving rise to the hypaxial musculature. To understand better the role thatPax3plays during development of paraxial mesoderm-derived structures, we followed the development of the musculature and skeleton in the murinePax3mutantSplotch.We found that the mutant dermomyotomes and myotomes failed to organize and to elongate medially and laterally, leading to the reduction and malformation of the entire trunk musculature. Mutants lacked ventral aspects of the body wall musculature and muscles derived from migratory myoblasts, suggesting a crucial function forPax3in the long-range migration of muscle precursors giving rise to the ventral hypaxial musculature. In addition, severe malformations were detected in the skeleton. The axial and appendicular skeleton displayed malformations and in particular multiple bone fusions
Tyrosine Kinase Inhibitors Block Sperm-Induced Egg Activation inXenopus laevis

AbstractFertilization ofXenopus laeviseggs triggers a wave of increased [Ca2+]i. The exact signal transduction pathway culminating in this Ca2+wave remains unknown. To determine whether increases in tyrosine kinase activity are part of this pathway, we microinjected tyrosine kinase inhibitors into unfertilized eggs. Upon fertilization, signs of activation were monitored, such as fertilization envelope liftoff and the Ca2+wave (for eggs microinjected with lavendustin A). Various concentrations of lavendustin A and tyrphostin B46 were microinjected, as well as inactive forms of these compounds (lavendustin B and tyrphostin A1) to provide negative controls. Peptide A, a 20-amino-acid peptide derived from the SH2 region of pp60v-srctyrosine kinase, was also microinjected. Peptide A inhibits tyrosine kinase activity but not PKA or PKG activity. Dose–response curves for lavendustin A, tyrphostin B46, and peptide A show clear inhibition of vitelline envelope liftoff by these three compounds. Confocal imaging of eggs coinjected with lavendustin A and Oregon Green–dextran showed that the Ca2+wave was inhibited under normal insemination conditions but that the block of the Ca2+wave could be overcome with very high sperm densities. A phenomenon of small local Ca2+increases termed “hot spots” seen in lavendustin A containing eggs is also described. Since this inhibition of egg activation by tyrosine kinase inhibitors can be overcome by Ca2+microinjection, the inhibitors must act on a step in the signal transduction cascade that is upstream of the Ca2+wave
Characterization of Protein Phosphatases in Mouse Oocytes

AbstractOkadaic acid (OA) enhances the resumption of meiosis in mouse oocytes, indicating that serine/threonine protein phosphatase-1 (PP1) and/or PP2A is involved. However, specific identification of PP1 and/or PP2A in mouse oocytes has not been reported. Here we demonstrate that fully grown germinal vesicle-intact (GVI) mouse oocytes contain mRNA corresponding to two isotypes of PP1, PP1αand PP1γ. In addition, the transcript for PP2A was also present. At the protein level only PP1αand PP2A were recognized in fully grown GVI oocytes by Western blot analysis. Neither of the PP1γspliced variant proteins, PP1γ1and PP1γ2, was detectable. Immunohistochemical analysis of ovarian tissue from gonadotropin-stimulated adult mice resulted in subcellular localization of both PP1αand PP2A, but not PP1γ, in oocytes from all stages of folliculogenesis. In primordial oocytes, PP1αand PP2A were present in the cytoplasm. In more advanced stages of oogenesis, PP1α, although still present in the cytoplasm, was highly concentrated in the nucleus, whereas PP2A was predominantly cytoplasmic with a distinct reduction in the nuclear area. Both PP1αand PP2A were immunodetectable in oocytes during the prepubertal period. Eleven-day-old mouse oocytes, considered OA-insensitive and germinal vesicle breakdown (GVB)-incompetent, displayed both PP1αand PP2A predominantly in the cytoplasm. By 15 days of age mouse oocytes, which are beginning to acquire OA sensitivity and GVB competence, showed a relocation of PP1αinto the nucleoplasm while PP2A remained predominantly cytoplasmic. This is the first specific identification of PP1αand PP2A in mouse oocytes. The differential localization of PP1αand PP2A, in addition to the relocation of PP1αduring the acquisition of meiotic competence, suggests that these PPs have distinct regulatory roles during the resumption of meiosis
Dynamic Behavior of the Cadherin-Based Cell–Cell Adhesion System duringDrosophilaGastrulation

AbstractDuringDrosophilagastrulation, morphogenesis occurs as a series of cell shape changes and cell movements which probably involve adhesive interactions between cells. In the present study, we examined the dynamic aspects of cadherin-based cell–cell adhesion in the morphogenetic events to assess its contribution to morphogenesis.DE- andDN-cadherin show complementary expression patterns in the presumptive ectoderm and mesoderm at the mRNA level. We found that switching of cadherin expression from theDE- to theDN-type in the mesodermal germ layer occurred downstream of the mesoderm-determination genestwistandsnail.However, examination of their protein expression patterns showed that considerable amounts ofDE-cadherin remained on the surfaces of mesodermal cells during invagination, whileDN-cadherin did not appear on the cell surfaces at this stage. Further immunocytochemical analysis of the localizations ofDE-cadherin and its associated proteins Armadillo (β-catenin) andDα-catenin revealed dynamic changes in their distributions which were accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Simultaneously, adherens junctions (AJs), based on the cadherin–catenin system, were shown to change their location, size, and morphology. These dynamic aspects of cadherin-based cell–cell adhesion appeared to be associated with the following: (1) initial establishment of the blastoderm epithelium, (2) acquisition of cell motility in the neuroectoderm, (3) cell sheet folding, and (4) epithelial to mesenchymal conversion of the mesoderm. These observations suggest that the behavior of theDE-cadherin–catenin adhesion system may be regulated in a stepwise manner during gastrulation to perform successive cell-morphology conversions. Moreover, the processes responsible for loss of epithelial cell polarity and elimination of preexistingDE-cadherin-based epithelial junctions during early mesodermal morphogenesis are discussed
The Cellular Mechanism of Epithelial Rearrangement during Morphogenesis of theCaenorhabditis elegansDorsal Hypodermis

AbstractThe mechanism by which epithelial cells rearrange is a process that is central to epithelial morphogenesis, yet remains poorly understood. We have investigated epithelial cell rearrangement in the dorsal hypodermis of theCaenorhabditis elegansembryo, in which two rows of epithelial cells rearrange in a morphogenetic process known as dorsal intercalation. The intercalating cells extend basal protrusions which squeeze between their opposing neighbors beneath their adherens junctions. As the intercalating cells move forward, these protruding tips become broader in the anterior–posterior and dorsoventral dimensions, effectively “plowing through” the adherens junctions and forcing an opening for the remainder of the intercalating cell to insert between the contralateral cells. These cell movements are dependent upon intact cytoarchitecture, since the pharmacological disruption of microtubules or actin filaments blocks cell rearrangement. The cells appear to intercalate independently of immediately adjacent neighboring hypodermal cells because dorsal intercalation is not blocked by the ablation of the progenitors for either half of the lateral hypodermal cells or the posterior half of the dorsal hypodermis. This is the first case in which the protrusive mechanism underlying epithelial cell rearrangement has been characterized, and we propose a model describing how epithelial cells rearrange within the confines of an epithelial monolayer, and discuss the mechanisms that may be guiding these directed cell movements
Apoptosis of Epaxial Myotome inDanforth's short-tail(Sd) Mice in Somites That Form Following Notochord Degeneration

AbstractDanforth's short-tail(Sd) mouse is a semidominant mutation that prevents completion of notochord development. In homozygous mutant mice, the notochord completely degenerates at embryonic day 9.5 (E9.5), whereas the neural tube and somites continue to form, permitting analysis of somite development in the absence of inductive signals from the notochord and floor plate. In the somites formed after notochord degeneration,Myf5expression initiates in a normal temporal sequence, but instead of the normal restriction to the dorsomedial somite, its expression extends into the ventral somite.MyoD, myogenin,andmyosin heavy chainare normally expressed in the ventral myotome and there is normal development of hypaxial muscles. In contrast, subsequent to initialMyf5expression, muscle gene expression was not detected in the dorsal myotome and a high level of apoptosis was observed with significantly decreased formation of epaxial muscles. The apoptosis of epaxial muscle in somites that formed after notochord degeneration is consistent with a role for the notochord in the survival and differentiation of the dorsal myotome
SH2 Domain-Mediated Activation of Phospholipase Cγ Is Not Required to Initiate Ca2+Release at Fertilization of Mouse Eggs

AbstractThe initiation of Ca2+release at fertilization of mammalian eggs requires inositol trisphosphate (Miyazakiet al.,1992,Science257, 251–255), indicating that an enzyme of the phospholipase C family is probably activated. Because Ca2+release at fertilization in echinoderm eggs is initiated by SH2 domain-mediated activation of phospholipase Cγ (Carrollet al.,1997,J. Cell Biol.138, 1303–1311), we examined the possible role of PLCγ in initiating Ca2+release at fertilization in mouse eggs. Both PLCγ isoforms, PLCγ1 and PLCγ2, are present in mouse eggs and sperm, and stimulation of these enzymes in the egg by way of an exogenously expressed PDGF receptor causes Ca2+release. Recombinant SH2 domains of PLCγ1 and PLCγ2 inhibit PLCγ1 and PLCγ2 activation by the PDGF receptor, completely preventing Ca2+release in response to PDGF when injected at an ≈20- to 40-fold excess over the concentrations of endogenous proteins. However, even at an ≈100- to 400-fold excess over endogenous protein levels, PLCγ1 and PLCγ2 SH2 domains do not inhibit Ca2+release at fertilization. These findings indicate that Ca2+release at fertilization of mouse eggs does not require SH2-domain-mediated activation of PLCγ. However, activation of PLCγ in the egg by an alternative pathway, or introduction of activated PLCγ from the sperm, may be important
The TATA Binding Protein in the Sea Urchin Embryo Is Maternally Derived

AbstractThe cDNA encoding the TATA binding protein was isolated from 8- to 16-cell and morula-stage embryonic libraries of two distantly related species of sea urchin,Strongylocentrotus purpuratusandLytechinus variegatus,respectively. The two proteins are 96% identical over both the N- and C-terminal domains, suggesting a conservation of transcriptional processes between the two species. The prevalence of SpTBP transcripts at several developmental time points was determined using the tracer excess titration method, and the corresponding number of TBP protein molecules was determined by quantitative Western blot analysis. Our results indicate that the amount of TBP mRNA and protein per embryo remains relatively constant throughout development. An initial large pool of TBP protein (>109) molecules in the egg becomes diluted as a consequence of cell division and decreases to about 2 × 106molecules per cell by the gastrula stage. We found byin situRNA hybridization that the oocyte contains a large amount of TBP mRNA which is depleted late in oogenesis so that the eggs and early embryos have extremely low levels of TBP mRNA. We conclude that the oocyte manufactures nearly all of the TBP protein necessary for embryogenesis
Distinct Regulatory Elements GovernFgf4Gene Expression in the Mouse Blastocyst, Myotomes, and Developing Limb

AbstractEmbryonic development requires a complex program of events which are directed by a number of signaling molecules whose expression must be rigorously regulated. We previously showed that expression ofFgf4,which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in EC cells by the synergistic interaction of Sox2 and Oct-3 with theFgf4EC cell-specific enhancer. To verify whether this mechanism was also operatingin vivo,and to identify new elements controllingFgf4gene expression in distinct developmental stages, we have analyzed the expression of LacZ reporter plasmids containing different fragments of theFgf4gene in transgenic mouse embryos. Utilizing these transgenic constructs we have been able to recapitulate, for the most part,Fgf4gene expression during embryonic development. We show here that most of thecis-acting regulatory elements determiningFgf4embryonic expression are located in conserved regions within the 3′ UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct–proteins binding sites. We were also able to identify specific and distinct enhancer elements that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Finally, we present evidence that the very restricted pattern of expression ofFgf4transcripts in the AER results from the combined action of positive and negative regulatory elements located 3′ of theFgf4coding sequences. Thus theFgf4gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression
Specification of Gut Cell Fate Differs Significantly between the NematodesAcrobeloides nanusandCaenorhabditis elegans

AbstractThe classic view of a strictly cell-autonomous development in nematode embryos has been overturned in recent years with the demonstration of various inductive interactions during early development ofCaenorhabditis elegans.To examine how conserved the pattern of embryonic cell specification is among nematodes, we have begun to study the pattern in other species after selective elimination of certain early blastomeres. Here we report considerable differences in specification of the gut lineage betweenC. elegansandAcrobeloides nanus,another free-living soil nematode belonging to the same order. InC. elegansnone of the early blastomeres is by itself able to establish a gut lineage for which an inductive interaction between the somatic EMS cell and its germline sister P2is required. In contrast, inA. nanusall blastomeres of the 3-cell stage carry the potential to generate gut cells. Our data suggest that repressive interactions take place among blastomeres to ensure that under normal conditions only one of them executes the gut fate. Thus, in related species of nematodes with a very conserved morphology, the assignment of cell fate during early embryogenesis appears to involve quite different strategies
Sperm-Activating Proteins Obtained from the Herring Eggs Are Homologous to Trypsin Inhibitors and Synthesized in Follicle Cells

AbstractThe activation of sperm motility by the egg is an ubiquitous phenomenon in the animal kingdom, but the molecules by which the egg activates sperm motility have been clarified in only a few invertebrate species. In the Pacific herring,Clupea pallasii,mature unfertilized eggs release the sperm-activating proteins which are prerequisite to successful fertilization. Complementary DNA clones encoding herring sperm-activating proteins were isolated from a herring ovarian complementary DNA library and amino acid sequences were deduced. The herring sperm-activating protein(s) is a secretory product(s) with a strong homology to Kazal-type trypsin inhibitors, such as mammalian acrosin inhibitors. The sperm-activating proteins were globally distributed in the outermost layer of the egg chorion and its gene was found to be expressed in the follicle cells which surround developing oocytes. These results suggest that in the Pacific herring, trypsin inhibitor-like proteins are synthesized in the follicle cells, secreted, accumulated in the egg chorion during oocyte development, and released into the milieu at spawning to activate the motility of spermatozoa at the time of gamete interaction
Calcium and Endoplasmic Reticulum Dynamics during Oocyte Maturation and Fertilization in the Marine WormCerebratulus lacteus

AbstractTo monitor calcium and endoplasmic reticulum (ER) dynamics during oocyte maturation and fertilization, oocytes of the marine wormCerebratulus lacteuswere injected with the calcium-sensitive indicator calcium green dextran and/or the ER-specific probe “DiI.” Based on time-lapse confocal imaging of such specimens, prophase-arrested immature oocytes failed to develop normally after insemination and typically produced non-wave-like calcium transients that were lower in amplitude and less persistent than the wave-like oscillations observed during fertilizations of mature oocytes. Accordingly, the ER of DiI-loaded immature oocytes lacked an obvious substructure, whereas ER clusters, or “microdomains,” began to form in maturing specimens at about the time that these oocytes became competent to undergo normal fertilization-induced calcium dynamics and cleavage. The ER microdomains of mature oocytes typically reached widths of 1–8 μm and disappeared approximately 1 h after fertilization, which in turn coincided with the termination of the calcium oscillations. Collectively, these findings indicate: (i) changes in ER structure are temporally correlated with the onset and cessation of the calcium oscillations required for subsequent cleavage, and (ii) such ER reorganizations may play an important role in early development by enabling mature oocytes to generate a normal calcium response
Juvenile Hormone Prevents Ecdysteroid-Induced Expression of Broad Complex RNAs in the Epidermis of the Tobacco Hornworm,Manduca sexta

AbstractA cDNA homolog of theDrosophila melanogaster Broad Complex (BRC)gene was isolated from the tobacco hornworm,Manduca sexta,which shows a predicted 88% amino acid identity withDrosophilaBRC in the N-terminal BTB domain. Three zinc finger domains encoding homologs of theDrosophilaZ2, Z3, and Z4 domains (93, 100, and 85% identity, respectively) were obtained by RT–PCR. InManducadorsal abdominal epidermis, BRC RNAs were not observed during the larval molt. Three BRC transcripts—6.0, 7.0, and 9.0 kb—first appeared at the end of the feeding stage of the fifth (final) instar when the epidermis is exposed to ecdysteroids in the absence of juvenile hormone (JH) and becomes committed to pupal differentiation. These RNAs were induced in day 2 fifth larval epidermisin vitroby 20-hydroxyecdysone (20E) in the absence of JH with dose–response and time courses similar to the induction of pupal commitment. This induction by 20Ein vitrowas prevented by the presence of JH I at levels seenin vivoduring the larval molt. In the wing discs, the BRC RNAs appeared shortly after ecdysis to the fifth instar and coincided with the onset of metamorphic competence of these discs. Application of a JH analogue pyriproxifen during the fourth instar molt delayed and reduced the levels of BRC mRNAs seen in the wing discs in the early fifth instar, but did not completely prevent their appearance in this tissue that first differentiates at metamorphosis. The expression of the BRC transcription factors thus appears to be one of the first molecular indications of the genetic reprogramming of the epidermis necessary for insect metamorphosis. How JH prevents BRC expression in this epidermis may provide the key to understanding how this hormone controls metamorphosis
Cellularization inDrosophila melanogasterIs Disrupted by the Inhibition of Rho Activity and the Activation of Cdc42 Function

AbstractRegulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis.Drosophila melanogasterembryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and inD. melanogaster.To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme fromClostridium botulinumor with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video records of DIC imaged embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. We conclude that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning