pinecone/core-2020-05-10-deduplication huggingface dataset:
Hox Group 3 Paralogous Genes Act Synergistically in the Formation of Somitic and Neural Crest-Derived Structures

AbstractHoxgenes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multipleHoxgenes is beginning to reveal a complex network of interactions among these closely related genes which is responsible for directing the formation of spatially restricted tissues and structures. In this report we present an analysis of the genetic interactions between all members of the third paralogous group,Hoxa3, Hoxb3,andHoxd3.Previous analysis has shown that although mice homozygous for loss-of-function mutations in eitherHoxa3orHoxd3have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of theHoxb3gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant forHoxa3andHoxd3orHoxb3andHoxd3show an indistinguishable defect, loss of the entire atlas. This suggests that the identity of the specificHoxgenes that are functional in a given region may not be as critical as the total number ofHoxgenes operating in that region
Cyr61, Product of a Growth Factor-Inducible Immediate-Early Gene, Regulates Chondrogenesis in Mouse Limb Bud Mesenchymal Cells

AbstractChondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene,cyr61.The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of thecyr61gene during embryogenesis is tissue specific and temporally regulated. Most notably,cyr61is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cellsin vitroand is likely to play a similar rolein vivobased on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesisin vivoandin vitro;(2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell–cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton
Assembly of AKAP82, a Protein Kinase A Anchor Protein, into the Fibrous Sheath of Mouse Sperm

AbstractThe assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of theA KinaseAnchorProtein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (Mr97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins ofMr109,000 (p109) andMr26,000 (p26, representing the “pro” domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82
Regulation, Localization, and Anchoring of Protein Kinase A Subunits during Mouse Sperm Capacitation

AbstractThe molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). We demonstrate in this report that capacitation of cauda epididymal mouse spermin vitrowas accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity did not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, were found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation was associated with enzyme activity found in the soluble fraction. Moreover, the regulatory and catalytic subunits of PK-A were observed by indirect immunofluorescence to be present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fraction of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum which anchors and compartmentalizes PK-A to the cytoskeleton via the RII subunit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein was mapped to a 57-amino-acid residue region at its N-terminus. Computer analysis revealed a 14-amino-acid region that resembled the RII-binding domains of otherA KinaseAnchorProteins. A synthetic peptide corresponding to this domain inhibited AKAP82–RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, to sperm function
Cells at the Center ofDictyosteliumAggregates Become Spores

AbstractThe cellular slime moldDictyostelium discoideumundergoes a developmental life cycle on starvation to generate a fruiting body consisting of a mass of spores supported on a stalk of dead, vacuolated cells. The choice between alternative cell fates is influenced by a variety of factors including cell cycle position at the onset of starvation. We present evidence to suggest that the cell cycle position influences cell fate by determining the position of cells in the early aggregate. The existence of a strain which cannot initiate development on its own but which can respond to signals generated by nonmutant cells has allowed us to investigate the eventual cell fate of the initiating cells which are, by definition, at the center of the early aggregate. Cells which have a propensity to become prespore cells show an increased efficiency in initiating development of this strain. Labeling the initiating cells by the expression of green fluorescent protein reveals that these cells become spores. The higher levels of expression of genes characteristic of early development in cells with a prespore tendency are consistent with the earlier expression of the components of relay in prespore cells
Imaginal Tissues ofDrosophila melanogasterExhibit Different Modes of Cell Proliferation Control

AbstractThe highly conserved regulatory mechanisms that control progression of a cell through the cell cycle do not, alone, explain the programmed control of cell proliferation during animal development. Additional controls must coordinate the cell cycle regulators with developmental regulatory events. Here we report studies of cell cycle control in the imaginal tissues ofDrosophila melanogaster,specifically in situations where cell cycle progression is regulated by varying the length of the G2phase. We show that G2-phase arrest in late larval wing imaginal disks requires transcriptional control ofstg,a mitotic inducer that encodes aD. melanogasterhomologue of theSchizosaccharomyces pombep80cdc25phosphatase. In a second study,stringtranscriptional regulation was also shown to be important for G2-phase regulation in eye disk cells posterior to the morphogenetic furrow. Finally, unlike all other situations described to date,stringtranscriptional regulation was found not to be the cause of G2arrest in abdominal histoblasts, these cells being refractory to ectopic expression ofstg.This study further establishesstringas an important regulator of G2phase duringD. melanogasterdevelopment, but also reveals that at least one additional mechanism is utilized to control G2-phase length and thus cell proliferation in different developmental contexts
NGF-Mediated Survival Depends on p21ras in Chick Sympathetic Neurons from the Superior Cervical but Not from Lumbosacral Ganglia

AbstractIn rat embryonic sympathetic neurons from the superior cervical ganglia (SCG) NGF-mediated survival depends on the activation of the trkA receptor tyrosine kinase and on the activity of the intracellular plasmamembrane-anchored small G-protein p21ras. In contrast, chick sympathetic neurons derived from the more caudally located lumbosacral chain ganglia (LSCG) do not respond to activated p21ras (G12V-Ha-ras mutant). In these neurons endogenous p21ras and its downstream effector MAP kinase are activated but are not essential for NGF-dependent survival. Here we show that also in chick sympathetic neurons of the SCG permanently activated p21ras protein does promote neuron survival. Consistently, their NGF-mediated survival is sensitive to Fab fragments blocking endogenous p21ras activity. These results suggest that sympathetic neurons derived from sympathoenteric (SCG) and sympathoadrenal (LSCG) lineages differ in their requirement for p21ras in the NGF-mediated survival pathways
Developmental Fates of Larval Tissues after Metamorphosis in AscidianHalocynthia roretzi

AbstractCell lineages during ascidian embryogenesis are invariant. In a previous study, the developmental fate of each blastomere during embryogenesis of the ascidianHalocynthia roretziwas analyzed in detail by intracellular injection of a tracer molecule, horseradish peroxidase (Nishida, H.,Dev. Biol.121, 526–541, 1987). In the present study, the developmental fates of various larval mesodermal tissues after metamorphosis inH. roretziwere traced by labeling each precursor blastomere of larval tissues. Mesenchyme cells of larvae gave rise to tunic cells of juveniles. Trunk ventral cells gave rise to body-wall (atrial siphon and latitudinal mantle) muscle, heart, and pericardium of juvenile. Trunk lateral cells gave rise to blood (coelomic) cells as well as body-wall (oral siphon and longitudinal mantle) muscle. Larval muscle and notochord cells did not contribute to juvenile tissues. It was revealed that it is predictable to which juvenile tissue a blastomere of the 110-cell embryo will give rise. Therefore, in ascidian development, developmental fates after metamorphosis were almost invariant with regard to cell type of descendants
Localized Maternal Proteins inXenopusRevealed by Subtractive Immunization

AbstractIt has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in theXenopusoocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within theXenopusegg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning
The Spemann Organizer ofXenopusIs Patterned along Its Anteroposterior Axis at the Earliest Gastrula Stage

AbstractThe Spemann organizer is largely responsible for organizing and patterning the anteroposterior axis during the development of amphibians. In this report, we examine the degree of anteroposterior pattern in the earliest gastrula organizer ofXenopususing a combination of embryological and molecular techniques. When we divide the earliest gastrula organizer, a region measuring 20 cells high by 25 cells wide, into stereotyped anterior (vegetal) and posterior (animal) halves, each half not only has a distinct fate and state of specification, but also induces a unique set of region-specific neural genes. When wrapped in animal cap ectoderm, the anterior half induces only anterior-specific genes (XAG-1andotxA), while the posterior half induces anterior (otxAand reduced levels ofXAG-1) and posterior (Hox B9) neural genes, revealing early localization of neural posteriorizing activity to posterior mesendoderm. This is the earliest demonstration of regionalized neural induction by theXenopusorganizer. Additionally, based on the expression ofgsc, Xbra,andXnot,we show that the organizer is patterned both at the early gastrula stage and prior to the appearance of bottle cells
Trabecular Myocytes of the Embryonic Heart Require N-Cadherin for Migratory Unit Identity

AbstractThe myocardial wall of the vertebrate heart changes from a simple epithelium to a trabeculated structure during embryogenesis. This process occurs when epithelioid cardiomyocytes migrate toward the endocardium, which we show is coincident with up-regulation of the cell adhesion molecule, N-cadherin. To study the role of N-cadherin expressed at the trabeculation stage, a replication-defective retrovirus expressing a dominant negative mutant of N-cadherin (ΔN-cadherin) was engineered. Control viruses were designed to express β-galactosidase or a full-length N-cadherin. Viruses were introduced into epithelioid presumptive myocytes at the time they initiate the epithelial–mesenchymal transformation. Individual cells infected with control viruses generated daughter myocytes which migrated toward endocardium as a tight cluster, thereby generating a clone that forms a single or at most two trabeculae. In contrast, myocytes expressing ΔN-cadherin were sparsely distributed within the myocardium and failed to form the ridge-shaped clone. Thus, in addition to its known roles in myocyte epithelialization and intercalated disc formation, N-cadherin appears to play a role in homotypic interactions between nonepithelial migratory myocytes during trabecular formation of the embryonic heart
Inactivation of the FGF-4 Gene in Embryonic Stem Cells Alters the Growth and/or the Survival of Their Early Differentiated Progeny

AbstractPrevious studies have shown that early mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, we prepared genetically engineered embryonic stem (ES) cells, which are unable to produce FGF-4. Specifically, we describe the isolation and characterization of ES cells with both FGF-4 alleles inactivated. The FGF-4−/−ES cells do not require FGF-4 to proliferatein vitro,and addition of FGF-4 to the medium has little or no effect on their growth. Thus, FGF-4 does not appear to act as an autocrine growth factor for cultured ES cells. We also demonstrate that FGF-4−/−ES cells, like their unmodified counterparts, are capable of forming highly complex tumors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, we demonstrate that the FGF-4−/−ES cells can differentiatein vitroafter exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importantly, addition of FGF-4 to the culture medium dramatically increases the number of differentiated cells derived from the FGF-4−/−ES cells, in particular cells with many of the properties of parietal extraembryonic endoderm. Finally, we demonstrate that there are differences in the RNA profiles expressed by the differentiated progeny formedin vitrofrom FGF-4−/−ES cells and FGF-4+/+ES cells when they are cultured with FGF-4. Taken together, the studies described in this report indicate that certain lineages formedin vitroare affected by the inactivation of the FGF-4 gene, in particular specific cells that form during the initial stage of ES cell differentiation. Thus, ES cells with both FGF-4 alleles inactivated should shed light on the important roles of FGF-4 during the early stages of mammalian development and help determine why FGF-4−/−embryos die shortly after implantation
Cell Death in the Avian Sclerotome

AbstractIn this study the occurrence of apoptotic cells in chick embryo trunk somites, between 2.5 and 4 days of development, has been examined using anin situnick-end-labeling method (TUNEL) to identify nuclei in which DNA is undergoing fragmentation. At 2.5 days of development, apoptotic cells were found in the sclerotome with a distribution that depended on the rostrocaudal level in the trunk. At the most rostral levels (somites 1–18), dying cells were present primarily in the rostral half of the ventral sclerotome; at midlevels (somites 19–26), they were present throughout the ventral sclerotome; and at caudal levels (somites 27–32), no dying cells were present. By 4 days of development, the number of dying cells in the sclerotome was sharply reduced, and those present were primarily distributed to the caudal side of the intrasclerotomal fissure. Double labeling of cells for both TUNEL and the HNK-1 epitope, at 2.5 days, indicated that the majority of the dying cells were not neural crest cells. Further, dying cells in the rostral somite half were present largely in regions of the sclerotome that labeled poorly for HNK-1. It was confirmed that apoptotic neural crest cells retain the HNK-1 epitope and therefore would have been observed if present. Neural crest cells only appeared to be apoptotic in relatively small numbers and only at the ventral border of the sclerotome. Examination of DiI-labeled neural crest cells confirmed that the dying cells in the body of the somite were not primarily neural crest cells. Two hypotheses regarding the TUNEL-positive cells in the sclerotome were experimentally tested. First, that they originate from the somitocoel compartment of the somite, because their distribution patterns at 4 days were similar to those of somitocoel cells. To test this, somitocoel cells were labeled with carboxyfluorescein and grafted into host embryosin ovo. Results showed that these cells did not become apoptotic and that the dying cells were therefore not derived from the somitocoel. Second, the hypothesis was tested that the distribution patterns of the dying cells in the sclerotome are determined by factors outside the somite itself. Somites and segmental plates were transplanted into hostsin ovowith reversed orientation, after which the patterns of dying cells were examined using nile blue sulfate staining. The results indicated that the patterns were unchanged after a further 2 days incubation, suggesting that the patterns of cell death in the sclerotome are not determined solely from within the somite. The distribution of the cell death-associated gene products, bcl-2, bax, and interleukin-1β converting enzyme, indicates that although these proteins are segmentally distributed in the dermomyotome and in the rostrodorsal quadrant of the sclerotome, their patterns are not directly correlated with the distribution of dying cells