pinecone/core-2020-05-10-deduplication huggingface dataset:
Significance of Bone Morphogenetic Protein-4 Function in the Initial Myofibrillogenesis of Chick Cardiogenesis

AbstractThe heart is the first organ to form and function during vertebrate embryogenesis. Using a secreted protein, noggin, which specifically antagonizes bone morphogenetic protein (BMP)-2 and -4, we examined the role played by BMP during the initial myofibrillogenesis in chick cultured precardiac mesoendoderm (mesoderm + endoderm; ME). Conditioned medium from COS7 cells transfected with Xenopus noggin cDNA inhibited the expression of sarcomeric proteins (such as sarcomeric α-actinin, Z-line titin, and sarcomeric myosin), and so myofibrillogenesis was perturbed in cultured stage 4 precardiac ME; however, it did not inhibit the expression of smooth muscle α-actin (the first isoform of α-actin expressed during cardiogenesis). In cultured stage 5 precardiac ME, noggin did not inhibit either the formation of I-Z-I components or the expression of sarcomeric myosin, but it did inhibit the formation of A-bands. Although BMP4 was required to induce expressions of sarcomeric α-actinin, titin, and sarcomeric myosin in cultured stage 6 posterolateral mesoderm (noncardiogenic mesoderm), smooth muscle α-actin was expressed without the addition of BMP4. Interestingly, in cultured stage 6 posterolateral mesoderm, BMP2 induced the expressions of sarcomeric α-actinin and titin, but not of sarcomeric myosin. These results suggest that (1) BMP4 function lies upstream of the initial formation of I-Z-I components and A-bands separately in a stage-dependent manner, and (2) at least two signaling pathways are involved in the initial cardiac myofibrillogenesis: one is an unknown pathway responsible for the expression of smooth muscle α-actin; the other is BMP signaling, which is involved in the expression of sarcomeric α-actinin, titin, and sarcomeric myosin
Essential and Redundant Functions of the MyoD Distal Regulatory Region Revealed by Targeted Mutagenesis

AbstractTransgenic analyses have defined two MyoD enhancers in mammals, the core enhancer and distal regulatory region (DRR); these enhancers exhibit complementary activities and together are sufficient to recapitulate MyoD expression in developing and mature skeletal muscle. DRR activity is restricted to differentiated muscle and persists postnatally, suggesting an important role in maintaining MyoD expression in myocytes and muscle fibers. Here, we use targeted mutagenesis in the mouse to define essential functions of the DRR in its normal chromosomal context. Surprisingly, deletion of the DRR resulted in reduced MyoD expression in all myogenic lineages at E10.5, at least 1 day prior to detection of DRR activity in limb buds and branchial arches of transgenic mice. At later embryonic and fetal stages, however, no defect in MyoD expression was observed, indicating that the DRR is dispensable for regulating MyoD during muscle differentiation. Expression analyses in wild-type and Myf-5 mutant embryos also indicate that the DRR is not an obligate target for Myf-5- and Pax-3-dependent regulation. In contrast to embryonic and fetal stages, deletion of the DRR resulted in a pronounced reduction in MyoD mRNA levels in adults, showing a functional requirement for DRR activity in mature muscle. These data reveal essential and redundant functions of the DRR and underscore the importance of loss-of-function enhancer analyses for understanding cis transcriptional circuitry
Distinct Enhancers Regulate Skeletal and Cardiac Muscle-Specific Expression Programs of the Cardiac α-Actin Gene in Xenopus Embryos

AbstractDuring vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the α-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac α-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)6GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac α-actin promoter, to confer appropriate cardiac α-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac α-actin gene promoter is to act solely as a high-affinity SRF binding site
Role of Runx Genes in Chondrocyte Differentiation

AbstractRunx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs
Analysis of Spatial and Temporal Gene Expression Patterns in Blastula and Gastrula Stage Chick Embryos

AbstractStudies on the genetic basis of rostral–caudal specification, neural induction, and head development require knowledge of the relevant gene expression patterns. Gaps in our understanding of gene expression have led us to examine the detailed spatiotemporal expression patterns of 19 genes implicated in early development, to learn more about their potential role in specifying and patterning early developmental processes leading to head formation. Here, we report the expression patterns of these markers in blastula- and gastrula-stage chick embryos, using whole-mount in situ hybridisation. Nodal, Fgf8, Bmp7, Chordin, Lim1, Hnf3β, Otx2, Goosecoid, Cerberus, Hex, Dickkopf1, and Crescent are all already expressed by the time the egg is laid. When the primitive streak has reached its full length, a later group of genes, including Ganf, Six3, Bmp2, Bmp4, Noggin, Follistatin, and Qin (BF1), begins to be expressed. We reassess current models of early rostral patterning based on the analysis of these dynamic spatiotemporal expression patterns
Truncation of c-fes via Gene Targeting Results in Embryonic Lethality and Hyperproliferation of Hematopoietic Cells

AbstractThe c-fes protooncogene encodes a nonreceptor tyrosine kinase (Fes) implicated in cytokine receptor signal transduction, granulocyte survival, and myeloid differentiation. To study the role of c-fes during myelopoiesis, we generated embryonic stem (ES) cells with a targeted disruption of the c-fes locus. Targeted mutagenesis deletes the C-terminal SH2 and tyrosine kinase domains of c-fes (referred to as c-fesΔc/Δc). We demonstrate that the c-fesΔc/Δc allele results in a truncated Fes protein that retains the N-terminal oligomerization domain, but lacks both the SH2 and the tyrosine kinase domain.In vitro differentiation of c-fesΔc/Δc ES cells results in hyperproliferation of an early myeloid cell. Generation of c-fesΔc/Δc mutant chimeric mice causes lethality by E13.5 with embryos exhibiting pleiotropic defects, the most striking being cardiovascular abnormalities. These results establish thatc-fes is an important regulator of myeloid cell proliferation and embryonic development
Integrin Signaling Regulates Blastocyst Adhesion to Fibronectin at Implantation: Intracellular Calcium Transients and Vesicle Trafficking in Primary Trophoblast Cells

AbstractAccumulating evidence indicates that the endometrial extracellular matrix (ECM) modulates trophoblast adhesion during mouse blastocyst implantation. In previous studies of adhesion-competent mouse blastocysts, we have demonstrated that integrin-mediated fibronectin (FN)-binding activity on the apical surface of trophoblast cells is initially low, but becomes strengthened after embryos are exposed to FN. In the present study, we have examined whether the ligand-induced upregulation of trophoblast adhesion to FN is mediated by integrin signaling. The strengthening of adhesion to FN required integrin ligation, which rapidly elevated cytoplasmic-free Ca2+. Chelation of intracellular Ca2+ using BAPTA-AM, or inhibition of the Ca2+-dependent proteins, protein kinase C or calmodulin, significantly attenuated the effect of FN on binding activity. Furthermore, direct elevation of cytoplasmic Ca2+ levels with ionomycin upregulated FN-binding activity, demonstrating that Ca2+ signaling is required and sufficient for strong adhesion to FN. Ca2+ signaling may induce protein trafficking, a known requirement for ligand-induced upregulation of FN-binding activity. Indeed, intracellular vesicles accumulated in adhesion-competent blastocysts, but were absent after exposure to either FN or ionomycin. These findings suggest that, during implantation, contact between peri-implantation blastocysts and FN elevates intracellular Ca2+, which strengthens trophoblast adhesion to ECM through protein redistribution
An Epithelial Precursor Is Regulated by the Ureteric Bud and by the Renal Stroma

AbstractKidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin α5) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma
DSF Nuclear Receptor Acts As a Repressor in Culture and in Vivo

AbstractLoss-of-function mutations affecting the dissatisfaction (DSF) nuclear receptor alter both sexual behavior and the sex-specific nervous system in Drosophila. As a step toward understanding the way DSF controls development and function of the nervous system, we have analyzed the regulatory activities of the DSF protein. DSF prefers an atypical DNA half site, AAGTCA. Wild-type DSF, but not the point mutant DSF7, monomerically binds and represses reporter constructs bearing this site. DSF also contains an atypically long, 356-amino-acid hinge separating its DNA-binding domain (DBD) and ligand-binding domain (LBD). The hinge contains at least two functions: a region that drastically lowers DNA-binding efficiency in vitro, and an amino-terminal repressive domain. The DBD and LBD of DSF, along with major portions of the hinge, are highly conserved in other insects. Ectopic expression of DSF in Drosophila imaginal discs results in developmental disruptions in disc-derived tissues, disruptions which are largely suppressed when DSF is fused to the VP16 activation domain, consistent with a repressive role for DSF. Finally, when tethered to DNA, DSF's hinge and LBD regions act as strong transcriptional repressors in multiple larval and pupal tissues, including many DSF-expressing tissues. These results suggest DSF can repress transcription in vivo, that repression is largely responsible for its ectopic expression phenotypes, and that repression may be a key component of normal DSF function
Dorsal Gradient Networks in the Drosophila Embryo

AbstractHere, we describe one of the major maternal regulatory gradients, Dorsal, and threshold outputs of gene expression that result from the graded distribution of this transcription factor. The analysis of a large number of authentic and synthetic target genes suggests that the Dorsal gradient directly specifies at least four, and possibly as many as seven, different thresholds of gene activity and tissue differentiation. These thresholds initiate the differentiation of the three primary embryonic tissues: the mesoderm, neurogenic ectoderm, and dorsal ectoderm. Moreover, primary readouts of the Dorsal gradient create asymmetries that subdivide each tissue into multiple cell types during gastrulation. Dorsal patterning thresholds represent the culmination of one of the most complete gene regulation network known in development, which begins with the asymmetric positioning of the oocyte nucleus within the egg chamber and leads to the localized activation of the Toll-Dorsal signaling pathway in ventral regions of the early embryo
Expression of Patella vulgata Orthologs of engrailed and dpp-BMP2/4 in Adjacent Domains during Molluscan Shell Development Suggests a Conserved Compartment Boundary Mechanism

AbstractThe engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. Here, we show that, in the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. Earlier it was shown that a compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. We conclude that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. We suggest that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains
Making Worm Guts: The Gene Regulatory Network of the Caenorhabditis elegans Endoderm

AbstractThe nematode Caenorhabditis elegans is a triploblastic ecdysozoan, which, although it contains too few cells during embryogenesis to create discernible germ “layers,” deploys similar programs for germ layer differentiation used in animals with many more cells. The endoderm arises from a single progenitor, the E cell, and is selected from among three possible fates by a three-state combinatorial regulatory system involving intersecting cell-intrinsic and intercellular signals. The core gene regulatory cascade that drives endoderm development, extending from early maternal regulators to terminal differentiation genes, is characterized by activation of successive tiers of transcription factors, including a sequential cascade of redundant GATA transcription factors. Each tier is punctuated by a cell division, raising the possibility that intercession of one cell cycle round, or DNA replication, is required for activation of the next tier. The existence of each tier in the regulatory hierarchy is justified by the assignment of a unique task and each invariably performs at least two functions: to activate the regulators in the next tier and to perform one other activity distinct from that of the next tier. While the regulatory inputs that initiate endoderm development are highly divergent, they mobilize a gene regulatory network for endoderm development that appears to be common to all triploblastic metazoans. Genome-wide functional genomic approaches, including identification of >800 transcripts that exhibit the same regulatory patterns as a number of endoderm-specific genes, are contributing to elucidation of the complete endoderm gene regulatory network in C. elegans. Dissection of the architecture of the C. elegans endoderm network may provide insights into the evolutionary plasticity and origins of this germ layer
Control of Intercalation Is Cell-Autonomous in the Notochord of Ciona intestinalis

AbstractDishevelled signaling plays a critical role in the control of cell intercalation during convergent extension in vertebrates. This study presents evidence that Dishevelled serves a similar function in the Ciona notochord. Embryos transgenic for mutant Dishevelled fail to elongate their tails, and notochord cells fail to intercalate, though notochord cell fates are unaffected. Analysis of mosaic transgenics revealed that the effects of mutant Dishevelled on notochord intercalation are cell-autonomous in Ciona, though such defects have nonautonomous effects in Xenopus. Furthermore, our data indicate that notochord cell intercalation in Ciona does not require the progressive signals which coordinate cell intercalation in the Xenopus notochord, highlighting an important difference in how mediolateral cell intercalation is controlled in the two animals. Finally, this study establishes the Ciona embryo as an effective in vivo system for the study of the molecular control of morphogenetic cell movements in chordates
Zebrafish SPI-1 (PU.1) Marks a Site of Myeloid Development Independent of Primitive Erythropoiesis: Implications for Axial Patterning

AbstractThe mammalian transcription factor SPI-1 (synonyms: SPI1, PU.1, or Sfpi1) plays a critical role in myeloid development. To examine early myeloid commitment in the zebrafish embryo, we isolated a gene from zebrafish that is a SPI-1 orthologue on the basis of homology and phylogenetic considerations. The zebrafish spi1 (pu1) gene was first expressed at 12 h postfertilization in rostral lateral plate mesoderm (LPM), anatomically isolated from erythroid development in caudal lateral plate mesoderm. Fate-mapping traced rostral LPM cells from the region of initial spi1 expression to a myeloid fate. spi1 expression was lost in the bloodless mutant cloche, but rostral spi1 expression and myeloid development were preserved in the mutant spadetail, despite its complete erythropoietic failure. This dissociation of myeloid and erythroid development was further explored in studies of embryos overexpressing BMP-4, or chordin, in bmp-deficient swirl and snailhouse mutants, and chordin-deficient chordino mutants. These studies demonstrate that, in zebrafish, spi1 marks a rostral population of LPM cells committed to a myeloid fate anatomically separated from and developmentally independent of erythroid commitment in the caudal LPM. Such complete anatomical and developmental dissociation of two hematopoietic lineages adds an interesting complexity to the understanding of vertebrate hematopoietic development and presents significant implications for the mechanisms regulating axial patterning
Laminin α5 Is Required for Lobar Septation and Visceral Pleural Basement Membrane Formation in the Developing Mouse Lung

AbstractLaminin α/β/γ heterotrimers are the major noncollagenous components of all basement membranes. To date, five α, three β, and three γ chains have been identified. Laminin α5 is expressed early in lung development and colocalizes with laminin α1. While laminin α1 expression in the lung is restricted to the embryonic period, laminin α5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin α5 gene Lama5 causes embryonic lethality at E14–E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5−/− mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin α4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin α5, which was found to be the only laminin α chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked