pinecone/core-2020-05-10-deduplication huggingface dataset:
Identification of Inducing, Responding, and Suppressing Regions in an Experimental Model of Notochord Formation in Avian Embryos

AbstractThe notochord normally arises from committed cells in the rostral tip of the primitive streak. After removal of these cells from the avian gastrula, embryos with notochords nevertheless develop in the majority of cases. A region required for the formation of this reconstituted notochord lies lateral to the primitive streak. In the present study we have determined that this region acts as an inducer for more lateral cells in the epiblast, which actually give rise to the reconstituted notochord. The strongest inducing region lies between 0–250 μm lateral to the streak and 500–750 μm caudal to the rostral end of the streak and chiefly contains cells normally fated to form lateral plate and somitic mesoderm. The responding region is located 250–500 μm lateral to the streak and 0–750 μm caudal to the rostral end of the streak. This area chiefly contains cells normally fated to form neural ectoderm, although cells normally fated to form lateral plate and somitic mesoderm are also within this area. The inducing and responding areas interact to form reconstituted notochord either when the primitive streak, including its rostral end (Hensen's node), is removed from the cultured blastoderm or when the inducer and responder are grafted together into an ectopic site. Grafting Hensen's node into isolates containing both inducer and responder blocks formation of reconstituted notochord, suggesting that Hensen's node suppresses formation of lateral notochords during normal development. These findings increase our understanding of the early interactions between mesoderm and ectoderm and provide a novel model system that is well defined and accessible for studying inductive events in higher vertebrates
Protein Tyrosine Kinase Activity during Egg Activation Is Important for Morphogenesis at Gastrulation in the Sea Urchin Embryo

AbstractFertilization triggers a series of preprogrammed events functioning to activate egg metabolism, incorporate the paternal genome, and initiate development. The activity of protein tyrosine kinases during egg activation is required for several steps leading up to the first cell division. We now present evidence for an additional protein tyrosine kinase-mediated event that occurs between 30 and 45 min after insemination and is not required until gastrulation, which occurs over 24 hr later. Eggs treated with protein tyrosine kinase inhibitors within this window of time cleaved and formed normal blastulae but could not gastrulate or undergo further development to the pluteus stage. These findings provide the first evidence that some of the control mechanisms used in later development are established during a brief period of time in the fertilized egg and require the action of one or more protein tyrosine kinases
Different Roles for Fibronectin in the Generation of Fore and Hind Limb Precartilage Condensations

AbstractFibronectin expression and spatiotemporal distribution were examined in relation to the distinctive patterns of mesenchymal condensation and chondrogenesis seen in high-density serum-free cultures of chicken wing and leg bud precartilage cells. More fibronectin protein was produced on a per cell basis by leg than by wing mesenchyme, both in freshly isolated tissue and during the prechondrogenic condensation period in culture, where the difference was twofold. The quantitative difference in fibronectin expression in freshly isolated wing and leg mesenchyme was also seen at the level of total and poly(A)+RNA. During the condensation phase, fibronectin was distributed in the wing and leg mesenchymal cultures in a way that prefigured the eventual distribution of cartilage in these cultures: in wing cultures condensations were broad and flat, and rich in diffusely organized fibronectin; in leg cultures, condensations were compact and spheroidal, and contained abundant deposits of fibronectin. In addition, the leg condensations were connected by long fibronectin-rich fibers. Transient treatment with TGF-β early during the culture period led to increase in fibronectin production and expansion of condensations in both wing and leg cultures. Leg mesenchyme was more responsive to transforming growth factor-β than wing mesenchyme with respect to fibronectin production, and this was reflected in a greater enhancement of cartilage formation in later cultures. Treatment of cultures with monoclonal antibody 304 directed against the amino-terminal heparin-binding domain of fibronectin inhibited condensation formation and reduced chondrogenesis in wing mesenchyme, but left these two processes unchanged in leg mesenchyme, despite disruption by the antibody of the leg-specific fibronectin fibers. These studies indicate that for both wing and leg mesenchyme the morphology, extent, and spatiotemporal regulation of precartilage condensation and subsequent chondrogenesis closely parallels the deposition of fibronectin. But whereas the interaction between cells and fibronectin in wing bud mesenchyme is mediated in part by the protein's amino-terminal domain, this domain does not appear to be involved in analogous interactions in leg bud mesenchyme
Cytoskeletal Control of Myogenesis: A Desmin Null Mutation Blocks the Myogenic Pathway during Embryonic Stem Cell Differentiation

AbstractA differentiating system based on embryonic stem (ES) cell-derived embryoid bodies (EBs) which recapitulates thein vivocardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation
TTF-1 Regulates Lung Epithelial Morphogenesis

AbstractTTF-1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain.In vitro,TTF-1 can activate the promoter of thyroid- and pulmonary-specific genes. We postulated that TTF-1 not only is essential for the activation of tissue-specific genes, but also may directly participate in epithelial cell morphogenesis. To test this postulate, we used an antisense oligonucleotide inhibitory strategy in anin vitromodel of embryonic mouse lung branching morphogenesis. This strategy suppressedTTF-1translation and inhibited lung branching morphogenesis. The resulting abnormal phenotype was characterized by hyperplastic and unorganized proliferation of epithelial cells in the airways. The mesenchymal compartment of the lung appeared to be unaffected. These results demonstrate, for the first time, that the expression of a homeoprotein transcriptional regulator is necessary for lung epithelial morphogenesis
The Receptor Tyrosine Kinase QEK5 mRNA Is Expressed in a Gradient within the Neural Retina and the Tectum

AbstractIn the retinotectal system, positional information has long been postulated to take the form of molecular gradients within both the retina and the tectum. Recent reports have implicated Mek4, a member of the Eph (also named class V) family of tyrosine kinase receptors (RTKs), and two ligands, RAGS and ELF-1, in this process. Here, we report the cloning and distribution pattern of QEK5, another member of the Eph family of RTKs, isolated from a quail cDNA library. During retinal differentiation, QEK5 transcripts accumulate in a ventral to dorsal gradient within the retinal neuroepithelium, where its expression becomes restricted to the ganglion and bipolar cell layers. Within the tectum, QEK5 transcripts are detectable in a posterior to anterior gradient in the ventricular layer and newly formed superficial layers. The pattern of QEK5 expression in the retina and tectum is distinct from that of Mek4, suggesting that complex patterns of Eph RTKs and their ligands may play a role in cell–cell interactions involved in retinotectal projections and differentiation of the central nervous system
The Sequence of Regulatory Events Controlling the Expression of the γD-crystallin Gene during Fibroblast Growth Factor-Mediated Rat Lens Fiber Cell Differentiation

AbstractThe transcriptional activation of tissue-specific genes during terminal differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed these events for the transcriptional activation of the rat γD-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced)in vitrodifferentiation of rat lens fiber cells.In vitro,in the presence of bFGF only, the endogenous γD mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and γD mRNA starts to accumulate at Day 8. Demethylation of the γD promoter region, as assessed by measuring the methylation state of theThaI site at −16, occurs much sooner, within 1 day. By genomic footprinting, the first protein interaction with the promoter region was visible at Day 8; full occupancy of the promoter region could be detected only at Day 12. The genomic footprint identified four putative regulatory regions: −141/−131, −88/−71, −55/−45, and −15/−4. Site-directed mutagenesis of the G residues at −55 and −46 resulted in a three- to fivefold decrease in promoter activity of transfected γD/CAT reporter genes and also abolished interaction with nuclear extract factor(s). A G→T mutation at −43 had no effect. The −55/−45 footprint thus derives from a proximal activator. The −88/−71 footprint identifies a silencer of the γD promoter in late fiber cell differentiation, as a tetramer of the −85/−67 sequence silenced a tk/CAT construct when transfected into fiber cells at a late stage, but not at an early stage, ofin vitrodifferentiation. To time the appearance of regulatory factors, the activity of a −73/+45 γD/CAT (containing the activator region) and of a −1100/+45 γD/CAT construct was measured during fiber cell differentiation. The −73/+45 construct was active between Day 5 and Day 14, with a maximum at Day 12. The additional sequence information present in the −1100/+45 construct constrained γD promoter activity to between Day 8 and Day 13, with a maximum at Day 10. We conclude that the phased appearance of transacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the γD-crystallin gene promoter
Progressive Spatial Restriction ofSek-1andKrox-20Gene Expression during Hindbrain Segmentation

AbstractAfter segmentation of the vertebrate hindbrain, expression of the zinc-finger geneKrox-20and the receptor tyrosine kinase geneSek-1is precisely restricted to rhombomeres (r) 3 and 5. This precise segmental expression is likely to reflect a critical requirement for these rhombomeres to acquire a distinct and homogeneous identity and raises the question as to how this relates to the intermingling and restriction of cell movement during segmentation. We have analysedKrox-20andSek-1expression in the mouse and chick hindbrain at single-cell resolution using whole-mountin situhybridisation and immunocytochemistry. We find that, in the mouse, the presumptive r3 and r5 expression domains each arise as narrow stripes that then broaden, suggestive of a recruitment of cells to an r3/r5 identity and/or a segmental regulation of cell proliferation. In addition, we find that expression of these genes initially occurs in fuzzy domains, and that these are progressively restricted to segmental domains, although occasional “violating” cells are observed even after segmentation. We propose that the establishment and maintenance of these segmental domains may involve both a dynamic regulation of r3/r5 identity and the restriction of cell movement across rhombomere boundaries
Left–Right Asymmetric Localization of Flectin in the Extracellular Matrix during Heart Looping

AbstractThe early embryo is initially bilaterally symmetrical. One of the first distinct indications of asymmetry in the embryo occurs during heart looping. The midline tubular heart begins to bend to the right to form a C-shaped structure around 30 hr of development in the avian model. A molecular basis for heart asymmetry and direction of looping is not known, although factors inherent to the myocardium are believed to underlie looping. A left–right asymmetric localization of a specific molecule in the bilateral heart forming regions has not been reported previously. One molecule that we are calling flectin (flectere,in L., to bend or to loop) shows a bilateral asymmetric localization early in the heart forming mesoderm and continues to be expressed asymmetrically in a highly organized manner in the cardiac jelly during heart looping. This large extracellular matrix molecule has been identified using a monoclonal antibody F-22 (Mieziewskaet al.,1994a,b). Flectin shows a discrete spatiotemporal pattern of extracellular matrix expression during avian heart development. An asymmetric expression of flectin is observed during heart development at stage 7+/8− (approximately at 24 hr of development around the 3-somite stage). It is predominantly expressed in the left precardiac mesoderm at this developmental period. Between stages 12 and 14, flectin continues to be asymmetrically expressed in the myocardium and is localized at high levels on the basal side of the myocardium and within the cardiac jelly extending to the endocardial cell surfaces. In the same plane of the looping part of the heart it is differentially organized within the cardiac jelly on the convex side and in the outer loop areas. A reduced expression is apparent anteriorly and posteriorly along the tubular heart. The initial asymmetry of localization is maintained throughout the tubular heart. At stage 22 (Embryonic Day 3.5), intensity of immunolocalization of flectin is significantly decreased, with left–right asymmetry becoming less discernible or absent. It again is expressed in Day 10 embryonic hearts. Flectin expression appears to be modulated by retinoids. In vitamin A-deficient quail embryonic hearts that do not loop (Dersch and Zile, 1993; Twalet al.,1995), flectin protein expression is decreased and disorganized, as are other extracellular matrix components comprising the cardiac jelly
Neurons and Ecdysteroids Promote the Proliferation of Myogenic Cells Cultured from the Developing Adult Legs ofManduca sexta

AbstractDuring metamorphosis in the hawkmothManduca sexta,larval leg motoneurons survive the degeneration of their target muscles to innervate new muscles that form during the development of the adult legs. Observations of muscle developmentin vivosuggest that there are close interactions between motor terminals and the muscle precursor cells at the earliest stages of muscle formation and surgical denervation compromises further development of adult muscles. Here we describe a nerve/muscle coculture system that allows further exploration of this critical developmental interaction. Muscle precursor cells derived from the developing thoracic legs of early pupae and cultured in the presence of neurons assumed a spindle-like morphology and fused to form multinucleate contractile myotubes. Contractile fibers did not form in cultures of muscle precursor cells alone. In the presence of neurons the rate of bromodeoxyuridine (BrdU) incorporation into myonuclei was significantly enhanced, suggesting that neurons promote the proliferation of myogenic cells. This effect was not unique to thoracic leg motoneurons of the early pupal stage, in that larval thoracic neurons as well as neurons from the pupal brain or abdominal ganglia were also effective at enhancing BrdU incorporation and the formation of contractile muscle fibers. Medium conditioned by neurons was ineffective at promoting BrdU incorporation, and in cocultures BrdU incorporation was enhanced only in regions of physical overlap between neurons and muscle precursor cells, suggesting that a very close-range interaction was involved. Tetrodotoxin-sensitive neuronal activity was not required for the effect on muscle development, but fixed neurons were ineffective. The insect steroid hormone 20-hydroxyecdysone enhanced BrdU incorporation into the nuclei of myogenic cells in both the presence and the absence of neurons. The results suggest that both neurons and ecdysteroids play an important regulatory role in adult muscle development, at least in part by enhancing the proliferation of myogenic cells
The Developmentally Restricted Pattern of Expression in the Male Germ Line of a MurineCyclin A, Cyclin A2,Suggests Roles in Both Mitotic and Meiotic Cell Cycles

AbstractWe have isolated cDNAs encoding a murine cyclin A, designatedcyclinA2,and have examined itsin vivoexpression at the level of both mRNA and protein, with particular focus on the male germ line.Cyclin A2is expressed in embryos and in a variety of adult tissues, including the testis. In the testis, however, a striking cellular specificity of expression was observed. At both the DNA and protein levels, the predominant sites ofcyclin A2expression were in the germ line stem cells, the spermatogonia, and in highest levels in preleptotene spermatocytes, cells in which premeiotic DNA synthesis occurs. The concurrent localization ofcyclin A2mRNA and protein further suggested thatcyclin A2is regulated at the level of transcription in these cells. The observed cellular specificity ofcyclin A2expression is consistent with its function during mitosis in the stem cell stage of this lineage, while the restricted meiotic stage localization suggests function in G1/S or S but not in the meiotic divisions per se
Initial Organization of Neurons and Tracts in the Embryonic Mouse Fore- and Midbrain

AbstractWe investigated the potential role of rostral–caudal and dorsal–ventral subdivisions of the early rostral brain by relating these subdivisions to the early patterning of neuron cell bodies and their axon projections. The earliest neurons were mapped using the lipophilic axon tracers diI and diO on embryos fixed on embryonic days 9.5–10.5 (E9.5–E10.5); neuromeric boundaries were marked by diO. The tracts were small in number, were organized orthogonally (2 dorsal–ventral and 4 rostral–caudal), and originated from groups of cell bodies which we term “sources.” Two parallel longitudinal axon systems, one dorsal (the tract of the postoptic commissure and the mesencephalic tract of the trigeminal nerve) and one ventral (the mammillotegmental tract and the medial longitudinal fasciculus), projected caudally from the prosencephalon into the rhombencephalon. We argue that the dorsal longitudinal pathway marked the boundary between the alar and basal plates along the entire neuraxis. The dorsal–ventral axons coursed circumferentially and either crossed the midline (forming the posterior and ventral tegmental commissures) or turned caudally without crossing the midline. The dorsal–ventral axons were not generally restricted to the interneuromeric boundaries, as others have suggested. Earlier, all neighboring neurons projected their axons together; later, nearby neurons projected into different pathways. Some tracts originated in single neuromeres, while other tracts had origins in two or more neuromeres. The dorsal longitudinal axons altered course at several of the borders, but the ventral longitudinal axons did not. In summary, the early subdivisions appeared to influence some, but not all, aspects of tract formation
Ontogeny of Basal and Regulated Secretion from POMC Cells of the Developing Anterior Lobe of the Rat Pituitary Gland

AbstractProopiomelanocortin (POMC)-producing cells are present in both the anterior (AL) and intermediate (IL) lobes of the adult rat pituitary. Both cell types are derived from a single embryonic rudiment, Rathke's pouch, and synthesize the same hormone precursor, POMC, but differ in the pattern of precursor processing and regulation of peptide secretion. Here we have used the reverse hemolytic plaque assay to determine the ontogeny of basal and regulated secretion by AL POMC cells. Basal secretion of β-endorphin was first observed at Embryonic Day 13.5 (e13.5), the age when POMC-derived peptides were first detected immunocytochemically. Peptide secretion stimulated by corticotropin releasing hormone (CRH; 10−8M) was first detected at e15.5. The CRH-stimulated secretion involved two different components: (1) an increase in the amount of hormone secreted per cell (increase in plaque size) as well as (2) an increase in the number of plaque-producing cells. The greatest stimulation by CRH was observed at e16.5, a time when AL POMC mRNA levels increase significantly and when CRH neurons are first detected immunocytochemically in the hypothalamus. CRH-stimulated secretion, but not basal release, was inhibited by preincubation with dexamethasone (DEX; 10−6M) as early as e15.5, while the dopamine agonist ergocryptine (ERG; 10−6M) did not alter basal or CRH-stimulated release at any age studied. These results demonstrate that AL POMC cells are capable of hormone secretion as early as POMC peptides are first detected immunocytochemically and that these cells respond in an adult-like manner to physiological regulators soon after their initial appearance. Moreover, these responses remain unaltered during the early postnatal stress-nonresponsive period, suggesting that deficits at this time must lie at a level other than the corticotroph. Taken together, these results show that AL POMC cells possess functional regulatory receptor systems prior to maturation of the hypothalamic–hypophyseal portal system