pinecone/core-2020-05-10-deduplication huggingface dataset:
Msx2 Is a Transcriptional Regulator in the BMP4-Mediated Programmed Cell Death Pathway

AbstractHomeobox-containing genes play an important role in patterning processes that occur during embryogenesis. Programmed cell death is a key process during pattern formation. The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, we see that P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with its proposed role in cell death in the embryo. Our data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process
Embryonic and Fetal Myogenic Programs Act through Separate Enhancers at the MLC1F/3F Locus

AbstractEmbryonic and fetal stages of skeletal muscle development are characterized by the differential expression of a number of muscle-specific genes. These include the products of independent promoters at the fast myosin light chain 1F/3F locus. In the mouse embryo MLC1F transcripts accumulate in embryonic skeletal muscle from E9, 4–5 days before high-level accumulation of MLC3F transcripts. A 3′ enhancer can activate MLC1F and MLC3F promoters in differentiated muscle cellsin vitroand in transgenic mice; both promoters, however, are activated at the time of MLC1F transcript accumulation. We now demonstrate the presence of a second muscle-specific enhancer at this locus, located in the intron separating the MLC1F and MLC3F promoters. Transgenic mice containing the intronic, but lacking the 3′ enhancer, express high levels of annlacZreporter gene from the MLC3F promoter in adult fast skeletal muscle fibers. In contrast to the 3′ enhancer, the intronic element is inactive both in embryonic muscle cellsin vivoand in embryonic myocyte cultures. The intronic enhancer is activated at the onset of fetal development in both primary and secondary muscle fibers, at the time of endogenous MLC3F transcript accumulation. Late-activated MLC3F transgenes thus provide a novelin totomarker of fetal myogenesis. These results suggest that temporal regulation of transcription at the MLC1F/3F locus is controlled by separate enhancers which are differentially activated during embryonic and fetal development
Involvement of the Bombyx Scr gene in development of the embryonic silk gland

Homeotic selector genes determine the identity of each segment and induce the differentiation of segment-specific organs. To analyze how the silk glands of the lepidopteran, Bombyx mori, develop, we cloned and identified two genes that encode the homeodomain and its flanking regions identical to the corresponding regions of Deformed and Sex combs reduced. Using in situ hybridization and immunohistochemistry, we analyzed the expression patterns of these genes during Bombyx embryogenesis. Bombyx Deformed is expressed in the mandibular and maxillary segments, whereas expression of Bombyx Sex combs reduced is first limited to the labial segment and at later stages extended to the anterior part of the prothoracic segment. The expression of Bombyx Sex combs reduced then disappears from the invaginating placodes of silk glands where expression of Bombyx fork head/SGF-1 follows. In the Nc/Nc mutant embryos, which lack the 3′ end region of Bombyx Antennapedia, in addition to the expression in the labial segment, the Bombyx Sex combs reduced is expressed ectopically in the thoracic and abdominal regions, and Bombyx fork head/SGF-1 is also ectopically expressed in the T1, T2, and T3 segments, resulting the ectopic induction of the silk gland invaginations. These results suggest that Bombyx homeobox genes such as the Bombyx Deformed and Sex combs reduced are associated with determination of the segment identities and Bombyx Sex combs reduced is involved in the induction of silk gland development
Transdifferentiation of muscle to electric organ: Regulation of muscle-specific proteins is independent of patterned nerve activity

Transdifferentiation is the conversion of one differentiated cell type into another. The electric organ of fishes transdifferentiates from muscle but little is known about how this occurs. To begin to address this question, we studied the expression of muscle- and electrocyte-specific proteins with immunohistochemistry during regeneration of the electric organ. In the early stages of regeneration, a blastema forms. Blastemal cells cluster, express desmin, fuse into myotubes, and then express α-actinin, tropomyosin, and myosin. Myotubes in the periphery of the blastema continue to-differentiate as muscle; those in the center grow in size, probably by fusing with each other, and lose their sarcomeres as they become electrocytes. Tropomyosin is rapidly down-regulated while desmin, α-actinin, and myosin continue to be diffusely expressed in newly formed electrocytes despite the absence of organized sarcomeres. During this time an isoform of keratin that is a marker for mature electrocytes is expressed. One week later, the immunoreactivities of myosin disappears and α-actinin weakens, while that of desmin and keratin remain strong. Since nerve fibers grow into the blastema preceding the appearance of any differentiated cells, we tested whether the highly rhythmic nerve activity associated with electromotor input plays a role in transdifferentiation and found that electrocytes develop normally in the absence of electromotor neuron activity
The ability to organize sperm DNA into functional chromatin is acquired during meiotic maturation in murine oocytes

Following fertilization of meiotically mature eggs, the chromatin of the sperm becomes biochemically and structurally remodeled within the egg cytoplasm. Despite the essential role of the paternal genome during embryogenesis, little is known of when the activities that regulate this chromatin remodeling appear during oogenesis. To determine whether these activities were acquired during meiotic maturation, we inseminated maturing oocytes of mice shortly after germinal vesicle breakdown. As previously shown, insemination at this stage did not activate the maturing oocytes, which became arrested at metaphase II. Immunofluorescent analysis revealed that at 1 hr postinsemination the sperm chromatin was dispersed and contained protamines but was devoid of core histones H2B and H3. At 4 hr postinsemination, both protamine and core histones were detectable on the sperm chromatin. By 8 hr postinsemination protamines were absent, and histones stained maximally. The appearance of immunoreactive histones was correlated with a morphological transition of the sperm chromatin from the dispersed to a condensed state, which suggests that the assembly of the histones reflected modification of the chromatin to a somatic-like state in which it was competent to respond to the metaphase-promoting factor activity of the oocyte. Both the assembly of histones and chromatin condensation were reversibly blocked when protein synthesis was inhibited, indicating that the remodeling process required proteins synthesized during maturation. Injection of core histones into protein synthesis-inhibited oocytes failed to induce condensation of the sperm chromatin, which implies that correct remodeling requires synthesis during maturation of nonhistone proteins. To test the functional capacity of remodeled sperm chromatin, maturing oocytes were inseminated, allowed to continue maturation for 17 hr and then parthenogenetically activated. Following activation, the sperm-derived chromatin as well as that of the oocyte became decondensed within pronuclei and underwent DNA replication, indicating that sperm chromatin remodeled in maturing oocyte cytoplasm was functionally normal. When the postinsemination incubation time was reduced to 11 hr; however, neither the female nor the male pronuclei underwent DNA replication, implying that factors synthesized late during maturation are required for DNA replication after activation. Taken together, these results indicate that the ability to organize sperm DNA into functional somatic-like chromatin develops in oocytes during meiotic maturation, requires proteins synthesized during maturation, and can be expressed independently of activation
A conserved cluster of homeodomain binding sites in the mouse Hoxa-4 intron functions in Drosophila embryos as an enhancer that is directly regulated by Ultrabithorax

The evolutionary conservation of the homeodomains suggests that their in vivo DNA binding sites may also be conserved between vertebrates and invertebrates. The regulatory function of the mouse Hoxa-4 and Hoxb-4 introns were analyzed in Drosophila since they both contain a cluster of three homeodomain binding sites, the HB1 element, which was also found in the introns of other Hox genes ranging from fish to humans as well as in the Ultrabithorax (Ubx) and decapentaplegic (dpp) genes of Drosophila. The enhancer of the Hoxa-4 intron was found to respond to several homeobox genes activating a lacZ reporter gene in particular cells of the epidermis in Drosophila embryos. The enhancer activity was found to be similar to previously described autoregulatory elements of Deformed (Dfd), the Drosophila homolog of Hoxa-4, but additional expression was observed in more posterior segments activated by Ubx and repressed by abdominal-A (abd-A). Point mutations in the homeodomain binding sites in HB1 abolished the enhancer activity. A second site suppression experiment showed that UBX interacts directly with the HB1 element. When the HB1 element in the Hoxa-4 intron was replaced by that of the mesodermal enhancer of dpp, which was previously shown to be directly controlled by Ubx, Ubx-dependent activation was retained, but repression by abd-A was lost. The same result was obtained when the third binding site of HB1 was altered, suggesting that this site is responsible for abd-A-dependent repression. Finally, deletion of potential cofactor binding sites flanking the HB1 element that are also conserved in the medaka, chicken, and mouse genes revealed that they are important for enhancer function in Drosophila and that the Dfd-dependent and the Ubx-dependent expression requires different sites. The evolutionary and functional conservation of the HB1 elements indicates that not only the homeodomains but also some of their in vivo binding sites are conserved between vertebrates and invertebrates
Muscle sensory innervation patterns in embryonic chick hindlimbs following dorsal root ganglion reversal

Previous studies suggest that sensory innervation of muscles is patterned by motor innervation. Muscle afferent projections mirror motor projections after various experimental manipulations and muscle afferents fail to project to muscle in the absence of motoneurons. It is not known, however, whether muscle afferents are specified with respect to the corresponding motoneurons or target muscles. To test this possibility we rotated three to four segments of neural crest in St. 15–17 chick embryos, leaving motoneurons intact, to reverse the rostrocaudal order of dorsal root ganglia (DRGs) T7/LS1–LS3. This caused sensory neurons derived from one segmental level to grow into the limb with motor axons from a different level. The resulting innervation patterns were assessed at St. 28–37 by injecting DiI and DiA into the sartorius and femorotibialis muscles or into the spinal cord and DRG. DiI labeling of crest prior to rotation showed that DRGs in the operated region were derived primarily from rotated cells. Muscle afferents from rotated DRGs grew to muscles in accord with their new rostrocaudal position, together with “inappropriate” motor axons from the same segmental level. The segmental distribution of sensory neurons innervating each muscle was more widespread in embryos operated at older than at younger stages. In contrast, sensory axons projected to the appropriate muscles in accord with their embryonic origin when segments of the whole neural tube, including motoneurons, were rotated, as reported previously. Thus, sensory neurons do not appear to be selectively matched with motoneurons or target muscles at stages when the corresponding motoneurons have clear identities
Regulated exocytosis and sequential construction of the extracellular matrix surrounding the sea urchin zygote

After fertilization most eggs become surrounded by a complex extracellular matrix. This study examines those matrix assembly processes that are triggered by fertilization of the sea urchin egg. The study uses antibodies that identify five different storage compartments in the egg. These compartments release their protein contents in a highly regulated fashion to assemble and modify the extraembryonic layers. The exocytosis sequence begins with a fertilization wave that progresses from the site of sperm entry and elevates the fertilization envelope above a water-filled perivitelline space. The immediate surface of the zygote then becomes covered by a newly secreted hyaline layer. Prior to fertilization some of the antigens are localized to cortical granules. Others are found in “basal laminar vesicles” that are released in a wave beginning at about 30 sec, or roughly at the same time as cortical granule exocytosis. The remaining antigens are exocytosed with a rather precise timing, but with a delay of several to tens of minutes relative to the first wave of exocytosis. “Apical vesicles,” so named because antigens from this class are preferentially exocytosed toward the apical cell surface of polarized cells, include antigens that are exocytosed beginning at about 5 min postfertilization. The fourth compartment, named “echinonectin vesicles” release echinonectin, a protein that is deposited to the inner side of the hyaline layer. Surface staining of echinonectin is first detected about 10–15 min following sperm contact. Finally, maternal cadherin, which is stored in yet a fifth distinct compartment, is not detected on the surface until at least 30 min following fertilization. The data are also consistent with the notion that the tightly regulated timing of exocytosis contributes to the ordered assembly of the hyaline layer and elevation of the fertilization envelope. Finally, two of the vesicle classes continue to exocytose after the cells become polarized. In polarized cells apical and basal laminar antigens are trafficked toward opposite sides of the same cell after passing through the same trans-Golgi network-like compartment
Identification of Metalloprotease/Disintegrins inXenopus laevisTestis with a Potential Role in Fertilization

AbstractProteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell–cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins fromXenopus laevistestis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibitX. laevisfertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein–receptor interactions are necessary for fertilization to occur
Postembryonic Development of the Midline Glia in the CNS ofDrosophila:Proliferation, Programmed Cell Death, and Endocrine Regulation

AbstractThe development ofDrosophilamidline glia during larval and pupal stages was characterized by localizing β-gal expression in enhancer trap lines, as well as with BrdU incorporation and pulse–chase experiments. At hatching about 40 to 50 glial cells are present along the midline of the ventral nerve cord (2 to 3 dorsal and 1 to 2 ventral cells per neuromere). The cells proliferate during the third larval instar and spread dorsoventrally within the midline, increasing in number to about 230 or more (around 20 cells per neuromere). Cell divisions cease shortly after pupariation, and the cells persist for the first half of pupal life with no apparent changes in numbers or positions. Between 50 and 80% of metamorphosis, however, virtually all of the midline glia undergo programmed cell death. Tissue culture experiments indicate that the peak of ecdysteroids occurring at pupariation is required for the cessation of proliferation of midline glia and their subsequent degeneration. Midline glia in central nervous systems (CNS) cultured with low or no ecdysteroids survive and continue to divide, whereas they cease proliferating and later degenerate with high ecdysteroids levels. The midline glial may play a role during CNS metamorphosis similar to that of their progenitors in the embryo, in stabilizing outgrowing neurites that cross or run along the midline
Genetic Analysis of Stomatogastric Nervous System Development inDrosophilaUsing Enhancer Trap Lines

AbstractThe stomatogastric nervous system (SNS) ofDrosophila melanogasteris a small, simply organized neural circuitry which innervates the anterior enteric system. It is responsible for regulating the passage of food through the pharynx and esophagus and into the midgut. Here we show that the development of the SNS is amenable to genetic dissection. We screened lines from a P-element mutagenesis, selecting those withlacZreporter gene expression and/or a phenotype in the SNS, associated glia, and garland cells. We report a collection of expression patterns and mutant phenotypes among lines found to have a mutation in genes required for the establishment of the larval SNS. Our results indicate that SNS development depends on pattern organizer genes including components of the Ras/Raf pathway
M-Spondin, a Novel ECM Protein Highly Homologous to Vertebrate F-Spondin, Is Localized at the Muscle Attachment Sites in theDrosophilaEmbryo

AbstractThe muscle attachment site (MAS) inDrosophilaprovides a unique and excellent model system to study the mechanism of cell–matrix adhesion in developing organisms. Here, we report on the isolation and characterization of a novel extracellular matrix (ECM) molecule localized at the MAS, encoded by theM-spondin(mspo) gene. M-spondin protein contains a thrombospondin type I repeat (TSR) previously found in a variety of ECM molecules. Furthermore, it shares two conserved domains with F-spondin, a vertebrate ECM molecule with TSRs. The presence of TSR(s) and the two homologous domains thus defines a novel gene family of ECM molecules. ThemspomRNA was expressed by a large subset of muscles in the embryonic body wall. Secreted M-spondin protein diffused and eventually became immobilized at the MAS in late embryos. When expressed in S2 cells, the protein was secreted and became concentrated in the matrix on the surface of the culture dish. Genetic analysis revealed that both deletion mutants and misexpression mutants suffered no obvious developmental defects. We propose that M-spondin, although its function is redundant, is a component of the ECM and mediates mechanical linkage between the muscles and apodemes
Neuroepithelial Stem Cells from the Embryonic Spinal Cord: Isolation, Characterization, and Clonal Analysis

AbstractAdherent cultures of E10.5 rat neuroepithelial cells (NEP cells) from the caudal neural tube require FGF (fibroblast growth factor) and CEE (chick embryo extract) to proliferate and maintain an undifferentiated phenotype in culture. Epidermal growth factor (EGF) does not support E10.5 NEP cells in adherent culture and NEP cells do not form EGF-dependent neurospheres. NEP cells, however, can be grown as FGF-dependent neurospheres. NEP cells express nestin and lack all lineage-specific markers for neuronal and glial sublineages, retain their pleuripotent character over multiple passages, and can differentiate into neurons, astrocytes, and oligodendrocytes when plated on laminin in the absence of CEE. In clonal culture, NEP cells undergo self-renewal and generate colonies that vary in size from single cells to several thousand cells. With the exception of a few single-cell clones, all other NEP-derived clones contain more than one identified phenotype, with over 40% of the colonies containing A2B5, β-111 tubulin, and GFAP-immunoreactive cells. Thus, NEP cells are multipotent and capable of generating multiple neural derivatives. NEP cells also differentiate into motoneurons immunoreactive for choline acetyl transferase (ChAT) and the low-affinity neurotrophin receptor (p75) in both mass and clonal culture. Double labeling of clones for ChAT and glial, neuronal, or oligodendrocytic lineage markers shows that motoneurons always arose in mixed cultures with other differentiated cells. Thus, NEP cells represent a common progenitor for motoneurons and other spinal cord cells. The relationship of NEP cells with other neural stem cells is discussed