pinecone/core-2020-05-10-deduplication huggingface dataset:
In Vitro Development of Growing Oocytes from Fetal Mouse Oocytes: Stage-Specific Regulation by Stem Cell Factor and Granulosa Cells

AbstractThe development of follicles in the mammalian ovary involves a bidirectional communication system between the follicular cells and oocyte that is now beginning to be characterized. Little is known about the mechanisms underlying the beginning of the oocyte growth and the acquisition of the competence to resume meiosis by the growing oocyte. In the present study, we devised a multistep culture system for mouse oocytes obtained from 15.5- to 16.5-days postcoitum embryos (mean diameter ± SEM, 9.7 ± 1.3 μm), allowing three stages of the oocyte growth to be identified: (i) an early stage in which the oocyte growth is induced by direct stimulation of a soluble growth factor, namely stem cell factor (SCF), independent of the formation of gap junctions with granulosa cells; (ii) a second phase in which the oocyte growth depends on the combined action of SCF and contacts with granulosa cells; and (iii) a third phase of granulosa cell-dependent, SCF-independent growth. At each stage, key events of oocyte development and differentiation, such as the c-kit reexpression, the early zona pellucida assembly, and the beginning of follicologenesis, were observed to occur independently by the presence of SCF. At the end of the in vitro growing phases, lasting 18–20 days, oocytes reached a size (50 ± 2.5 μm) and a chromatin differentiation (stage I-II) equivalent to those of 9- to 10-day-old preantral oocytes and were unable to complete the growth phase. About 50% of the in vitro-grown oocytes were induced to resume meiosis by okadaic acid (OA) treatment. However, a significant fraction of them (48%) showed inability to maintain the chromosome condensation in M-phase. When in vitro-grown oocytes were treated with UO126, a specific MEK inhibitor that prevents activation of mitogen-activated protein kinases (ERK-1 and ERK-2), for 1 h before, during, and following OA treatment, only 22% of oocytes underwent germinal vesicle breakdown after 24 h from the OA treatment. These studies demonstrate that SCF alone can induce the onset of the oocyte growth. This is, however, not sufficient to fully activate the mechanisms governing the acquisition of the meiotic competence previously described as a 15-day oocyte-autonomous clock starting at the onset of growth. The inability of oocytes to progress into the last stages of growth and the lack of synchrony between nuclear and cytoplasm maturation showed by a subset of them resemble the characteristics of oocytes from connexin-37- and -43-deficient mice and indicate the preantral/antral transition point as a critical stage of oocyte development requiring the coordinated differentiation of the oocyte with granulosa cells and the maintenance of adequate communication between these two cell types to assure the correct oocyte meiotic maturation
Joint Development in the Drosophila Leg: Cell Movements and Cell Populations

AbstractFlexible joints separate the rigid sections of the insect leg, allowing them to move. In Drosophila, the initial patterning of these joints is apparent in the larval imaginal discs from which the adult legs will develop. Here, we describe the later patterning and morphogenesis of the joints, which occurs after pupariation (AP). In the tibial/tarsal joint, the apodeme insertion site provides a fixed marker for the boundary between proximal and distal joint territories (the P/D boundary). Cells on either side of this boundary behave differently during morphogenesis. Morphogenesis begins with the apical constriction of distal joint cells, about 24 h AP. Distal cells then become columnar, causing distal tissue nearest the P/D boundary to fold into the leg. In the last stage of joint morphogenesis, the proximal joint cells closest to the P/D boundary align and elongate to form a “palisade” (a row of columnar cells) over the distal joint cells. The proximal and distal joint territories are characterised by the differential organisation of cytoskeletal and extracellular matrix proteins, and by the differential expression of enhancer trap lines and other gene markers. These markers also define a number of more localised territories within the pupal joint
The Expression of the let-7 Small Regulatory RNA Is Controlled by Ecdysone during Metamorphosis in Drosophila melanogaster

AbstractIn Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects
Functional Comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 Homeoboxes

AbstractA number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, we describe the results of two homeobox swaps. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5′ cluster positions were generally dominant over more 3′ homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development
Cooperation of Hoxa5 and Pax1 Genes during Formation of the Pectoral Girdle

AbstractHox and Pax transcription factors are master regulators of skeletal and organ morphogenesis. Some skeletal malformations encountered in Hoxa5 mutants are shared by the undulated (un) mice, which bear a point mutation in the Pax1 gene. To investigate whether Hoxa5 and Pax1 act in common pathways during skeletal development, we analyzed Hoxa5;un compound mutants. Our genetic studies show that Hoxa5 and Pax1 cooperate in the vertebral patterning of the cervicothoracic transition region and in acromion morphogenesis. The dynamics of expression of Hoxa5 and Pax1 in the pectoral girdle region suggest that both genes function in a complementary fashion during acromion formation. Whereas Pax1 is required for the recruitment of acromion precursor cells, Hoxa5 may provide regional cues essential for the correct formation of the acromion by ensuring Pax1 expression at the proper time and position during morphogenesis of the pectoral girdle. Hoxa5 also has a distinctive role in specifying the fate of perichondrial and chondrogenic cell lineages in a Sox9-dependent way
Efficient Recombination in Diverse Tissues by a Tamoxifen-Inducible Form of Cre: A Tool for Temporally Regulated Gene Activation/Inactivation in the Mouse

AbstractIn recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ERTM) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ERTM is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration
Zic1 Promotes the Expansion of Dorsal Neural Progenitors in Spinal Cord by Inhibiting Neuronal Differentiation

AbstractThe role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differention is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick
Enhanced Branching Morphogenesis in Mammary Glands of Mice Lacking Cell Surface β1,4-Galactosyltransferase

AbstractDevelopment of the mammary gland is influenced both by the systemic hormonal environment and locally through cell–cell and cell–extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of β1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell–ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin α1 and α3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin γ2 was elevated 2.3-fold. The expression and cleavage of laminin γ2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in γ2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin γ2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system
Egg Activation at Fertilization: Where It All Begins

AbstractA centrally important factor in initiating egg activation at fertilization is a rise in free Ca2+ in the egg cytosol. In echinoderm, ascidian, and vertebrate eggs, the Ca2+ rise occurs as a result of inositol trisphosphate-mediated release of Ca2+ from the endoplasmic reticulum. The release of Ca2+ at fertilization in echinoderm and ascidian eggs requires SH2 domain-mediated activation of a Src family kinase (SFK) and phospholipase C (PLC)γ. Though some evidence indicates that a SFK and PLC may also function at fertilization in vertebrate eggs, SH2 domain-mediated activation of PLCγ appears not to be required. Much work has focused on identifying factors from sperm that initiate egg activation at fertilization, either as a result of sperm–egg contact or sperm–egg fusion. Current evidence from studies of ascidian and mammalian fertilization favors a fusion-mediated mechanism; this is supported by experiments indicating that injection of sperm extracts into eggs causes Ca2+ release by the same pathway as fertilization
Interaction of the Essential Drosophila Nuclear Protein YA with P0/AP3 in the Cytoplasm and in Vitro: Implications for Developmental Regulation of YA's Subcellular Location

AbstractThe Drosophila nuclear lamina protein YA is essential for the transition from female meiosis to embryo mitosis. Its localization and, hence, function is under developmental and cell cycle controls. YA protein is hyperphosphorylated and cytoplasmic in ovaries. Upon egg activation, YA is partially dephosphorylated and acquires the ability to enter nuclei. Its function is first detected at this time. To investigate the cytoplasmic retention machinery that keeps YA from entering nuclei, we used affinity chromatography and blot overlay assays to identify cytoplasmic proteins that associate with YA. Drosophila P0/AP3, a ribosomal protein that is also an apurinic/apyrimidinic endonuclease, binds to YA in ovary and embryo cytoplasms. P0 and YA bind specifically and directly in vitro and are present in a 20S complex in the cytoplasmic extracts. YA protein can be phosphorylated by MAPK, but not by p34Cdc2 kinase, in vitro. This phosphorylation increases YA's binding to P0. We propose that the P0-containing 20S cytoplasmic complex retains hyperphosphorylated ovarian YA in the cytoplasm. In response to egg activation, YA is partially dephosphorylated and its binding to the 20S complex is reduced. Hence, some YA dissociates from the complex and enters nuclei. Consistent with this model, decreasing P0 levels partially suppress a hypomorphic Ya mutant allele
Xenopus Cdc42 Regulates Convergent Extension Movements during Gastrulation through Wnt/Ca2+ Signaling Pathway

AbstractRho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. We have isolated the Xenopus homolog of Rho GTPase Cdc42 and examined its potential role during gastrulation movements in early Xenopus embryos. XCdc42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCdc42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCdc42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-α, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCdc42, implying that XCdc42 acts downstream of the Wnt/Ca2+ signaling pathway involving PKC activation. XCdc42 also lies downstream of XWnt-5a in the regulation of Ca2+-dependent cell adhesion. Taken together, our results suggest that XCdc42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca2+ pathway
Cardiac-Specific Activity of an Nkx2–5 Enhancer Requires an Evolutionarily Conserved Smad Binding Site

AbstractHeart formation in vertebrates and fruit flies requires signaling by bone morphogenetic proteins (BMPs) to cardiogenic mesodermal precursor cells. The vertebrate homeobox gene Nkx2–5 and its Drosophila ortholog, tinman, are the earliest known markers for the cardiac lineage. Transcriptional activation of tinman expression in the cardiac lineage is dependent on a mesoderm-specific enhancer that binds Smad proteins, which activate transcription in response to BMP signaling, and Tinman, which maintains its own expression through an autoregulatory loop. Here, we show that an evolutionarily conserved, cardiac-specific enhancer of the mouse Nkx2–5 gene contains multiple Smad binding sites, as well as a binding site for Nkx2–5. A single Smad site is required for enhancer activity at early and late stages of heart development in vivo, whereas the Nkx2–5 site is not required for enhancer activity. These findings demonstrate that Nkx2–5, like tinman, is a direct target for transcriptional activation by Smad proteins; however, the independence of this Nkx2–5 enhancer of Nkx2–5 binding suggests a fundamental difference in the transcriptional circuitry for activation of Nkx2–5 and tinman expression during cardiogenesis in vertebrates and fruit flies
Nkx-2.5 Gene Induction in Mice Is Mediated by a Smad Consensus Regulatory Region

AbstractIn the forming vertebrate heart, bone morphogenetic protein signaling induces expression of the early cardiac regulatory gene nkx-2.5. A similar regulatory interaction has been defined in Drosophila embryos where Dpp signaling mediated by the Smad homologues Mad and Medea directly regulates early cardiac expression of tinman. A conserved cluster of Smad consensus binding sequences was identified in early cardiac regulatory sequences of the mouse nkx-2.5 gene. The importance of the nkx-2.5 Smad consensus region in early cardiac gene expression was examined in transgenic mice and in cultured mouse embryos. In transgenic mice, deletion of the Smad consensus region delays induction of embryonic ΔSmadnkx-2.5/lacZ gene expression during early heart formation. Induction of ΔSmadnkx-2.5/lacZ expression is also delayed in the outflow tract myocardium and visceral mesoderm. Targeted mutation of the three Smad consensus sequences inhibited nkx-2.5/lacZ expression in the cardiac crescent, demonstrating a specific requirement for the Smad consensus sites in early cardiac gene induction. Cultured ΔSmadnkx-2.5/lacZ transgenic mouse embryos also exhibit delayed induction of transgene expression. In the four-chambered heart, deletion of the Smad consensus region resulted in expanded ΔSmadnkx-2.5/lacZ transgene expression. Thus, the nkx-2.5 Smad consensus region can have positive or negative regulatory function, depending on the developmental context and cellular environment
The IGF Pathway Regulates Head Formation by Inhibiting Wnt Signaling in Xenopus

AbstractThe insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating head formation in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of β-catenin. Thus, the IGF pathway is required for head formation during embryogenesis