pinecone/core-2020-05-10-deduplication huggingface dataset:
Connexin31-Deficiency in Mice Causes Transient Placental Dysmorphogenesis but Does Not Impair Hearing and Skin Differentiation

AbstractMutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3−/−) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3+/−), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3−/− embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3−/− embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3−/− mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse
Dermal Fibroblast-Derived Growth Factors Restore the Ability of β1 Integrin-Deficient Embryonal Stem Cells to Differentiate into Keratinocytes

AbstractEmbryonal stem (ES) cells that are homozygous null for the β1 integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/β1-null chimeric mice. The failure of β1-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, deepidermized human dermis (DED) we showed that epidermal basement membrane did not induce β1-null ES cells to undergo keratinocyte differentiation and did not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also had no effect. However, when human dermal fibroblasts were incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increased dramatically, and small groups of keratin 14-positive cells differentiated from β1-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and β1-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFα all stimulated differentiation of keratin 14-positive β1-null cells, and KGF and FGF10 were shown to be produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells were much less pronounced, suggesting that the concentrations of inducing factors already present in the medium were not limiting for wild-type cells. We conclude that the lack of β1 integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation
Regulation of dharma/bozozok by the Wnt Pathway

AbstractThe zebrafish homeobox gene dharma/bozozok (boz) is required for the formation and/or function of the Nieuwkoop center and the subsequent induction of the Spemann organizer. dharma is expressed soon after the midblastula transition in the dorsal blastomeres and the dorsal yolk syncytial layer (YSL). We found that the expression of dharma was upregulated or ectopically induced by misexpression of a Wnt protein and cytoplasmic components of the Wnt signaling pathway and downregulated by the expression of dominant-negative Tcf3. A 1.4-kbp fragment of the dharma promoter region contains consensus sequences for Tcf/Lef binding sites. This promoter region recapitulated the Wnt-dependent and dorsal dharma expression pattern when it was fused to luciferase or GFP. Deletion and point mutant analyses revealed that the Tcf/Lef binding sites were required to drive this expression pattern. These data established that dharma/boz functions between the dorsal determinants-mediated Wnt signals and the formation of the Nieuwkoop center
Paternal Chromosome Incorporation into the Zygote Nucleus Is Controlled by maternal haploid in Drosophila

Abstractmaternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternal chromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia
Temporal and Spatial Effects of Sonic Hedgehog Signaling in Chick Eye Morphogenesis

AbstractProper dorsal–ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal–distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal–ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium
Autocrine Expression and Ontogenetic Functions of the PACAP Ligand/Receptor System during Sympathetic Development

AbstractThe superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor (PAC1) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via PAC1 receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via PAC1 receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular Ca2+, the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis
Growth Cones Utilize both Widespread and Local Directional Cues in the Zebrafish Brain

AbstractThe distribution of cues that provide directional information for specific growth cones in the zebrafish brain was functionally assayed by transplanting epiphysial neurons to ectopic locations in the embryonic brain followed by determining the pathways taken by the donor axons. Epiphysial axons normally first extend ventrally from their position in the dorsal diencephalon and then turn and extend anteriorly in the ventral diencephalon. When transplanted to ectopic sites at other axial levels of the brain, where in principle the axons could extend in any direction, epiphysial axons consistently extended ventrally. Furthermore, following initial ventral extension ectopic epiphysial axons turned randomly in the anterior and posterior directions. These results suggest that the cues for ventral extension are widely distributed along the rostrocaudal axis of the zebrafish brain, but the cues for subsequent anterior extension are restricted to the site where the epiphysial axons normally turn longitudinally
The Golgi Apparatus Segregates from the Lysosomal/Acrosomal Vesicle during Rhesus Spermiogenesis: Structural Alterations

AbstractThe acrosome is an acidic secretory vesicle containing hydrolytic enzymes that are involved in the sperm's passage across the zona pellucida. Imaging of the acrosomal vesicle and the Golgi apparatus in live rhesus monkey spermatids was accomplished by using the vital fluorescent probe LysoTracker DND-26. Concurrently, the dynamics of living spermatid mitochondria was visualized using the specific probe MitoTracker CMTRos and LysoTracker DND-26 detected the acrosomal vesicle from its formation through spermatid differentiation. LysoTracker DND-26 also labeled the Golgi apparatus in spermatogenic cells. In spermatocytes the Golgi is spherical and, in round spermatids, it is localized over the acrosomal vesicle, as confirmed by using polyclonal antibodies against Golgin-95/GM130, Golgin-97, and Golgin-160. Using both live LysoTracker DND-26 imaging and Golgi antibodies, we found that the Golgi apparatus is cast off from the acrosomal vesicle and migrates toward the sperm tail in elongated spermatids. The Golgi is discarded in the cytoplasmic droplet and is undetectable in mature ejaculated spermatozoa. The combined utilization of three vital fluorescent probes (Hoechst 33342, LysoTracker DND-26, and MitoTracker CMTRos) permits the dynamic imaging of four organelles during primate spermiogenesis: the nucleus, the mitochondria, the acrosomal vesicle, and the Golgi apparatus
P Granules in the Germ Cells of Caenorhabditis elegans Adults Are Associated with Clusters of Nuclear Pores and Contain RNA

AbstractThe germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA
A Novel Drosophila, mef2-Regulated Muscle Gene Isolated in a Subtractive Hybridisation-Based Molecular Screen Using Small Amounts of Zygotic Mutant RNA

AbstractIt is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. I have therefore undertaken a novel molecular screen in Drosophila in a systematic search for such genes. The approach utilised subtractive hybridisation coupled to directional cDNA library construction. Libraries were made from as little as 20 μg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increased the subtraction efficiency. It resulted in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure and then DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, that encodes a putative nuclear protein and fulfils the screen's aims is described. It is novel and its expression is mesoderm-specific, twi-dependent, and early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression revealed new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation
Snail-Related Transcriptional Repressors Are Required in Xenopus for both the Induction of the Neural Crest and Its Subsequent Migration

AbstractThe neural crest is a transient population of precursor cells that arises at the border between the neural plate and prospective epidermis in vertebrate embryos. The earliest known response to neural-crest-inducing signals is the expression of the zinc-finger transcription factors slug and snail. Although it is widely believed that these transcription factors play an essential role in neural crest development, relatively little is understood about their mechanism of action during this process. We have previously shown that overexpression of XSlug leads to expanded expression of neural crest markers and an excess of at least one neural crest derivative, melanocytes. In order to further investigate XSlug function, we overexpressed mutant constructs in which the DNA-binding domain was fused to either the activation domain from Gal4 or the repressor domain from Drosophila Engrailed. The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. In contrast, overexpression of either the activation domain fusion or the DNA-binding domain alone was found to inhibit XSlug function. Using a hormone-inducible inhibitory mutant, we show that inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration, demonstrating for the first time that this transcriptional repressor is required during multiple stages of neural crest development
BMPs Are Required at Two Steps of Limb Chondrogenesis: Formation of Prechondrogenic Condensations and Their Differentiation into Chondrocytes

AbstractFormation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205–217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes
Influence of FGF4 on Digit Morphogenesis during Limb Development in the Mouse

AbstractMuch of what we currently know about digit morphogenesis during limb development is deduced from embryonic studies in the chick. In this study, we used ex utero surgical procedures to study digit morphogenesis during mouse embryogenesis. Our studies reveal some similarities; however, we have found considerable differences in how the chick and the mouse autopods respond to experimentation. First, we are not able to induce ectopic digit formation from interdigital cells as a result of wounding or TGFβ-1 application in the mouse, in contrast to what is observed in the chick. Second, FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. We demonstrate with cell marking studies that this bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. We propose that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event. Finally, we discuss the relationship between the digit IV bifurcation restriction and the placement of the metapterygial axis in the evolution of the tetrapod limb
Translational Control of Cyclin B1 mRNA during Meiotic Maturation: Coordinated Repression and Cytoplasmic Polyadenylation

AbstractTranslational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3′UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3′UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration