pinecone/core-2020-05-10-deduplication huggingface dataset:
A Germline-Specific Splicing Generates an Extended Ovo Protein Isoform Required for Drosophila Oogenesis

AbstractMost regulatory genes are employed multiple times to control different processes during development. The Drosophila Ovo/Shavenbaby (Svb) transcription factor is required both for germline and epidermal differentiation, two roles also found for its ortholog m-ovo1 in mice. In Drosophila, these two distinct functions are contributed by separate control regions directing the expression of Ovo/Svb in the germline (ovo) and soma (svb), respectively. We report here that alternative splicing represents an additional level of the regulation of Ovo/Svb functional specificity. Characterization of the ovoD1rv23 mutation revealed that the intragenic insertion of a novel retrotransposon,romano, inactivates ovo without altering svb. We provide evidence that this insertion disrupts a germline-specific alternative exon, exon 2b, which encodes a 178-amino-acid internal extension (2B). While both isoforms, Ovo+2B and Ovo−2B, accumulate during oogenesis, only Ovo+2B is able to fulfill germinal ovo functions. Ovo−2B is unable, even when overexpressed, to fully rescue oogenic defects resulting from the absence of wild type ovo product. By contrast, either Ovo+2B or Ovo−2B germline protein can substitute for Svb in the epidermis. Our results emphasize the specific features of splicing in the germline, and reveal its functional importance for the control of ovo/svb-dependent ovarian and epidermal differentiation
The Single Amphioxus Mox Gene: Insights into the Functional Evolution of Mox Genes, Somites, and the Asymmetry of Amphioxus Somitogenesis

AbstractMox genes are members of the “extended” Hox-cluster group of Antennapedia-like homeobox genes. Homologues have been cloned from both invertebrate and vertebrate species, and are expressed in mesodermal tissues. In vertebrates, Mox1 and Mox2 are distinctly expressed during the formation of somites and differentiation of their derivatives. Somites are a distinguishing feature uniquely shared by cephalochordates and vertebrates. Here, we report the cloning and expression of the single amphioxus Mox gene. AmphiMox is expressed in the presomitic mesoderm (PSM) during early amphioxus somitogenesis and in nascent somites from the tail bud during the late phase. Once a somite is completely formed, AmphiMox is rapidly downregulated. We discuss the presence and extent of the PSM in both phases of amphioxus somitogenesis. We also propose a scenario for the functional evolution of Mox genes within chordates, in which Mox was co-opted for somite formation before the cephalochordate–vertebrate split. Novel expression sites found in vertebrates after somite formation postdated Mox duplication in the vertebrate stem lineage, and may be linked to the increase in complexity of vertebrate somites and their derivatives, e.g., the vertebrae. Furthermore, AmphiMox expression adds new data into a long-standing debate on the extent of the asymmetry of amphioxus somitogenesis
Directionality of Heart Looping: Effects of Pitx2c Misexpression on Flectin Asymmetry and Midline Structures

AbstractA critical regulatory laterality gene expressed in the left side of the straight heart tube during development is Pitx2, which when mutated in humans underlies Rieger's Syndrome. Previously reported results have indicated that, when using gain-of-function and loss-of function approaches of the chick cPitx2c isoform, this results in randomization of heart looping. To determine whether Pitx2c misexpression affects downstream morphogenesis by altering the expression of specific proteins in the myocardium during looping, after experimental manipulations, we analyzed immunohistochemically for the extracellular matrix molecule flectin that normally is expressed predominantly in the left lateral plate mesoderm (LPM) and left side of the straight chick heart tube before and during looping. We show here that the left-side predominance of flectin is due to a delay in the timing of expression in one heart field vs the other. Experimental results indicate that misexpression of Pitx2c in the heart fields using antisense or retroviral delivery perturbs the normal temporal pattern of flectin expression in the left LPM relative to the right: abnormally leftward looping hearts show predominate right-sided flectin expression in the dorsal mesocardial regions around the foregut ventral midline. Additionally, Pitx2c misexpression affects the positioning of the developing foregut to more lateral areas, either on the right or left side of the embryonic midline. The position of the heart with respect to the embryo midline is defined by the position of the foregut. Incubating embryos in the presence of flectin antibody caused randomization of heart looping or no looping
lon-1 Regulates Caenorhabditis elegans Body Size Downstream of the dbl-1 TGFβ Signaling Pathway

AbstractIn Caenorhabditis elegans, two well-characterized TGFβ signaling cascades have been identified: the Small/Male tail abnormal (Sma/Mab) and Dauer formation (Daf) pathways. The Sma/Mab pathway regulates body size morphogenesis and male tail development. The ligand of the pathway, dbl-1, transmits its signal through two receptor serine threonine kinases, daf-4 and sma-6, which in turn regulate the activity of the Smads, sma-2, sma-3, and sma-4. In general, Smads have been shown to both positively and negatively regulate the transcriptional activity of downstream target genes in various organisms. In C. elegans, however, target genes have remained elusive. We have cloned and characterized lon-1, a gene with homology to the cysteine-rich secretory protein (CRISP) family of proteins. lon-1 regulates body size morphogenesis, but does not affect male tail development. lon-1 is expressed in hypodermal tissues, which is the focus of body size determination, similar to sma-2, sma-4, and sma-6. Using genetic methods, we show that lon-1 lies downstream of the Sma/Mab signaling cascade and demonstrate that lon-1 mRNA levels are up-regulated in sma-6-null mutant animals. This provides evidence that lon-1 is negatively regulated by Sma/Mab pathway signaling. Taken together, these data identify lon-1 as a novel downstream target gene of the dbl-1 TGFβ-like signaling pathway
Disruption of Testis Cords by Cyclopamine or Forskolin Reveals Independent Cellular Pathways in Testis Organogenesis

AbstractMost studies to date indicate that the formation of testis cords is critical for proper Sertoli cell differentiation, inhibition of germ cell meiosis, and regulation of Leydig cell differentiation. However, the connections between these events are poorly understood. The objective of this study was to dissect the molecular and cellular relationships between these events in testis formation. We took advantage of the different effects of two hedgehog signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effect of Dhh−/− on formation of testis cords without influencing Sertoli cell differentiation. However, they exhibited different effects on other cellular events during testis development. Treatment with cyclopamine did not affect inhibition of germ cell meiosis and mesonephric cell migration but caused defects in Leydig cell differentiation. In contrast, forskolin treatment induced germ cell meiosis, inhibited mesonephric cell migration, and had no effect on Leydig cell differentiation. By carefully contrasting the different effects of these two hedgehog inhibitors, we demonstrate that, although formation of testis cords and development of other cell types normally take place in a tightly regulated sequence, each of these events can occur independent of the others
Modeling a Hox Gene Network in Silico Using a Stochastic Simulation Algorithm

AbstractThe amount of molecular information that has been gathered about Hox cis-regulatory mechanisms allows us to take the next important step: integrating the results and constructing a higher-level model for the interaction and regulation of the Hox genes. Here, we present the results of our investigation into a cis-regulatory network for the early Hox genes. Instead of using conventional differential equation approaches for analyzing the system, we have adopted the use of a stochastic simulation algorithm (SSA) to model the network. The model allows us to track in detail the behavior of each component of a biochemical pathway and to produce computerized movies of the time evolution of the system that is a result of the dynamic interplay of these various components. The simulation is able to reproduce key features of the wild-type pattern of gene expression, and in silico experiments yield results similar to their corresponding in vivo experiments. This analysis shows the utility of using stochastic methods to model biochemical networks. In addition, the model has suggested several intriguing new results that are currently being investigated in vivo
Control of Cardiac Development by an Evolutionarily Conserved Transcriptional Network

AbstractFormation of the heart is dependent on an intricate cascade of developmental decisions. Analysis of the molecules and mechanisms involved in the specification of cardiac cell fates, differentiation and diversification of cardiac muscle cells, and morphogenesis and patterning of different cardiac cell types has revealed an evolutionarily conserved network of signaling pathways and transcription factors that underlies these processes. The regulatory network that controls the formation of the primitive heart in fruit flies has been elaborated upon to form the complex multichambered heart of mammals. We compare and contrast the mechanisms involved in heart formation in fruit flies and mammals in the context of a network of transcriptional interactions and point to unresolved questions for the future
Elements of Transcription Factor Network Design for T-Lineage Specification

AbstractThe complex spectrum of cell types produced in mammalian hematopoiesis can be understood as the output of highly combinatorial transcription factor action. The generation of multiple diverse combinations of transcription factors from the common starting state of the hematopoietic stem cell must be explained through the cross-regulatory interactions of these transcription factors at several levels. Here, the operation of such a network is addressed through a focus on murine T cell development, where we have recently established regulatory linkages between GATA-3 and PU.1 and multiple other factors essential to this differentiation pathway. The action of both essential/rate-limiting factors and factors with effects that shift qualitatively with dose and time of action can be traced through the regulatory interaction network. Hypothetical models are proposed to indicate the network nodes that are differentially activated in normal T cell lineage progression and in cells diverted to other potential fates
Sonic hedgehog Signaling from the Urethral Epithelium Controls External Genital Development

AbstractExternal genital development begins with formation of paired genital swellings, which develop into the genital tubercle. Proximodistal outgrowth and axial patterning of the genital tubercle are coordinated to give rise to the penis or clitoris. The genital tubercle consists of lateral plate mesoderm, surface ectoderm, and endodermal urethral epithelium derived from the urogenital sinus. We have investigated the molecular control of external genital development in the mouse embryo. Previous work has shown that the genital tubercle has polarizing activity, but the precise location of this activity within the tubercle is unknown. We reasoned that if the tubercle itself is patterned by a specialized signaling region, then polarizing activity may be restricted to a subset of cells. Transplantation of urethral epithelium, but not genital mesenchyme, to chick limbs results in mirror-image duplication of the digits. Moreover, when grafted to chick limbs, the urethral plate orchestrates morphogenetic movements normally associated with external genital development. Signaling activity is therefore restricted to urethral plate cells. Before and during normal genital tubercle outgrowth, urethral plate epithelium expresses Sonic hedgehog (Shh). In mice with a targeted deletion of Shh, external genitalia are absent. Genital swellings are initiated, but outgrowth is not maintained. In the absence of Shh signaling, Fgf8, Bmp2, Bmp4, Fgf10, and Wnt5a are downregulated, and apoptosis is enhanced in the genitalia. These results identify the urethral epithelium as a signaling center of the genital tubercle, and demonstrate that Shh from the urethral epithelium is required for outgrowth, patterning, and cell survival in the developing external genitalia
Colony Stimulating Factor-1 Is Required to Recruit Macrophages into the Mammary Gland to Facilitate Mammary Ductal Outgrowth

AbstractMammary gland development initiates postnatally with the development of terminal end buds (TEBs) at the end of the rudimentary ducts. These grow out through the fat pad and bifurcate to lay down the rudimentary ductal tree. At the initiation of their development, TEBs recruit to their surrounding stroma a substantial population of macrophages. Using mice homozygous for a null mutation in the gene for the macrophage growth factor, colony stimulating factor-1 (CSF-1), that are severely depleted in macrophages, we demonstrated that CSF-1-regulated macrophages are required for normal branching morphogenesis in the mammary gland. However, these mice have a pleiotropic phenotype as a result of the generalized macrophage deficiency. To test that the effect of the mutation observed in the mammary gland was organ-autonomous, we developed a tetracycline-binary system whereby CSF-1 was specifically expressed in the mammary epithelium under the regulation of the MMTV-promoter. This restored mammary macrophage populations but not those in other tissues and corrected the branching morphogenesis defect. Inhibition of CSF-1 expression by tetracycline treatment for varying periods suggested that CSF-1-regulated macrophages are required throughout early mammary gland development. These data show that macrophages acting locally are required for branching morphogenesis of the mammary gland
Membrane-Bound Neuregulin1 Type III Actively Promotes Schwann Cell Differentiation of Multipotent Progenitor Cells

AbstractMany steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation
Spermatogonia-Dependent Expression of Testicular Genes in Mice

AbstractSpermatogenesis is initiated by the interaction of germ cells and somatic cells in seminiferous tubules. We used cDNA microarrays and representational difference analysis to identify genes that are expressed in the testis of the jsd/jsd mutant mouse, which contains only type A spermatogonial germ cells and Sertoli cells, but not in the testis of the W/Wv mutant mouse, where Sertoli cells but few germ cells are present. We isolated 20 known genes and 4 novel genes, including 2 genes encoding lipocalin family members (prostaglandin D synthetase and 24p3) and 2 tumor suppressors (protein tyrosine phosphatase TD14 and Sui1). All 24 of thesejsd/jsd-derived genes were highly expressed in the cryptorchid testis as well as in the jsd/jsd testis. This indicates that their selective expression is not directly caused by the as-yet-uncharacterized jsd gene product, but is rather correlated to the cessation of spermatogonial differentiation. In situ hybridization analysis and flow cytometric sorting followed by reverse transcriptase-PCR revealed that these genes are expressed in both the spermatogonial germ cells and the somatic cells in the developing gonads and adult testes. As the mRNAs of these jsd/jsd-derived genes were barely detectable in the W/Wv testis, we propose that early spermatogonial germ cells regulate the expression of a group of testicular genes
The Planar Cell Polarity Gene Strabismus Regulates Convergence and Extension and Neural Fold Closure in Xenopus

AbstractWe cloned Xenopus Strabismus (Xstbm), a homologue of the Drosophila planar cell or tissue polarity gene. Xstbm encodes four transmembrane domains in its N-terminal half and a PDZ-binding motif in its C-terminal region, a structure similar to Drosophila and mouse homologues. Xstbm is expressed strongly in the deep cells of the anterior neural plate and at lower levels in the posterior notochordal and neural regions during convergent extension. Overexpression of Xstbm inhibits convergent extension of mesodermal and neural tissues, as well as neural tube closure, without direct effects on tissue differentiation. Expression of Xstbm(ΔPDZ-B), which lacks the PDZ-binding region of Xstbm, inhibits convergent extension when expressed alone but rescues the effect of overexpressing Xstbm, suggesting that Xstbm(ΔPDZ-B) acts as a dominant negative and that both increase and decrease of Xstbm function from an optimum retards convergence and extension. Recordings show that cells expressing Xstbm or Xstbm(ΔPDZ-B) fail to acquire the polarized protrusive activity underlying normal cell intercalation during convergent extension of both mesodermal and neural and that this effect is population size-dependent. These results further characterize the role of Xstbm in regulating the cell polarity driving convergence and extension in Xenopus
Definition of Regulatory Network Elements for T Cell Development by Perturbation Analysis with PU.1 and GATA-3

AbstractPU.1 and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Rα expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects
Mnb/Dyrk1A Is Transiently Expressed and Asymmetrically Segregated in Neural Progenitor Cells at the Transition to Neurogenic Divisions

AbstractThe Minibrain (Mnb) gene encodes a new family of protein kinases that is evolutionarily conserved from insects to humans. In Drosophila, Mnb is involved in postembryonic neurogenesis. In humans, MNB has been mapped within the Down's Syndrome (DS) critical region of chromosome 21 and is overexpressed in DS embryonic brain. In order to study a possible role of Mnb on the neurogenesis of vertebrate brain, we have cloned the chick Mnb orthologue and studied the spatiotemporal expression of Mnb in proliferative regions of the nervous system. In early embryos, Mnb is expressed before the onset of neurogenesis in the three general locations where neuronal precursors are originated: neuroepithelia of the neural tube, neural crest, and cranial placodes. Mnb is transiently expressed during a single cell cycle of neuroepithelial progenitor (NEP) cells. Mnb expression precedes and widely overlaps with the expression of Tis21, an antiproliferative gene that has been reported to be expressed in the onset of neurogenic divisions of NEP cells. Mnb transcription begins in mitosis, continues during G1, and stops before S-phase. Very interestingly, we have found that Mnb mRNA is asymmetrically localized during the mitosis of these cells and inherited by one of the sibling cells after division. We propose that Mnb defines a transition step between proliferating and neurogenic divisions of NEP cells
Cnidarian and Bilaterian Promoters Can Direct GFP Expression in Transfected Hydra

AbstractComplete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken β-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated
Distinct Patterns of Downstream Target Activation Are Specified by the Helix–Loop–Helix Domain of Proneural Basic Helix–Loop–Helix Transcription Factors

AbstractBoth gain- and loss-of-function analyses indicate that proneural basic/helix–loop–helix (bHLH) proteins direct not only general aspects of neuronal differentiation but also specific aspects of neuronal identity within neural progenitors. In order to better understand the function of this family of transcription factors, we have used hormone-inducible fusion constructs to assay temporal patterns of downstream target regulation in response to proneural bHLH overexpression. In these studies, we have compared two distantly related Xenopus proneural bHLH genes, Xash1 and XNgnr1. Our findings indicate that both Xash1 and XNgnr1 induce expression of the general neuronal differentiation marker, N-tubulin, with a similar time course in animal cap progenitor populations. In contrast, these genes each induce distinct patterns of early downstream target expression. Both genes induce expression of the HLH-containing gene, Xcoe2, at early time points, but only XNgnr1 induces early expression of the bHLH genes, Xath3 and XNeuroD. Structure:function analyses indicate that the distinct pattern of XNgnr1-induced downstream target activation is linked to the XNgnr1 HLH domain, demonstrating a novel role for this domain in mediating the differential function of individual members of the proneural bHLH gene family