pinecone/core-2020-05-10-deduplication huggingface dataset:
Independent Regulatory Elements in the Upstream Region of theDrosophila β3 tubulinGene (βTub60D) Guide Expression in the Dorsal Vessel and the Somatic Muscles

AbstractTheβ3 tubulingene (βTub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for theDrosophilamyocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of β3 tubulin inD-mef2mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearbycisregulatory elements. These data show that the upstream control region of theβ3 tubulingene is an early target of the D-MEF2 transcriptional activator
EctopicorthodenticleExpression Alters Segment Polarity Gene Expression but Not Head Segment Identity in theDrosophilaEmbryo

AbstractThe cephalic gap genes specify anterior head development in theDrosophilaembryo. However, the mechanisms of action of these genes remain poorly understood. Here, we focused on the cephalic gap geneorthodenticle (otd),which establishes a specific region of the anterior head. It has been proposed thatotdacts in a combinatorial fashion with the cephalic gap genesempty spiracles (ems)andbuttonhead (btd)to assign segmental identities in this region. To test this model, we used a heat-inducible transgene to generate pulses of ubiquitousotdexpression during embryonic development. Ectopicotdexpression caused significant defects in head formation, including the duplication of sensory structures derived fromotd-dependent segments. However, these defects do not appear to result from the transformation of head segment identities predicted by the combinatorial model. Instead, they correlate with specific regulatory effects ofotdon the expression of the segment polarity genesengrailed (en)andwingless (wg).Ectopicotdexpression also caused the loss of head structures derived from the maxillary segment, which lies posterior to theotddomain. We show that this effect is associated withotdrepression of the homeotic selector geneDeformed (Dfd)
Development of the Avian Iris and Ciliary Body: The Role of Activin and Follistatin in Coordination of the Smooth-to-Striated Muscle Transition

AbstractAlthough general principles have been established in the regulation of vetebrate organogenesis, the specific molecules responsible for such signaling are just being identified. We have studied differentiation in the avian iris and ciliary body which undergoes a transition from smooth to striated muscle. Using heterochronic cocultures, we have found that striated muscle differentiation in pretransition (E8) cells is induced by midtransition (E11) cells through a secreted and soluble activity. In addition, contact-mediated mechanisms among pretransition cells prevented precocious striated muscle differentiation. We have tested the role of activin and its antagonist follistatin, as candidate regulators of this muscle transition. Activin induced smooth muscle differentiation while repressing striated muscle development. Conversely, follistatin promoted the emergence of striated muscle, while inhibiting smooth muscle differentiation. Significantly, secreted follistatin activity was found to increase during the smooth-to-striated muscle transition. Moreover, the striated muscle inducing activity from midtransition iris and ciliary body cell conditioned medium was depleted with an activin-affinity column which binds follistatin. These results suggest that activin and follistatin coordinate differentiation in the avian iris and ciliary body
cMeso-1, a Novel bHLH Transcription Factor, Is Involved in Somite Formation in Chicken Embryos

AbstractThe segmentation of somites from the paraxial mesoderm is a crucial event in vertebrate embryonic development; however, the mechanisms underlying this process are not well understood. In a yeast two-hybrid screen we have identified the novel basic-helix-loop-helix (bHLH) protein cMeso-1 which is expressed in the presomitic mesoderm of early chicken embryos. Initially the gene is activated in the epiblast and transcripts concentrate later in and around the primitive streak. When the segmental plate is laid down the cMeso-1 expression domain successively retracts toward the caudal end but a second domain appears in bilateral stripes in the anterior paraxial mesoderm. This highly dynamic domain of cMeso-1 transcripts demarcates the area immediately posterior to the next prospective pair of somites in cyclic waves which apparently correspond to the formation of new somites. Loss of cMeso-1 function by antisense RNA or oligonucleotides results in severe attenuation of somitogenesis suggesting that it plays an important role in setting up the segmentation process. The dynamic and periodically reiterated expression of cMeso-1 along the anteroposterior axis is not dependent on anterior structures or the propagation of a signal along the anteroposterior axis but seems to follow an intrinsic patterning program which is already set up in the segmental plate
Dispensability of the Actin-Binding Site and Spectrin Repeats for Targeting Sarcomeric α-Actinin into Maturing Z Bandsin Vivo:Implications forin VitroBinding Studies

AbstractTo explore the roles of specific domains of sarcomeric α-actinin (s-α-actinin) in the assembly and maintenance of striated myofibrils, myogenic cultures were transfected with four MYC-tagged s-α-actinin peptides. They were: (1) full-length sarcomeric α-actinin, (2) an N-terminal deletion that removed the actin-binding site only (MYC/A−), (3) a peptide that consisted of the actin-binding site only (MYC/A+), and (4) an N-terminal deletion that removed the EF-hands and titin-binding domains (MYC/EFT−). While cytotoxic in replicating myogenic cells, as they were in PtK2 cells, the four MYC peptides were not cytotoxic in postmitotic myotubes. In myotubes each of the four different MYC peptides were promptly and selectively incorporated into normal Z bands. The incorporation of MYC/A−, MYC/A+, and MYC/EFT−into Z bands suggests that (a) the actin-binding site, (b) the spectrin-repeats believed to be responsible for anti-parallel dimerization, and (c) the C-terminal EF-hands and titin-binding domains are each dispensable for targeting s-α-actinin/MYC peptides into Z bands. These findings could not have been predicted from the behavior of α-actinin (a) in binding assays in cell-free systems or (b) when expressed in transfected nonmuscle cells
MouseAlx3:Anaristaless-like Homeobox Gene Expressed during Embryogenesis in Ectomesenchyme and Lateral Plate Mesoderm

AbstractMouseAlx3is a homeobox gene that is related to theDrosophila aristalessgene and to a group of vertebrate genes includingPrx1, Prx2, Cart1,andAlx4.The protein encoded contains a diverged variant of a conserved peptide sequence present near the carboxyl terminus of at least 15 different paired-class-homeodomain proteins.Alx3is expressed in mouse embryos from 8 days of gestation onward in a characteristic pattern, predominantly in neural crest-derived mesenchyme and in lateral plate mesoderm. We detected prominent expression in frontonasal head mesenchyme and in the first and second pharyngeal arches and some of their derivatives. High expression was also seen in the tail and in many derivatives of the lateral plate mesoderm including the limbs, the body wall, and the genital tubercle. aristaless-related genes likeAlx3, Cart1,andPrx2are expressed in overlapping proximodistal patterns in the pharyngeal arches. Similar, but more lateral patterns have been described for theDistal-less-related (Dlx) genes. Intriguingly, expression and to some extent function ofaristalessandDistal-lessinDrosophilaalso have overlapping as well as complementary aspects.Alx3was localized to chromosome 3, near the droopy-ear (de) mutation
GFP Expression inDrosophilaTissues: Time Requirements for Formation of a Fluorescent Product

AbstractWe analyzed the acquisition of fluorescence of a GFP fusion protein expressed from gene constructs designed to regulate either the time of translation of the protein or the time of gene transcription. Both gene constructs expressed a fusion protein with wild-type GFP appended to the N-terminus of the Bicoid (Bcd) transcription factor. One gene construct, P[gfp-bcd] expressed an mRNA that was initially translated at the time of egg deposition. GFP–Bcd fluorescence could first be detected in anterior nuclei in the embryo at syncytial blastoderm stage, within 1.5–2.5 h after translation commenced. The second gene construct, P[hs-gfp-bcd], utilized thehsp70promoter to regulate the induction of gene expression. In larval salivary glands, GFP–Bcd fluorescence could first be detected in nuclei after a 40-min recovery period following a 10-min heat shock induction of gene expression. These estimates of the time required for the acquisition of a fluorescent GFP product inDrosophilatissues are less than suggested by previously published reports
Timing of Gene Expression and Oolemma Localization of Mouse α6and β1Integrin Subunits during Oogenesis

AbstractThe sperm antigen fertilin α/β and the integrin complex α6β1present on the oolemma are two of the most promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach the size of 25–30 μm in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine the timing of (1) gene expression of α6and β1integrins and (2) localization of these integrin subunits on the plasma membrane in primordial germ cells and in oocytes during oogenesis. We found that both α6and β1genes are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p.c., during oocyte growth, and in ovulated eggs. The alternatively spliced isoform α6Bis expressed from 10.5 d.p.c., whereas α6Abegins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants.In situimmunodetection of α6or β1shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5 d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals and only when oocytes during growth reach the size of about 25–30 μm in diameter does the fluorescence become homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an involvement of α6and β1integrins in sperm–egg fusion
The Dynamics and Regulation of Mesenchymal Cell Fusion in the Sea Urchin Embryo

AbstractCell–cell fusion occurs in a wide variety of developmental contexts, yet the mechanisms involved are just beginning to be elucidated. In the sea urchin embryo, primary mesenchyme cells (PMCs) fuse to form syncytial filopodial cables within which skeletal spicules are deposited. Taking advantage of the optical transparency and ease of micromanipulation of sea urchin embryos, we have developed methods for directly observing the dynamics of PMC fusionin vivo.A fraction of the PMCs was labeled with fluorescent dextran and transfer of the dye to unlabeled PMCs was followed by time-lapse, fluorescence microscopy. Fusion was first detected about 2 h after PMCs began to migrate within the blastocoel. Fusion proceeded in parallel with the assembly of the PMC ring pattern and was complete by the early gastrula stage. The formation of a single, extensive PMC syncytium was confirmed by DiI labeling of fixed embryos. When single micromeres were isolated and cultured in unsupplemented seawater, they divided and their progeny underwent fusion. This shows that the capacity to fuse is autonomously programmed in the micromere–PMC lineage by the 16-cell stage. PMC transplantations at late embryonic stages revealed that these cells remain fusion-competent long after their fusion is complete. At late stages, other mesenchyme cells (blastocoelar cells) are also present within the blastocoel and are migrating and fusing with one another. Fusion-competent blastocoelar cells and PMCs come into contact but do not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. When secondary mesenchyme cells convert to a skeletogenic fate they alter their fusogenic properties and join the PMC syncytium, as shown by transfer of fluorescent dextran. Our analysis has provided a detailed picture of the cellular basis and regulation of mesodermal cell fusion and has important implications regarding molecular mechanisms that underlie fusion
Ventral and Lateral Regions of the Zebrafish Gastrula, Including the Neural Crest Progenitors, Are Established by abmp2b/swirlPathway of Genes

AbstractA bone morphogenetic protein (BMP) signaling pathway is implicated in dorsoventral patterning inXenopus.Here we show that three genes in the zebrafish,swirl, snailhouse,andsomitabun,function as critical components within a BMP pathway to pattern ventral regions of the embryo. The dorsalized mutant phenotypes of these genes can be rescued by overexpression ofbmp4, bmp2b,an activated BMP type I receptor, and the downstream functioningSmad1gene. Consistent with a function as a BMP ligand,swirlfunctions cell nonautonomously to specify ventral cell fates. Chromosomal mapping ofswirland cDNA sequence analysis demonstrate thatswirlis a mutation in the zebrafishbmp2bgene. Interestingly, our analysis suggests that the previously described nonneural/neural ectodermal interaction specifying the neural crest occurs through a patterning function ofswirl/bmp2bduring gastrulation. We observe a loss in neural crest progenitors inswirl/bmp2bmutant embryos, whilesomitabunmutants display an opposite, dramatic expansion of the prospective neural crest. Examination of dorsally and ventrally restricted markers during gastrulation reveals a successive reduction and reciprocal expansion in nonneural and neural ectoderm, respectively, insnailhouse, somitabun,andswirlmutant embryos, withswirl/bmp2bmutants exhibiting almost no nonneural ectoderm. Based on the alterations in tissue-specific gene expression, we propose a model wherebyswirl/bmp2bacts as a morphogen to specify different cell types along the dorsoventral axis
Target-Dependent Development of the Vesicular Acetylcholine Transporter in Rodent Sweat Gland Innervation

AbstractDescriptive studies have delineated a developmental change in neurotransmitter phenotype from noradrenergic to cholinergic in the sympathetic innervation of sweat glands in rodent footpads. Transplantation and culture experiments provide evidence that interactions with the target tissue induce this change. Recent studies with an antiserum that recognizes the vesicular acetylcholine transporter (VAChT) suggest, however, that the development of cholinergic function in sympathetic neurons, including those that innervate sweat glands, occurs prior to and does not require target contact. To clarify these apparently contradictory findings, we directly compared the appearance of VAChT immunoreactivity in the sympathetic neurons that innervate sweat glands with the time that axons contact this target. We find that VAChT immunoreactivity is not detectable in either the axons or cell bodies of sweat gland neurons until several days after target innervation. Before and during VAChT acquisition, the developing sweat gland innervation contains vesicular stores of catecholamines. An analysis of mutant mice that lack sweat glands was undertaken to determine whether VAChT expression requires target interactions and revealed that VAChT does not appear in the absence of glands. These findings, together with previous studies, confirm the target dependence of cholinergic function in the sympathetic neurons that innervate sweat glands
Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

AbstractOocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity
Developmental Expression of the 84-kDa ODF Sperm Protein: Localization to both the Cortex and Medulla of Outer Dense Fibers and to the Connecting Piece

AbstractOuter dense fibers (ODF) are specialized cytoskeletal elements of the mammalian sperm tail which are composed of several prominent proteins. We previously reported the isolation of a cDNA (111-450) encoding a putative 84-kDa ODF protein. Here we demonstrate by independent cDNA isolations and by translational/immunoprecipitation of testicular mRNAs using anti-ODF 84 antibodies that 111-450 cDNA encodes the 84-kDa protein. We then analyzed the testicular expression of the ODF 84 mRNA and protein. Riboprobes generated from the clones recognized four testicular-specific transcripts of 1.6, 2.2, 2.4, and 2.8 kb in both rat and bull of which the immunoprecipitable product of the 2.4-kb mRNA comigrates with ODF 84 protein. Developmental Northerns indicated that the 2.2- and 2.4-kb mRNAs are first transcribed during meiotic prophase while the other two species are first expressed in round spermatids. The levels of all the transcripts steadily increased up to elongated spermatids. Immunocytochemistry revealed that the anti-84 reactive ODF proteins were synthesized and assembled in the cytoplasm of elongated spermatids (steps 9–18) with peak activity occurring in step 16 of spermiogenesis. Immunogold labeling was selective to the assembling ODF and connecting piece of the tail and to granulated bodies of the cytoplasmic lobe. Both the striated collar and capitulum of the connecting piece were immunolabeled as well as the basal plate of the implantation fossa. A combination of pre- and postembedding immunogold labeling provided evidence that the 84-kDa ODF protein is localized to both the cortex and medulla of the ODF in contrast to the sole medullary localization of the major 27-kDa ODF protein. Thus the 84-kDa ODF protein, encoded by the 2.4 transcript, is translationally regulated, packaged after synthesis into granulated bodies, assembled in a proximal to distal direction along the axoneme and may interact by means of leucine zippers specifically with the 27-kDa ODF protein during assembly. Its localization to both the cortex and medulla of the ODF, as opposed to exclusive medullary localization of the 27-kDa ODF protein, and the presence of two leucine zippers, only one of which interacts with the 27-kDa ODF, suggests that it could act as a link between proteins of the two regions of the ODF
lagC-Null andgbf-Null Cells Define Key Steps in the Morphogenesis ofDictyosteliumMounds

AbstractThe transition to multicellularity is a key feature of theDictyosteliumlife cycle, and two genes,gbfandlagC, are known to play pivotal roles in regulating this developmental switch.lagC-null andgbf-null cells fail to induce cell-type-specific genes ordinarily expressed during multicellular development. The null mutants also share a similar morphological phenotype: mutant cells repeatedly aggregate to form a loose mound, disperse, and reform a mound, rather than proceeding to form a tip. To characterize defects in morphogenesis in these mutants, we examined cell motion in the mutant mounds. In analogy with the failed transition in gene expression, we found thatlagC-null andgbf-null mounds failed to make a morphogenetic transition from random to rotational motion normally observed in the parent strain. One reason for this was the inability of the mutant mounds to establish a single, dominant signaling-wave center. This defect oflagC-null orgbf-null cells could be overcome by the addition of adenosine, which alters cAMP signaling, but then even in the presence of apparently normal signaling waves, cell motility was still aberrant. This motility defect, as well as the signaling-wave defect, could be overcome inlagC-null cells by overexpression of GBF, suggesting thatlagCis dispensable if GBF protein levels are high enough. This set of morphogenetic defects that we have observed helps define key steps in mound morphogenesis. These include the establishment of a dominant signaling-wave center and the capacity of cells to move directionally within the cell mass in response to guidance cues