pinecone/core-2020-05-10-deduplication huggingface dataset:
A Retinoic Acid-Inducible Modular Protease in Budding Ascidians

AbstractRetinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein–protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase–TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species
Suppression of Muscle Fate by Cellular Interaction Is Required for Mesenchyme Formation during Ascidian Embryogenesis

AbstractThe tadpole larva of the ascidian Halocynthia roretzi has several hundred mesenchyme cells in its trunk. Mesenchyme cells are exclusively derived from the B8.5 and B7.7 blastomere pairs of the 110-cell embryo. It has been believed that specification of mesenchyme cells is an autonomous process. In the present study, we have demonstrated that presumptive-mesenchyme blastomeres isolated from early 32-cell embryos did not express mesenchyme-specific features, whereas those isolated after the late 64-cell stage developed mesenchyme markers autonomously. Results of experiments involving coisolation and recombination of blastomeres showed that cellular interaction with adjacent presumptive-endoderm blastomeres during the late 32- and early 64-cell stages is required for mesenchyme formation. When such interaction was absent, the presumptive-mesenchyme blastomeres developed into muscle cells. Therefore, a signal from endoderm precursor blastomeres promotes mesenchyme fate, suppressing the muscle fate that is specified by ooplasmic muscle determinants. In Halocynthia, the muscle actin gene was precociously activated in mesenchyme–muscle precursor blastomeres at the 32-cell stage, and the mesenchyme and muscle fates were separated into two daughter blastomeres at the next cleavage. In presumptive-mesenchyme blastomeres at the 64-cell stage, expression of the muscle actin gene was immediately down-regulated by the signal from the neighboring endoderm precursor blastomeres. Thus, mesenchyme formation involves a novel mechanism of fate specification in ascidians, where formation of mesenchyme cells requires cellular interaction that suppresses muscle fate in the mesenchyme precursor blastomeres
Ascidian Tail Formation Requires caudal Function

AbstractAlthough the tail is one of the major characteristics of animals of the phylum Chordata, evolutionary aspects of the molecular mechanisms involved in its formation are not clear. To obtain insights into these issues, we have isolated and investigated the caudal gene of an ascidian, one of the lower animal groups among chordates. Ascidian caudal is expressed from the midgastrula stage onward in the lateral walls of the posterior neural tube cell lineage and also in the posterior epidermal cells from the neurula stage. Thus, ascidian caudal expression is restricted to the ectoderm of a tail-forming region throughout embryogenesis. Suppression of caudal function by an antisense oligonucleotide or a dominant negative construct caused inhibition of the cell movement required for tail formation. Overexpression of wild-type caudal mRNA in an ascidian animal cap, an animal half explant prepared at the eight-cell stage, caused elongation of the cap. Furthermore, Xenopus embryos injected with dominant negative ascidian caudal exhibited defects in elongation, suggesting a conserved caudal function among chordates. These results indicate that caudal function is required for chordate tail formation and may play a key role in its evolution
Loss of Ectodermal Competence for Lateral Line Placode Formation in the Direct Developing Frog Eleutherodactylus coqui

AbstractIn the direct-developing frog Eleutherodactylus coqui neuromasts and ganglia of the lateral line system never develop. We show here that this absence of the lateral line system, which is evolutionarily derived in anurans, is due to very early changes in development. Ectodermal thickenings, which are typical of lateral line placodes, and from which neuromasts and ganglion cells of the lateral line originate, never form in E. coqui, although other neurogenic placodes are present. Moreover, although NeuroD is expressed in the lateral line placodes of Xenopus laevis, corresponding expression sites are lacking in E. coqui. Heterospecific transplantation experiments show that axolotl ectoderm can be induced to form lateral line placodes after transplantation to E. coqui hosts but that E. coqui ectoderm does not form lateral line placodes on axolotl hosts. This suggests that the loss of the lateral line system in E. coqui is due to the specific loss of ectodermal competence to form lateral line placodes in response to inductive signals. Our results (1) indicate that the competence for lateral line placode formation is distinct and dissociable from the competence to form other neurogenic placodes and (2) support the idea that the lateral line system acts as a module in development and evolution
Embryonic Expression and Function of the Chemokine SDF-1 and Its Receptor, CXCR4

AbstractDirected cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events
Restricted Expression of Cardiac Myosin Genes Reveals Regulated Aspects of Heart Tube Assembly in Zebrafish

AbstractThe embryonic vertebrate heart is divided into two major chambers, an anterior ventricle and a posterior atrium. Although the fundamental differences between ventricular and atrial tissues are well documented, it is not known when and how cardiac anterior–posterior (A–P) patterning occurs. The expression patterns of two zebrafish cardiac myosin genes, cardiac myosin light chain 2 (cmlc2) and ventricular myosin heavy chain (vmhc), allow us to distinguish two populations of myocardial precursors at an early stage, well before the heart tube forms. These myocardial subpopulations, which may represent the ventricular and atrial precursors, are organized in a medial–lateral pattern within the precardiac mesoderm. Our examinations of cmlc2 and vmhc expression throughout the process of heart tube assembly indicate the important role of an intermediate structure, the cardiac cone, in the conversion of this early medial–lateral pattern into the A–P pattern of the heart tube. To gain insight into the genetic regulation of heart tube assembly and patterning, we examine cmlc2 and vmhc expression in several zebrafish mutants. Analyses of mutations that cause cardia bifida demonstrate that the achievement of a proper cardiac A–P pattern does not depend upon cardiac fusion. On the other hand, cardiac fusion does not ensure the proper A–P orientation of the ventricle and atrium, as demonstrated by the heart and soul mutation, which blocks cardiac cone morphogenesis. Finally, the pandora mutation interferes with the establishment of the early medial–lateral myocardial pattern. Altogether, these data suggest new models for the mechanisms that regulate the formation of a patterned heart tube and provide an important framework for future analyses of zebrafish mutants with defects in this process
Silencing of Developmental Genes in Hydra

AbstractNumerous developmental control genes have been isolated in a variety of organisms by either homology cloning or system-specific strategies. Functional genetic tests, however, are available for only a few model organisms and particularly are missing in a number of animals that occupy key positions for understanding the evolution of development and gene function. Double-stranded RNA-mediated interference (RNAi) opens a way to perform functional studies in such “nongenetic” organisms. Here we show that RNAi can be used to test the function of developmental genes in the cnidarian Hydra, a classical model for developmental studies. Introduction of double-stranded RNA corresponding to the head-specific gene ks1 caused strong depletion of ks1 transcripts. ks1 loss-of-function polyps exhibited severe defects in head formation, indicating an important role of ks1 in Hydra head development. Our results demonstrate for the first time efficient gene silencing in Hydra. RNAi provides an entry point for a variety of functional studies and a direct approach for analyzing the hierarchy of regulatory genes in Hydra, which until now has not been amenable to loss-of-function genetics
Role of the Amnioserosa in Germ Band Retraction of the Drosophila melanogaster Embryo

AbstractAs the germ band shortens in Drosophila melanogaster embryos, cell shape changes cause segments to narrow anteroposteriorly and to lengthen dorsoventrally. One of the genes required for this retraction process is the hindsight (hnt) gene. hnt encodes a nuclear Zinc-finger protein that is expressed in the extraembryonic amnioserosa and the endodermal midgut prior to and during germ band retraction (M. L. R. Yip, M. L. Lamka, and H. D. Lipshitz, 1997, Development 124, 2129–2141). Here we show, through analysis of hnt genetic mosaic embryos, that hnt activity in the amnioserosa—particularly in those cells that are adjacent to the epidermis—is necessary for germ band retraction. In hnt mutant embryos the amnioserosa undergoes premature cell death (L. C. Frank and C. Rushlow, 1996, Development 122, 1343–1352). We demonstrate that prevention of premature apoptosis in hnt mutants does not rescue retraction. Thus, failure of this process is not an indirect consequence of premature amnioserosal apoptosis; instead, hnt must function in a pathway that controls germ band retraction. We show that the Krüppel gene is activated by hnt in the amnioserosa while the Drosophila insulin receptor (INR) functions downstream of hnt in the germ band. We present evidence against a physical model in which the amnioserosa “pushes” the germ band during retraction. Rather, it is likely that the amnioserosa functions in production, activation, or presentation of a diffusible signal required for retraction
Synapse Formation and Agrin Expression in Stratospheroid Cultures from Embryonic Chick Retina

AbstractStratospheroids are three-dimensional cellular spheres which develop in vitro through the proliferation and differentiation of retinal neuroepithelial precursor cells. We investigated synapse formation in stratospheroids by analyzing the development of aggregates of synapse-associated molecules and of electron microscopically identifiable synaptic specializations. Our results show that the first aggregates of the GABAA receptor, the glycine receptor, and gephyrin appear in the inner plexiform layer after 8 days in culture simultaneously with the development of the first active zones and postsynaptic densities. In contrast, presynaptic molecules including synaptophysin could be detected in the inner plexiform layer before synaptogenesis, suggesting functions for these molecules in addition to neurotransmitter exocytosis at mature synapses. Similar to the retina in vivo, synapses were not found in the nuclear layers of stratospheroids. We also analyzed the isoform pattern, expression, and distribution of the extracellular matrix molecule agrin, a key regulator during formation, maintenance, and regeneration of the neuromuscular junction. In stratospheroids, several agrin isoforms were expressed as highly glycosylated proteins with an apparent molecular weight of approximately 400 kDa, similar to the molecular weight of agrin in the retina in vivo. The expression specifically of the neuronal isoforms of agrin was concurrent with the onset of synaptogenesis. Moreover, the neuronal agrin isoforms were exclusively found in the synapse-containing inner plexiform layer, whereas other agrin isoforms were associated also with the inner limiting membrane and with Müller glial cells. These results show that synapse formation is very similar in stratospheroids and in the retina in vivo, and they suggest an important role for agrin during CNS development
Interspecies Chimeric Sperm Lysins Identify Regions Mediating Species-Specific Recognition of the Abalone Egg Vitelline Envelope

AbstractAbalone sperm lysin is a nonenzymatic, 16-kDa protein that creates a hole in the egg vitelline envelope (VE) through which the sperm swims to fuse with the egg. The dissolution of isolated VE by lysin is species specific. Interspecies comparisons show that the most divergent region of lysin is the N-terminal segment of residues 1–12 which is always species-unique. The C-terminus and three internal segments are moderately variable between species, but not species unique. Analysis of nucleotide substitutions shows that lysin evolves rapidly by positive Darwinian selection, suggesting that there is adaptive value in altering its amino acid sequence. The results reported here, in which segments of lysin were exchanged between two species, prove by direct experimentation that the interspecies variable termini play major roles in the species-specific recognition between sperm lysin and the egg VE
The Early Expression Control of Xepsin by Nonaxial and Planar Posteriorizing Signals in Xenopus Epidermis

AbstractThe control mechanism of the anteroposterior axis specification in Xenopus epidermis was studied by comparing the expression of a novel anterior marker, Xepsin, with that of a panepidermal marker, type I keratin. Xepsin mRNA, which encodes a novel Xenopus serine protease, is transcribed zygotically with the expression peak in neurula stages. In normal development, its expression is limited to the anterior and anterior–dorsal portions within epidermis during neurula and tailbud stages, respectively. In UV-irradiated ventralized embryos (dorsoanterior index, DAI 0 and 1), an expression boundary for Xepsin is apparently formed within the epidermis. In contrast, Xepsin expression was observed throughout the epidermis in LiCl-treated dorsalized embryos (DAI 10), as seen from an expression pattern indistinguishable from that of type I keratin. These data suggest that posteriorizing signals which suppress the transcription of Xepsin are present in nonaxial regions and absent in the anterior dorsal mesoderm. That posteriorizing signals were present in nonaxial regions was also supported by a conjugation experiment in which Xepsin expression was suppressed in ectodermal explants conjugated with lateral or ventral marginal zone. Moreover, the partly suppressed expression of Xepsin in the epidermal region of exogastrulae indicates that the signals may travel horizontally within the plane of the epidermis. We also present data showing that both treatment with retinoic acid and the overexpression of a constitutively active form of a retinoic acid receptor caused the suppression of Xepsin mRNA transcription, suggesting that anterior–posterior patterning in the central nervous system and in the epidermis may share common endogenous factors, i.e., retinoids, in the Xenopus embryo
Patterns of Programmed Cell Death in Populations of Developing Spinal Motoneurons in Chicken, Mouse, and Rat

AbstractDuring embryonic development, approximately one-half of the spinal motoneurons initially generated are lost during a wave of programmed cell death (PCD). Classical studies in this system laid the basis of much work on the role and control of neuronal cell death during development. However, we have little information concerning the timing of cell death in motoneuron pools at different rostrocaudal levels, especially in rodents. We developed a novel protocol for whole-mount TUNEL labeling that allows apoptotic nuclei to be visualized in whole-mount preparations of embryonic spinal cord; double labeling with antibodies to Islet 1/2 showed that nearly all TUNEL-positive cells were motoneurons. In chicken and mouse embryos, the density of TUNEL-positive nuclei was specifically increased following target ablation. The pattern of naturally occurring motoneuron PCD was studied in spinal cords from different species and ages: chick (E4.5–E9.0), mouse (E11.5–E15.5), and rat (E13.5–E16.5). In all species, motoneuron PCD is first apparent at cervical levels and last at sacral levels. However, motoneuron PCD does not follow a strict rostrocaudal sequence. Following cervical motoneuron PCD, TUNEL profiles are first observed at lumbar levels in chick but at thoracic levels in rat. At a given rostrocaudal level, medial motoneurons tend to die before lateral populations, but here too there are exceptions. Motoneuron cell death is thus regulated in a highly stereotyped manner during development of vertebrate spinal cord. Our technique will provide a basis for the monitoring even localized changes in this pattern
Synergism between Pax-8 and lim-1 in Embryonic Kidney Development

AbstractPax genes encode a family of highly conserved DNA-binding transcription factors. These proteins play key roles in regulating a number of vertebrate and invertebrate developmental processes. Mutations in Pax-6 result in eye defects in flies, mice, and humans, and ectopic expression of this gene can trigger the development of ectopic compound eyes in flies. Likewise, mutation of other Pax genes in vertebrates results in the failure of specific differentiation programs—Pax-1 causes skeletal defects; Pax-2, kidney defects; Pax-3 or Pax-7, neural crest defects; Pax-4, pancreatic β-cell defects; Pax-5, B-cell defects; Pax-8, thyroid defects; and Pax-9, tooth defects. Although this class of genes is obviously required for the normal differentiation of a number of distinct organ systems, they have not previously been demonstrated to be capable of directing the embryonic development of organs in vertebrates. In this report, it is demonstrated that Pax-8 plays such a role in the establishment of the Xenopus embryonic kidney, the pronephros. However, in order to efficiently direct cells to form pronephric kidneys, XPax-8 requires cofactors, one of which may be the homeobox transcription factor Xlim-1. These two genes are initially expressed in overlapping domains in late gastrulae, and cells expressing both genes will go on to form the kidney. Ectopic expression of either gene alone has a moderate effect on pronephric patterning, while coexpression of XPax-8 plus Xlim-1 results in the development of embryonic kidneys of up to five times normal complexity and also leads to the development of ectopic pronephric tubules. This effect was synergistic rather than additive. XPax-2 can also synergize with Xlim-1, but the expression profile of this gene indicates that it normally functions later in pronephric development than does XPax-8. Together these data indicate that the interaction between XPax-8 and Xlim-1 is a key early step in the establishment of the pronephric primordium
Calcium Release at Fertilization of Xenopus Eggs Requires Type I IP3 Receptors, but Not SH2 Domain-Mediated Activation of PLCγ or Gq-Mediated Activation of PLCβ

AbstractElevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm–egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP3 receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP3. We then examine how IP3 production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCγ and PLCβ. The Src-homology 2 (SH2) domains of PLCγ were used to inhibit SH2-mediated activation of PLCγ, and an antibody against Gq family G-proteins was used to inhibit Gq-mediated activation of PLCβ. Though the PLCγ SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the Gq family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCγ SH2 domains and the Gq antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP3-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCγ or Gq-mediated activation of PLCβ
Ubiquitous Expression of Marker Transgenes in Mice and Rats

AbstractThe ability to unambiguously mark a cell's genotype is essential for studies in which genetically distinct cell populations must be distinguished from one another in vivo. One approach to this challenge has been the creation of transgenic mice expressing a transgene marker that is easily detectable, with no background staining. Multiple transgenic mouse strains bearing constructs with different combinations of promoter elements and coding sequences have been described, each with its own advantages and limitations. In this report we describe the use of an 800-bp promoter fragment isolated from the βgeo integration site in ROSA26 mice to target expression of two marker genes. We demonstrate that the ROSA26 promoter directs ubiquitous expression of human placental alkaline phosphatase and enhanced green fluorescent protein during embryonic and postnatal development in mouse and rat. We further demonstrate the general utility of these transgenes for marking donor cells in transplantation studies