pinecone/core-2020-05-10-deduplication huggingface dataset:
Zebrafish Dkk1 Functions in Forebrain Specification and Axial Mesendoderm Formation

AbstractWe identified a zebrafish homologue of Dickkopf-1 (Dkk1), which was previously identified in Xenopus as a Wnt inhibitor with potent head-inducing activity. Zebrafish dkk1 is expressed in the dorsal marginal blastoderm and also in the dorsal yolk syncytial layer after mid-blastula transition. At later blastula stages, the expression expands to the entire blastoderm margin. During gastrulation, dkk1-expressing cells are confined to the embryonic shield and later to the anterior axial mesendoderm, prospective prechordal plate. Embryos, in which dkk1 was ectopically expressed, exhibited enlarged forebrain, eyes, and axial mesendoderm such as prechordal plate and notochord. dkk1 expression in the dorso-anterior mesendoderm during gastrulation was prominently reduced in zebrafish mutants bozozok (boz), squint (sqt), and one-eyed pinhead (oep), which all display abnormalities in the formation and function of the Spemann organizer and axial mesendoderm. dkk1 expression was normal in these embryos during the blastula period, indicating that zygotic functions of these genes are required for maintenance but not establishment of dkk1 expression. Overexpression of dkk1 suppressed defects in the development of forebrain, eyes, and notochord in boz mutants. Overexpression of dkk1 promoted anterior neuroectoderm development in the embryos injected with antivin RNA, which lack most of the mesoderm and endoderm, suggesting that Dkk1 can affect regionalization of neuroectoderm independently of dorso-anterior mesendoderm. These data indicate that Dkk1, expressed in dorsal mesendoderm, functions in the formation of both the anterior nervous system and the axial mesendoderm in zebrafish
Positional Regulation of Krox-20 and mafB/kr Expression in the Developing Hindbrain: Potentialities of Prospective Rhombomeres

AbstractKrox-20 and mafB/kr encode transcription factors involved in the control of hindbrain development and are expressed in rhombomeres (r) 3 and 5 and 5 and 6, respectively. To analyse the regulation of the expression of these genes by positional cues, focusing on the stages just preceding the formation of rhombomeres, we have performed ectopic grafts involving single prospective rhombomeres (pr) or couples of pr on 4–6 somite avian embryos. Transplantation of pr6 in the pr5 position leads to Krox-20 activation and grafting of pr7 in the pr5 position results in mafB/kr activation. Furthermore, pr6 grafted in the pr5 position develops an r5-like cytoarchitecture. These data establish that rostral transplantation can lead to anteriorization within the hindbrain. However, additional experiments indicate that the competence of the transplanted tissue for such anteriorization appears limited and that transformations corresponding to shifts of a single rhombomere are favoured. We also show that caudal transplantation of pr5 into the pr6 position can lead to a down-regulation of Krox-20 expression consistent with posteriorization, suggesting that caudalizing influences are present within the nonsomitic hindbrain after the 4- to 6-somite stage. Finally, combinations of extirpation and grafting experiments suggest that the regulation of mafB/kr expression in the r6–r7 region may involve anteriorizing influences in addition to previously identified posteriorizing signals from the somitic region
Double-Stranded RNA Injection Produces Null Phenotypes in Zebrafish

AbstractZebrafish is a simple vertebrate that has many attributes that make it ideal for the study of developmental genetics. One feature that has been lacking in this model system is the ability to disable specifically targeted genes. Recently, double-stranded RNA has been used to silence gene expression in the nematode Caenorhabditis elegans. We have found that expression of the green fluorescent protein (GFP) from a microinjected plasmid vector can be suppressed in zebrafish embryos by the coinjection of a double-stranded RNA that is specifically targeted to GFP. To determine that double-stranded RNA can attenuate endogenous gene expression, single-cell zebrafish embryos were injected with double-stranded RNA specifically targeted to Zf-T and Pax6.1. We found that microinjection of double-stranded Zf-T RNA resulted in a high incidence of a phenotype similar to that of ntl. Furthermore, Zf-T gene expression could not be detected by in situ hybridization and the message was decreased by 75% by semiquantitative RT-PCR in 12-h embryos that had been injected with the double-stranded RNA. Expression of the zebrafish genes sonic hedgehog and floating head was altered in the embryos microinjected with the Zf-T double-stranded RNA in a manner that is remarkably similar to the zebrafish no-tail mutant. Microinjection of double-stranded RNA targeted to Pax6.1 was associated with depressed expression of Pax6.1 and resulted in absent or greatly reduced eye and forebrain development, similar to the phenotype seen in mouse mutants. Simultaneous injection of Pax6.1 and Zf-T resulted in embryos lacking notochords, eyes, and brain structures
Expression Patterns of Anterior Hox Genes in the Polychaete Chaetopterus: Correlation with Morphological Boundaries

AbstractExpression patterns for five Hox genes were examined by whole-mount in situ hybridization in larvae of Chaetopterus, a polychaete annelid with a tagmatized axial body plan. Phylogenetic analysis demonstrates that these genes are orthologs of the Drosophila genes labial, proboscipedia, zen, Deformed, and Sex combs reduced and are termed CH-Hox1, CH-Hox2, CH-Hox3, CH-Hox4, and CH-Hox5, respectively. Expression studies reveal a biphasic expression pattern. In early larval stages, well before any indications of segmental organization exist, a novel pattern of expression in bilateral posterior proliferating cell populations, corresponding to the teloblasts, was detected for each of the genes, with CH-Hox1 and CH-Hox2 expressed before the remaining three. In middle larval stages, all five genes are expressed in bilateral strips along the ventral midline, corresponding with the developing ventral nerve cord. In addition, CH-Hox1 and CH-Hox2 show strong expression at the foregut-midgut boundary. By late larval stages the expression is generally confined to the ventral CNS and ectoderm of the anterior parapodia. Anterior boundaries of expression are “colinear,” at later larval stages, with CH-Hox2 expressed most rostrally, in the first segment, and anterior expression boundaries for CH-Hox1, CH-Hox3, CH-Hox4, and CH-Hox5 in segments 2, 3, 4, and 5, respectively. Like vertebrates and spiders, but unlike insects, CH-Hox3 participates in this colinear axial expression pattern. CH-Hox1 and CH-Hox2 have distinct posterior boundaries of expression in the ninth segment, which corresponds to a major morphological boundary, and may reflect a reorganization of Hox gene regulation related to the evolutionary reorganization of the Chaetopterus body plan
Presence and Roles of Calcium Gradients along the Dorsal-Ventral Axis in Drosophila Embryos

AbstractDorsal-ventral specification of the Drosophila embryo is mediated by signaling pathways which have been very well described in genetic terms. However, little is known about the physiology of Drosophila development. By imaging patterns of free cytosolic calcium in Drosophila embryos, we found that several calcium gradients are generated along the dorsal-ventral axis. The most pronounced gradient is formed during stage 5, in which calcium levels are high dorsally. Manipulation of the stage 5 calcium gradient affects specification of the amnioserosa, the dorsal-most region of the embryo. We further show that this calcium gradient is inhibited in pipe, Toll, and dorsal mutants, but is unaltered in decapentaplegic (dpp) or punt mutants, suggesting that the stage 5 calcium gradient is formed by a suppression of ventral calcium concentrations. We conclude that calcium plays a role in specification of the dorsal embryonic region
Expression Pattern of GDNF, c-ret, and GFRαs Suggests Novel Roles for GDNF Ligands during Early Organogenesis in the Chick Embryo

AbstractWe have cloned a partial cDNA of chicken glial cell line-derived neurotrophic factor (GDNF) and systematically examined its expression pattern as well as that of GDNF-binding components (GDNF family receptor alpha-1 and 2: GFRα-1 and 2) and a common signal transduction receptor (c-ret protooncogene: RET) during very early developmental stages. In addition, we also examined the expression pattern of an apparent avian-specific binding component, GFRα-4. The cloned chicken cDNA for GDNF had approximately 80% homology to mammalian counterparts. The expression of GDNF mRNA occurred in many spatially and temporally discrete regions such as the intermediate mesoderm, the floor plate of the spinal cord, pharyngeal endoderm contacting the epibranchial placodes, distal ganglia of cranial nerves, subpopulations of mesenchyme cells in the craniofacial region, and in the mesodermal wall of the digestive tract. Both a GDNF receptor signal transduction component (RET) and a binding component (GFRα-1 or GFRα-2) were independently expressed in nearby interacting tissues such as the somites, peripheral and central nervous system, and mesenchyme cells in the craniofacial region. These observations suggest that possible combinations of novel unidentified receptors acting with RET or with GFRαs may mediate GDNF-derived signals and indicate that GDNF or other family members may have previously unidentified actions in early organogenesis in the chick embryo
Cardiac Expression of the Ventricle-Specific Homeobox Gene Irx4 Is Modulated by Nkx2-5 and dHand

AbstractWe report the isolation and characterization of the cDNAs encoded by the murine and human homeobox genes, Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0–8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5–11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression was also assessed in embryos with aberrant cardiac development: mice lacking RXRα or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development, which is downstream of Nkx2-5 and dHand
DCC Plays a Role in Navigation of Forebrain Axons across the Ventral Midbrain Commissure in Embryonic Xenopus

AbstractIn the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain
Severe Limb Defects in Hypodactyly Mice Result from the Expression of a Novel, Mutant HOXA13 Protein

AbstractHypodactyly (Hoxa13Hd) mice have a 50-bp deletion in the coding region of exon 1 of theHoxa13 gene and have more severe limb defects than mice with an engineered deletion of the entire gene (Hoxa13−/−). Increased cell death is observed in the autopod of Hoxa13Hd/Hd but not Hoxa13−/− limb buds. In addition, compound heterozygotes for oneHd allele and a Hoxa13− allele have a more severe limb phenotype than mice homozygous for the engineered null allele, suggesting a dominant-negative effect of theHd mutation. The Hoxa13Hd deletion does not interfere with steady-state mRNA levels; however, its consequences on translation are unknown. In this paper, we characterize theHoxa13 transcription initiation site in limbs and determine the initiator methionine of HOXA13. We show that the Hoxa13Hd deletion results in a translational frame shift that leads to the loss of wild-type HOXA13 protein and the simultaneous production of a novel, stable protein in the limb buds of mutant mice. The mutant Hd protein (HOXA13Hd) consists of the first 25 amino acids of wild-type HOXA13 sequence, followed by 275 amino acids of arginine- and lysine-rich, novel sequence, and lacks the homeodomain. Like wild-type HOXA13, HOXA13Hd is localized to the nucleus in transfected COS-7 cells, perhaps mediated by the arginine- and lysine-rich peptide sequences created by the translational frame shift. To determine whether HOXA13Hd could alter limb morphogenesis, we misexpressed the mutant mRNA throughout the developing limb bud using aPrx-1 promoter–Hd gene construct in transgenic mice. Three of 15 transgenic founder animals displayed reduction or absence of proximal and distal limb structures. We propose that the expression of HOXA13Hd plays a role in the profound failure of digit formation in Hoxa13Hd/Hd mice and explains the morphologic differences between these twoHoxa13 alleles
TCF Is the Nuclear Effector of the β-Catenin Signal That Patterns the Sea Urchin Animal–Vegetal Axis

AbstractThe mechanism of animal–vegetal (AV) axis formation in the sea urchin embryo is incompletely understood. Specification of the axis is thought to involve a combination of cell–cell signals and as yet unidentified maternal determinants. In Xenopus the Wnt pathway plays a crucial role in defining the embryonic axes. Recent experiments in sea urchins have shown that at least two components of the Wnt signaling pathway, GSK3β and β-catenin, are involved in embryonic AV axis patterning. These results support the notion that the developmental network that regulates axial patterning in deuterostomes is evolutionarily conserved. To further test this hypothesis, we have examined the role of β-catenin nuclear binding partners, members of the TCF family of transcriptional regulators, in sea urchin AV axis patterning. To test the role of TCFs in mediating β-catenin signals in sea urchin AV axis development we examined the consequences of microinjecting RNAs encoding altered forms of TCF on sea urchin development. We show that expression of a dominant negative TCF results in a classic “animalized” embryo. In contrast, microinjected RNA encoding an activated TCF produces a highly “vegetalized” embryo. We show that the transactivational activity of endogenous sea urchin TCF is potentiated by LiCl treatment, which vegetalizes embryos by inhibiting GSK3, consistent with an in vivo interaction between endogenous β-catenin and TCF. We also provide evidence indicating that all of β-catenin's activity in patterning the sea urchin AV axis is mediated by TCF. Using a glucocorticoid-responsive TCF, we show that TCF transcriptional activity affects specification along the AV axis between fertilization and the 60-cell stage
Mammalian Oocyte Activation by the Synergistic Action of Discrete Sperm Head Components: Induction of Calcium Transients and Involvement of Proteolysis

AbstractSperm-borne oocyte-activating factor (SOAF) elicits activation sufficient for full development and originates from sperm head submembrane matrices. SOAF comprises discrete, heat-sensitive and -stable components (referred to here respectively as SOAF-I and -II) which are each necessary but not sufficient to activate oocytes. The heat-sensitive SOAF component, SOAF-Im, becomes solubilized from the perinuclear matrix under reducing conditions (the SOAF transition) to generate SOAF-Is. Although calcium transients likely play an important role in oocyte activation at fertilization, the question is open as to whether demembranated heads or SOAF-Is and/or SOAF-II can induce calcium transients. We now report that injection of demembranated sperm heads into mouse oocytes efficiently induced Ca2+ oscillations. When injected independently, SOAF-Is and demembranated heads heated to 48°C failed to generate Ca2+ oscillations. However, co-injection of SOAF-Is and 48°C-heated heads induced oscillations, mirroring their synergistic ability to activate oocytes. This suggests that SOAF-mediated activation proceeds via pathways resembling those at fertilization and provides the first direct evidence that multiple sperm components are required to induce Ca2+ oscillations. We probed the SOAF-Is liberation at the center of this activation and show that in vitro it was sensitive to a profile of serine protease inhibitors. These findings support a model in which mammalian oocyte activation, including the induction of calcium transients, involves proteolytic processing of SOAF from sperm head submembrane compartments
Animal–Vegetal Axis Patterning Mechanisms in the Early Sea Urchin Embryo

AbstractWe discuss recent progress in understanding how cell fates are specified along the animal–vegetal axis of the sea urchin embryo. This process is initiated by cell-autonomous, maternally directed, mechanisms that establish three unique gene-regulatory domains. These domains are defined by distinct sets of vegetalizing (β-catenin) and animalizing transcription factor (ATF) activities and their region of overlap in the macromeres, which specifies these cells as early mesendoderm. Subsequent signaling among cleavage-stage blastomeres further subdivides fates of macromere progeny to yield major embryonic tissues. Zygotically produced Wnt8 reinforces maternally regulated levels of nuclear β-catenin in vegetal derivatives to down regulate ATF activity and further promote mesendoderm fates. Signaling through the Notch receptor from the vegetal micromere lineages diverts adjacent mesendoderm to secondary mesenchyme fates. Continued Wnt signaling expands the vegetal domain of β-catenin's transcriptional regulatory activity and competes with animal signaling factors, including BMP2/4, to specify the endoderm–ectoderm border within veg1 progeny. This model places new emphasis on the importance of the ratio of maternally regulated vegetal and animal transcription factor activities in initial specification events along the animal–vegetal axis
The Targeting of Xcat2 mRNA to the Germinal Granules Depends on a cis-Acting Germinal Granule Localization Element within the 3′UTR

AbstractThe germ cell lineage is specified by the germ plasm, which in Xenopus laevis contains putative determinants called germinal granules. The pathway through which these structures form and how their components are assembled remain unclear. Using a combination of electron microscopy and in situ hybridization with the germinal granule-associated Xcat2 mRNA we demonstrated that the granules were derived from a branching network of granulofibrillar material within the mitochondrial cloud. Targeting of Xcat2 mRNA to the germinal granules depended on a 164-nt 3′UTR germinal granule localization element (GGLE; nt 631–795) that was distinct from the previously defined mitochondrial cloud localization element (MCLE; nt 403–630; Y. Zhou and M. L. King, 1996, Development 122, 2947–2953). This demonstrated that the Xcat 3′UTR contains a compound localization element consisting of a general element (MCLE) targeting the RNA to the mitochondrial cloud and a second element (GGLE) responsible for targeting to the germinal granules within the cloud. The GGLE when fused to Xlsirt RNA was sufficient to target this nongranule mitochondrial cloud-associated RNA to the germinal granules. This is the first example of a localization element involved in targeting an mRNA to a specific subcellular target such as the germinal granules and suggests that cis-acting elements on RNAs play an important role in the assembly of germinal granules and, therefore, the establishment of the germ cell lineage
A Critical Period for Conversion of Ectodermal Cells to a Neural Crest Fate

AbstractPreviously, we found that interactions between neural and nonneural ectoderm can generate neural crest cells, with both the ectodermal and the neuroepithelial cells contributing to induced population (M. A. J. Selleck and M. Bronner-Fraser, 1995, Development 121, 525–538). To further characterize the ability of ectodermal cells to form neural crest, we have challenged their normal fate by transplanting them into the neural tube. To ensure that the ectoderm was from nonneural regions, we utilized extraembryonic ectoderm (the proamnion) and transplanted it into the presumptive midbrain of 1.5-day-old chick embryos. We observed that the grafted ectoderm has the capacity to adopt a neural crest fate, responding within a few hours of surgery by turning on neural crest markers HNK-1 and Slug. However, the competence of the ectoderm to respond to neural crest-inducing signals is time limited, declining rapidly in donors older than the 10-somite stage. Similarly, the inductive capacity of the host midbrain declines in a time-dependent fashion. Our results show that extraembryonic ectoderm has the capacity to form neural crest cells given proper inducing signals, expressing both morphological and molecular markers characteristic of neural crest cells