bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integrin α subunit mRNAs are differentially expressed in early Xenopus embryos
Authors: Whittaker Charles A. , DeSimone Douglas W. 
Published: April 2021

Abstract
Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin sub-units from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits α2, α3, α4, α5, α6 and an αIIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins α2, α3, α4, α5 and α6 each expressed by the end of gastrulation. Both α3 and α5 are expressed as maternal mRNAs. Zygotic expression of α2, α3, α4 and α6 transcripts begins during gastrulation. Integrin α5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin α1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990)Development 108, 289–298.). Here we report that integrin α3, α4 and α6 mRNA levels increase following induction with 10 U/ml activin-A whereas α5, α1 and α3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that α3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, α3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, α3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.
Neurotrophin-4 is a target-derived neurotrophic factor for neurons of the trigeminal ganglion
Authors: Ibáñez Carlos F. , Ernfors Patrik , Timmusk Tõnis , Ip Nancy Y. , Arenas Ernest , Yancopoulos George D. , Persson Håkan 
Published: April 2021

Abstract
The cellular localization of mRNA for neurotrophin-4 (NT-4), a novel neurotrophic factor, in the developing whisker follicles and skin of the embryonic rat is demonstrated by in situ hybridization. Levels of NT-4 mRNA in the whisker pad decrease between embryonic day 13 (E13) and E20, correlating in time with the onset of naturally occurring neuronal death in the innervating trigeminal ganglion. In addition to NT-4, brain-derived neuotrophic factor (BDNF) mRNA is also shown to be expressed in the rat embryonic whisker follicles although in a different cellular localization, which combined with previous data on the expression of NGF and NT-3 mRNAs, shows that all four neurotrophins are expressed during development of this structure. NT-4 protein is shown to elicit neurite outgrowth from explanted embryonic trigeminal ganglia and to promote neuronal survival of dissociated trigeminal ganglion neurons when cultured during the phase of cell death. NT-4 and NT-3 mainly support different neuronal sub-populations, whereas some NT-4-responsive cells appear to respond also to NGF and BDNF. Analysis of mRNAs for members of the Trk family of neurotrophin receptors in neurons rescued by different neurotrophins demonstrates the presence of distinct neuronal subpopulations that respond to specific combinations of these factors. Based on these results we propose that NT-4, together with the other three neurotrophins, orchestrate the innervation of the different structures of the developing whisker pad by the trigeminal ganglion, acting as target-derived neurotrophic factors for different sub-populations of trigeminal ganglion neurons.
Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos
Authors: Sousa Paul A. De , Valdimarsson Gunnar , Nicholson Bruce J. , Kidder Gerald M. 
Published: April 2021

Abstract
Gap junction assembly in the preimplantation mouse embryo is a temporally regulated event, beginning a few hours after the third cleavage during the morphogenetic event known as compaction. Recently, we demonstrated that both mRNA and protein corresponding to connexin43, a gap junction protein, accumulate through preimplantation development beginning at least as early as the 4-cell stage. Using an antibody raised against a synthetic C-terminal peptide of connexin43, this protein was shown to assemble into gap junction-like plaques beginning at compaction (G. Valdimarsson, P. A. De Sousa, E. C. Beyer, D. L. Paul and G. M. Kidder (1991). Molec. Reprod. Dev. 30, 18-26). The purpose of the present study was to follow the fate of nascent connexin43 during preimplantation development, from synthesis to plaque insertion, and to learn more about the control of gap junction assembly during compaction. Cell fractionation and reverse transcription-polymerase chain reaction were employed to show that connexin43 mRNA is in polyribosomes at the 4-cell stage, suggesting that synthesis of connexin43 begins at least one cell cycle in advance of when gap junctions first form. The fate of nascent connexin43 was then followed throughout preimplantation development by means of laser confocal microscopy, using two other peptide (C-terminal)-specific antibodies. As was reported previously, connexin43 could first be detected in gap junction-like plaques beginning in the 8-cell stage, at which time considerable intracellular immunoreactivity could be seen as well. Later, connexin43 becomes differentially distributed in the apposed plasma membranes of morulae and blastocysts: a zonular distribution predominates between outside blastomeres and trophectoderm cells whereas plaque-like localizations predominate between inside blastomeres and cells of the inner cell mass. The cytoplasmic immunoreactivity in morulae was deemed to be nascent connexin en route to the plasma membrane since it could be abolished by treatment with cycloheximide, and redistributed by treatment with monensin or brefeldin-A, known inhibitors of protein trafficking. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appearance of gap junction-like structures and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the regulated step in the onset of gap junction assembly during compaction is downstream of transcription and translation and involves mobilization of connexin43 through trafficking organelles to plasma membranes.
Defects of embryonic organogenesis resulting from targeted disruption of the N-myc gene in the mouse
Authors: Sawai Shoji , Shimono Akihiko , Wakamatsu Yoshio , Palmes Cynthia , Hanaoka Kazunori , Kondoh Hisato 
Published: April 2021

Abstract
The highest expression of the N-myc gene occurs during embryonic organogenesis in the mouse ontogeny, with the peak of expression around embryonic day 9.5. Homozygous N-myc-deficient mice, produced by germline transmission of a disrupted allele in ES cells, developed normally to day 10.5, indicating dispensability of N-myc expression in the earlier period, but later accumulated organogenic abnormalities and died around day 11.5. The most notable abnormalities were found in the limb bud, visceral organs (lung, stomach, liver and heart) and the central/peripheral nervous systems, and were highly correlated with the site of N-myc expression. The limb buds and the lungs excised from N-myc-deficient mutant embryos were placed in culture to allow their development to stages beyond the point of death of the embryos. Analyses indicated that the mutant limbs failed to develop distal structures and the development of bronchi from the trachea was defective in the lungs. The latter defect was largely corrected by addition of fetal calf serum to the culture medium, suggesting that an activity missing in the mutant lung was replenished by a component of the serum. The phenotype of N-myc-deficient mutant embryos indicated requirement of the N-myc function in many instances of tissue interactions in organogenesis and also in cell-autonomous regulation of tissue maturation.
Localized regions of egg cytoplasm that promote expression of endodermspecific alkaline phosphatase in embryos of the ascidian Halocynthia roretzi
Authors: Nishida Hiroki 
Published: April 2021

ABSTRACT
Embryogenesis in ascidians is known to be of the mosaic type, a property that suggests the presence of cytoplasmic factors in the egg which are responsible for specification of the developmental fates of early blastomeres. Endoderm cells are present in the trunk region of tadpole larvae, and these cells specifically express alkaline phosphatase (AP). Endoderm cells originate exclusively from blastomeres of the vegetal hemisphere of early embryos. To obtain direct evidence for cytoplasmic determinants of endoderm specification, we carried out cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various regions. Initially, presumptive-epidermis blastomeres (blastomeres from the animal hemisphere) were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi, and development of endoderm cells was monitored by histochemical staining for AP. AP activity was observed only when presumptive-epidermis blastomeres were fused with cytoplasmic fragments from the presumptive-endoderm blastomeres. The results suggest that cytoplasmic factors that promote the initial event of endoderm differentiation (endoderm determinants) are present in endoderm-lineage blastomeres. Next, to examine the presence and localization of endoderm determinants in the egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the presumptive-epidermis blastomeres. The results suggest that endoderm determinants are already present in unfertilized eggs, and that they are segregated by movements of the ooplasm after fertilization. Initially, these determinants move to the vegetal pole of the egg. Then, prior to the first cleavage, their distribution extends in the equatorial direction, namely, to the entire vegetal hemisphere from which future endoderm-lineage blastomeres are formed.
Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase
Authors: Melnick Michael B. , Perkins Lizabeth A. , Lee Mark , Ambrosio Linda , Perrimon Norbert 
Published: April 2021

ABSTRACT
Formation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase corkscrew, controls the domains of expression of the transcription factors tailless and huckebein. In this paper, we report the molecular and developmental characterization of mutations in the Draf gene. We show that mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that Draf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wildtype amount of D-raf protein.
Anterior mesendoderm induces mouse Engrailed genes in explant cultures
Authors: Ang Siew-Lan , Rossant Janet 
Published: April 2021

ABSTRACT
We have developed germ layer explant culture assays to study the role of mesoderm in anterior-posterior (A-P) patterning of the mouse neural plate. Using isolated explants of ectodermal tissue alone, we have demonstrated that the expression of Engrailed-1 (En-1) and En-2 genes in ectoderm is independent of mesoderm by the midto late streak stage, at least 12 hours before their onset of expression in the neural tube in vivo at the early somite stage. In recombination explants, anterior mesendoderm from headfold stage embryos induces the expression of En-1 and En-2 in preto early streak ectoderm and in posterior ectoderm from headfold stage embryos. In contrast, posterior mesendoderm from embryos of the same stage does not induce En genes in preto early streak ectoderm but is able to induce expression of a general neural marker, neurofilament 160×103Mr. These results provide the first direct evidence for a role of mesendoderm in induction and regionalization of neural tissue in mouse.
Non-neuronal cells inhibit catecholaminergic differentiation of primary sensory neurons: role of leukemia inhibitory factor
Authors: Fan Guoping , Katz David M. 
Published: April 2021

ABSTRACT
Although some sensory ganglion cells in mature animals are catecholaminergic, most mammalian sensory neurons that express the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) do so only transiently during early gangliogenesis in vivo. The lack of TH expression at later stages appears to be due to modulation of this catecholaminergic potential. A previous study showed that the phenotype reappears, for example, when E16.5 and older sensory ganglia are dissociated in culture into single cells, suggesting that extracellular influences can modulate TH expression. Moreover, TH expression in dissociate cultures is celldensity dependent, as a four-fold increase in plating density led to a 30% decrease in the percentage of TH neurons. The present study demonstrates that inhibition of TH expression in high density cultures is mediated by ganglionic non-neuronal cells (NNC), as removal of NNC abolished density-dependent inhibition. Moreover, plating E16.5 trigeminal neurons at low density on top of NNC monolayers resulted in an 85% decrease in the percentage of TH neurons. Treatment of cultures with non-neuronal cell conditioned medium (NNC–CM) reproduced the effect of coculture with NNC, suggesting that diffusible factors from NNC were involved in the inhibition of TH. The inhibitory effect of NNC–CM was mimicked by treatment of dissociate cultures with ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). However, immunoprecipitation of NNC–CM with antibodies against LIF or CNTF showed that only anti-LIF antibodies were able partially to remove the TH inhibitory activity of NNC–CM. Therefore, LIF is one, but not the only, factor mediating NNC inhibition of TH expression in cultured sensory neurons. In summary, these data indicate that ganglionic NNC can regulate sensory transmitter phenotype in culture by inhibiting expression of specific molecular traits. The finding that LIF can partially account for the inhibitory effect of ganglionic NNC on TH expression suggests a novel role for this cytokine in regulating differentiation of catecholaminergic properties in sensory neurons.
Evolutionary change in neural development within the arthropods: axonogenesis in the embryos of two crustaceans
Authors: Whitington Paul M. , Leach David , Sandeman Renate 
Published: April 2021

ABSTRACT
It has been previously suggested that there is a conservative program for neural development amongst the arthropods, on the basis that a stereotyped set of cells involved in establishing the axon tracts in the CNS of insect embryos is also present in crayfish embryos. We have examined the spatiotemporal pattern of axon growth from a set of early differentiating central neurons in the embryo of two crustaceans, the woodlouse Porcellio scaber and the freshwater crayfish Cherax destructor, and drawn comparisons with insect neurons whose somata lie in corresponding positions within the CNS. While many of the woodlouse and crayfish neurons show a similar pattern of axon growth to their insect counterparts, the axon trajectories taken by others differ from those seen in insects. We conclude that this aspect of early neural development has not been rigidly conserved during the evolution of the crustaceans and insects. However, the extent of similarity between the insects and the crustaceans is consistent with the idea that these groups of arthropods share a common evolutionary ‘Bauplan’ for the construction of their nervous systems. While the pattern of early axon growth in the woodlouse and crayfish embryos is sufficiently similar that many neurons could be confidently recognised as homologues, several differences were noted in both the relative order of axon outgrowth and axon morphologies of individual neurons.
Xenopus axis formation: induction of goosecoid by injected Xwnt-8 and activin mRNAs
Authors: Steinbeisser Herbert , Robertis Eddy M. De , Ku Min , Kessler Daniel S. , Melton Douglas A. 
Published: April 2021

ABSTRACT
In this study, we compare the effects of three mRNAs goosecoid, activin and Xwnt-8 that are able to induce partial or complete secondary axes when injected into Xenopus embryos. Xwnt-8 injection produces complete secondary axes including head structures whereas activin and goosecoid injection produce partial secondary axes at high frequency that lack head structures anterior to the auditory vesicle and often lack notochord. Xwnt-8 can activate goosecoid only in the deep marginal zone, i.e., in the region in which this organizerspecific homeobox gene is normally expressed on the dorsal side. Activin B mRNA, however, can turn on goosecoid in all regions of the embryo. We also tested the capacity of these gene products to restore axis formation in embryos in which the cortical rotation was blocked by UV irradiation. Whereas Xwnt-8 gives complete rescue of anterior structures, both goosecoid and activin give partial rescue. Rescued axes including hindbrain structures up to level of the auditory vesicle can be obtained at high frequency even in the absence of notochord structures. The possible functions of Wnt-like and activin-like signals and of the goosecoid homeobox gene, and their order of action in the formation of Spemann’s organizer are discussed.
Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila
Authors: Huet François , Ruiz Claude , Richards Geoff 
Published: April 2021

ABSTRACT
The steroid hormone ecdysone orchestrates insect development by regulating gene networks. In Drosophila the most detailed description of ecdysone action is the sequential activation of early and late puffs in the polytene chromosomes of the late larval salivary gland. A number of these early puffs (2B5, 74EF and 75B) contain complex transcription units (Broad-Complex, E74 and E75 respectively) encoding families of regulatory proteins which are expressed in most if not all tissues. In vitro, transcripts of the different isoforms of these early genes as well as the ecdysone receptor (EcR) present varying dose response characteristics (Karim and Thummel, 1992, EMBO J. 11, 4083–4093). We have developed an in vivo approach using a reverse transcription-polymerase chain reaction assay (RT-PCR) so as to visualise these transcripts in the RNA extracted from a single salivary gland. Using one salivary gland lobe for developmental puff staging and the sister lobe for RT-PCR, we have obtained precise developmental profiles for these transcripts and have extended our study to other tissues and stages where puffing studies were not possible. In the salivary gland we have characterised three distinct ecdysone responses. For the mid and late third larval instar responses our results confirm and extend the conclusions of the in vitro studies concerning the temporal expression of the early gene isoforms. The relatively brief prepupal response contains elements in common with each of the larval responses and all three can be explained by the profiles of the respective ecdysone peaks. Interestingly EcR transcripts respond differently during each response. The analysis of different tissues of the same animal reveals subtle differences in the timing of the ecdysone response and isoform expression and suggests that this may reflect tissue differences in the ecdysone profiles. As these molecules have homologues in vertebrates, our analysis may have general implications for the organisation of hormonal responses in vivo.
W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis
Authors: Duttlinger Regina , Manova Katia , Chu Tang Y. , Gyssler Corina , Zelenetz Andrew D. , Bachvarova Rosemary F. , Besmer Peter 
Published: April 2021

ABSTRACT
The receptor tyrosine kinase c-kit and its cognate ligand KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis (erythrocytes and mast cells). The W-sash mutation differs from most W mutations in that it affects primarily mast cells and melanogenesis but not other cellular targets of W and Sl mutations. Thus, Wsh/Wsh mice are fertile and not anemic, but they lack mast cells in their skin and intestine and are devoid of coat pigment. Heterozygotes are black with a broad white sash/belt in the lumbar region. In order to determine the basis for the phenotypes of W-sash mice, we investigated c-kit RNA and protein expression patterns in adult Wsh/Wsh mice and during embryonic development. We show that c-kit expression is absent in bone-marrow-derived Wsh/Wsh mast cells, the fetal and the adult lung, and the digestive tract at embryonic day , tissues that normally express c-kit. Unexpectedly, in and Wsh/Wsh embryos, we found c-kit expression in the dermatome of the somites, the mesenchyme around the otic vesicle and the floorplate of the neural tube, structures known to express the c-kit ligand in wild-type embryos. The ectopic c-kit expression in Wsh homozygous embryos does not affect c-kit ligand expression. The presumed Wsh/Wsh melanoblasts appeared to be normal and, at similar numbers were found in normal and homozygous mutant embryos. At embryos had a graded distribution of melanoblasts from cranial to caudal with a minimum in the lumbar region. Whereas homozygous Wsh/Wsh embryos essentially lacked c-kit-positive cells in the skin, heterozygous Wsh/+ embryos had reduced numbers of melanoblasts compared to +/+ with few or none in the lumbar region (future sash). It is proposed that ectopic c-kit expression in the somitic dermatome affects early melanogensis in a dominant fashion. Molecular analysis of Wsh chromosomal DNA revealed a deletion or rearrangement in the vicinity of the c-kit gene. These results provide an explanation for the Wsh phenotype and have implications for the control of c-kit expression.