bluuebunny/crossref_metadata_2025_split huggingface dataset:
EGF, TGF-α and EGFR expression in human preimplantation embryos
Authors: Chia Choy May , Winston Robert M. L. , Handyside Alan H. 
Published: April 2021

ABSTRACT
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) through their common receptor, epidermal growth factor receptor (EGFR) are known to enhance mitogenesis, development and implantation in several species. In the mouse, co-culture of grouped embryos in microdrops increases the cell number and pro-portion developing to the blastocyst stage. A similar effect is observed with culture of single embryos in medium sup-plemented with EGF or TGF-α highlighting their embry-otrophic effects.
To study the role of EGF, TGF-α and EGFR in early human development, two methods applicable for analysis of expression at the single embryo level have been employed. In the first method, reverse transcription-poly-merase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for speci-ficity, were used for amplification from the cDNA equiva-lent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. In the second method, immunocytochemistry has been used to co-localise the expressed proteins. Individual embryos were paraffin embedded and serial sectioned, allowing adjacent sections to be examined with different antibodies and controls. Monoclonal TGF-α and polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry with the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth factors and/or their receptors in the same human embryos.
Transcripts for EGF, TGF-α and EGFR were detected in unfertilized oocytes and embryos between 8-cell and blastocyst stages on day 3 to 6 post-insemination. Similarly, at the protein level, all three were detected in unfertilized oocytes and throughout preimplantation development to day 8. At the blastocyst stage, expression was observed in both the trophectoderm and inner cell mass but decreased in more advanced blastocysts except in the polar trophec-toderm and inner cell mass. This pattern of expression contrasts with the murine and bovine species in which TGF-α and EGFR but not EGF are expressed at preim-plantation stages. The concurrent co-expression of these growth factors and their receptor suggests a role for autocrine stimulation in preimplantation development. In the human, reduced protein levels of EGF, TGF-α and EGFR in advanced blastocysts may reflect a switch to dependence on paracrine stimulation. This may explain the inadequacy of simple culture media to maintain the devel-opment of human embryos at later stages and the benefits of co-culture with somatic cells. The continuous expression of EGF, TGF-α and EGFR in the polar trophectoderm may be significant for implantation.
Target-induced neurogenesis in the leech CNS involves efferent projections to the target
Authors: Becker Tom , Berliner Alyson J. , Nitabach Michael N. , Gan Wen-Biao , Macagno Eduardo R. 
Published: April 2021

ABSTRACT
During a critical period in leech embryogenesis, the sex nerves that connect the 5th and 6th midbody ganglia (MG5 and MG6) to the primordium of the male sexual organ carry a spatially localized signal that induces the birth of several hundred neurons specific to these ganglia. We examined particular cellular elements (afferents, efferents, non-neuronal components) within these nerves as potential conveyors of the inductive signal. We show that axons of peripheral sensory neurons in the male genitalia travel along the sex nerves and into MG5 and MG6, but reach the CNS after the critical period has elapsed and cannot, therefore, be involved in the induction. Of the six sex nerves, four contain non-neuronal cells that span the entire distance between the male genitalia and the sex ganglia. However, when male genitalia were transplanted to ectopic locations close to MG6, induction occurred frequently but only in MG6, mediated by ectopic nerves that do not contain these cells. Thus, non-neuronal cells specific to the normal sex nerves are not necessary for induction. In addition, dye injections into the target during the critical period failed to reveal migrating cells in the sex nerves that could convey the inductive signal to the CNS. Finally, we show that 11 pairs of central neurons in each ganglion project to the male organ early during the critical period. In the adult, at least 3 additional pairs of neurons in MG6 also innervate this target. We conclude that the only components of the sex nerves that connect the sex ganglia to the target during the critical period that could be associated with induced central mitogenesis are the axons of central neurons that innervate the male genitalia.
Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice
Authors: Kwee Lia , Baldwin H. Scott , Shen Hong Min , Stewart Colin L. , Buck Catherine , Buck Clayton A. , Labow Mark A. 
Published: April 2021

ABSTRACT
VCAM-1 is a cytokine-inducible cell surface protein capable of mediating adhesion to leukocytes expressing α4 integrins. Mice deficient in VCAM-1 expression were produced by targeted homologous recombination in ES cells. VCAM-1-deficient embryos were not viable and exhibited either of two distinct phenotypes. Approximately half of the embryos died before embryonic day 11.5 and exhibited a severe defect in placental development in which the allantois failed to fuse with the chorion. The remaining VCAM-1-deficient embryos survived to embryonic day 11.5-12.5 and displayed several abnormalities in their developing hearts including a reduction of the compact layer of the ventricular myocardium and intraventricular septum. The hearts also contained significant amounts of blood in the pericardial space and lacked an epicardium. α4 and VCAM-1 were found to be expressed in wild-type embryos in a reciprocal fashion in the chorion and allantois and in the epicardium and the underlying myocardium, although VCAM-1 was expressed in the intraventricular septum in the absence of adjacent α4-expressing cells. These data suggest important roles for VCAM-1 and α4 in the development of the placenta and the heart.
Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal
Authors: Lampron Carmen , Rochette-Egly Cécile , Gorry Philippe , Dollé Pascal , Mark Manuel , Lufkin Thomas , LeMeur Marianne , Chambon Pierre 
Published: April 2021

ABSTRACT
We have disrupted the CRABPII gene using homologous recombination in embryonic stem cells, and shown that this disruption results in a null mutation. CRABPII null mutant mice are essentially indistinguishable from wild-type mice as judged by their normal development, fertility, life span and general behaviour, with the exception of a minor limb malformation. Moreover, CRABPI−/−/CRABPII−/− double mutant mice also appear to be essentially normal, and both CRABPII−/− single mutant and CRABPI−/−/CRABPII−/− double mutant embryos are not more sensitive than wild-type embryos to retinoic acid excess treatment in utero. Thus, CRABPI and CRABPII are dispensable both during mouse development and adult life. Our present results demonstrate that CRABPs are not critically involved in the retinoic acid signaling pathway, and that none of the functions previously proposed for CRABPs are important enough to account for their evolutionary conservation.
Cell adhesion events mediated by α4 integrins are essential in placental and cardiac development
Authors: Yang Joy T. , Rayburn Helen , Hynes Richard O. 
Published: April 2021

ABSTRACT
α4 integrins are cell surface receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesions by interacting with fibronectin (FN) and vascular cell adhesion molecule 1 (VCAM-1), respectively. We have generated a null mutation in the gene for the α4 integrin subunit. Homozygous null embryos express no α4 integrins and show two unexpected defects, both of which lead to embryonic lethality. The first defect is failure of fusion of the allantois with the chorion during placentation. The second is in the development of the epicardium and coronary vessels leading to cardiac hemorrhrage. Both processes clearly involve α4 integrin interactions that were previously unsuspected. α4 integrin and VCAM-1 are expressed at the sites of these interactions. These results raise the possibility of abortifacients targeting α4 integrins, and raise serious questions about potential side effects of drugs currently being designed to block α4 integrin functions in inflammation.
Identification of a germ-cell-specific transcriptional repressor in the promoter of Tctex-1
Authors: O’Neill Michael J. , Artzt Karen 
Published: April 2021

ABSTRACT
The Tctex-1 gene family maps to the t complex of the mouse and consists of four copies on chromosome 17 in both wild-type and t-haplotypes. Tctex-1 mRNA is eightfold overexpressed in male and female germ cells in t-haplotype compound heterozygotes (tx/ty). In order to determine the cause of this aberrant expression and the role of this gene family in spermatogenesis and oogenesis it was subjected to extensive molecular analysis. We find that Tctex-1 protein is present in sperm tails and oocytes and that it is present at equal levels in wild-type and t-haplotype testis. Surprisingly, the excess message in t-haplotypes is not translated. Sequence analysis of the gene family reveals that one copy in t-haplotypes has a mutated start codon. This same copy is deleted for a protein-binding motif in its promoter. This motif, GIM (Germ cell Inhibitory Motif) has strong homology to the Xenopus AP-2-binding site but does not appear to be a binding site for mammalian AP-2. A factor(s) present in testis and ovary, but absent in other mouse tissues binds specifically to this site. Transfection assays using Tctex-1 promoter constructs suggest that GIM functions as a transcriptional repressor. The possible role of Tctex-1 in t complex transmission ratio distortion and sterility is discussed.
The biogenesis of the coiled body during early mouse development
Authors: Ferreira João , Carmo-Fonseca Maria 
Published: April 2021

ABSTRACT
The coiled body is an ubiquitous nuclear organelle that contains essential components of the pre-mRNA splicing machinery as well as the nucleolar protein fibrillarin. Here we have studied the biogenesis of the coiled body in early mouse embryos. The results show that coiled bodies form and concentrate splicing snRNPs as early as in the maternal and paternal pronuclei of 1-cell embryos. This argues that the coiled body is likely to play a basic role in the nucleus of mammalian cells. In order to correlate the appearance of coiled bodies with the onset of transcriptional activity, embryos were incubated with brominated UTP and the incorporated nucleotide was visualized by fluorescence microscopy. In agreement with previous studies, transcriptional activity was first observed during the 2-cell stage. Thus, coiled bodies form before activation of embryonic gene expression.
The appearance of coiled bodies in 1-cell embryos was preceded by the formation of morphologically distinct structures that also contain coilin and which we therefore refer to as pre-coiled bodies. At the electron microscopic level pre-coiled bodies have a compact fibrillar structure, whereas coiled bodies resemble a tangle of coiled threads. Although both pre-coiled bodies and coiled bodies contain the nucleolar protein fibrillarin, the assembly of coiled bodies is separated both in time and in space from ribosome synthesis. Our results suggest that the embryonic ‘nucleolus-like body’ is a structural scaffold that nucleates independently the formation of the coiled body and the assembly of the machinery responsible for ribosome biosynthesis.
Repression of Pax-2 by WT1 during normal kidney development
Authors: Ryan Gabriella , Steele-Perkins Victoria , Morris Jennifer F. , Rauscher Frank J. , Dressler Gregory R. 
Published: April 2021

ABSTRACT
The developmental, regulatory gene Pax-2 is activated during early kidney morphogenesis and repressed in mature renal epithelium. Persistent Pax-2 expression is also observed in a variety of kidney tumors. Yet, little is known about the signals regulating this transient expression pattern in the developing kidney. We have examined the spatial and temporal expression patterns of Pax-2 and the Wilms’ tumor suppresser protein WT1 with specific antibodies in developing mouse kidneys. A marked increase in WT1 protein levels coincided precisely with down-regulation of the Pax-2 gene in the individual precursor cells of the visceral glomerular epithelium, suggesting a direct effect of the WT1 repressor protein on Pax-2 regulatory elements. To examine whether WT1 could directly repress Pax-2 transcription, binding of WT1 to three high affinity sites in the 5’ untranslated Pax-2 leader sequence was demonstrated by DNAseI footprinting analysis. Furthermore, co-transfection assays using CAT reporter constructs under the control of Pax-2 regulatory sequences demonstrated WT1-dependent transcriptional repression. These three WT1 binding sites were also able to repress transcription, in a WT1-dependent manner, when inserted between a heterologous promoter and the reporter gene. The data indicate that Pax-2 is a likely target gene for WT1 and suggest a direct link, at the level of transcriptional regulation, between a developmental control gene, active in undifferentiated and proliferating cells, and a known tumor suppresser gene.
Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons
Authors: Groves Andrew K. , George Kathleen M. , Tissier-Seta Jean-Phillipe , Engel James Douglas , Brunet Jean-François , Anderson David J. 
Published: April 2021

ABSTRACT
We have examined the regulation of transcription factor gene expression and phenotypic markers in developing chick sympathetic neurons. Sympathetic progenitor cells first express the bHLH transcriptional regulator Cash-1 (a chicken achaete-scute homologue), followed by coordinate expression of Phox2, a paired homeodomain protein, and GATA-2, a zinc finger protein. SCG10, a pan-neuronal membrane protein, is first detected one stage later, followed by the catecholaminergic neurotransmitter enzyme tyrosine hydroxylase (TH). We have used these markers to ask two questions: (1) is their expression dependent upon inductive signals derived from the notochord or floor plate?; (2) does their sequential expression reflect a single linear pathway or multiple parallel pathways? Notochord ablation experiments indicate that the floor plate is essential for induction of GATA-2, Phox2 and TH, but not for that of Cash-1 and SCG10. Taken together these data suggest that the development of sympathetic neurons involves multiple transcriptional regulatory cascades: one, dependent upon notochord or floor plate-derived signals and involving Phox2 and GATA-2, is assigned to the expression of the neurotransmitter phenotype; the other, independent of such signals and involving Cash-1, is assigned to the expression of pan-neuronal properties. The parallel specification of different components of the terminal neuronal phenotype is likely to be a general feature of neuronal development.
Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes
Authors: Erickson Carol A. , Goins Tina L. 
Published: April 2021

ABSTRACT
Neural crest cells are conventionally believed to migrate arbitrarily into various pathways and to differentiate according to the environmental cues that they encounter. We present data consistent with the notion that melanocytes are directed, by virtue of their phenotype, into the dorsolateral path, whereas other neural crest derivatives are excluded.
In the avian embryo, trunk neural crest cells that migrate ventrally differentiate largely into neurons and glial cells of the peripheral nervous system. Neural crest cells that migrate into the dorsolateral path become melanocytes, the pigment cells of the skin. Neural crest cells destined for the dorsolateral path are delayed in their migration until at least 24 hours after migration commences ventrally. Previous studies have suggested that invasion into the dorsolateral path is dependent upon a change in the migratory environment. A complementary possibility is that as neural crest cells differentiate into melanocytes they acquire the ability to take this pathway. When quail neural crest cells that have been grown in culture for 12 hours are labeled with Fluoro-gold and then grafted into the early migratory pathway at the thoracic level, they migrate only ventrally and are coincident with the host neural crest. When fully differentiated melanocytes (96 hours old) are back-grafted under identical conditions, however, they enter the dorsolateral path and invade the ectoderm at least one day prior to the host neural crest. Likewise, neural crest cells that have been cultured for at least 20 hours and are enriched in melanoblasts immediately migrate in the dorsolateral path, in addition to the ventral path, when back-grafted into the thoracic level. A population of neural crest cells depleted of melanoblasts – crest cells derived from the branchial arches – are not able to invade the dorsolateral path, suggesting that only pigment cells or their precursors are able to take this migratory route. These results suggest that as neural crest cells differentiate into melanocytes they can exploit the dorsolateral path immediately.
Even when 12-hour crest cells are grafted into stage 19-21 embryos at an axial level where host crest are invading the dorsolateral path, these young neural crest cells do not migrate dorsolaterally. Conversely, melanoblasts or melanocytes grafted under the same circumstances are found in the ectoderm. These latter results suggest that during normal development neural crest cells must be specified, if not already beginning to differentiate, as melanocytes in order to take this path. Thus melanocyte precursors are one subpopulation of the neural crest that has been documented to exploit a specific pathway on the basis of their phenotype.
Disruption of intermediate filament organization leads to structural defects at the intersomite junction in Xenopus myotomal muscle
Authors: Cary Robert B. , Klymkowsky Michael W. 
Published: April 2021

ABSTRACT
In mature striated muscle, intermediate filaments (IFs) are associated with the periphery of Z-discs and sites of myofibril-membrane attachment. Previously T. Schultheiss, Z. X. Lin, H. Ishikawa, I. Zamir, C. J. Stoeckert and H. Holtzer (1991) J. Cell Biol. 114, 953)reported that the disruption of IF organization in cultured chick myotubes had no detectable effect on muscle cell structure. Cultured muscle is not, however, under the mechanical loads characteristic of muscle in situ. The dorsal myotomal muscle (DMM) of the Xenopus tadpole provides an accessible model system in which to study the effects of mutant IF proteins on an intact, functional muscle. DNAs encoding truncated forms of Xenopus vimentin or desmin were injected into fertilized Xenopus eggs. Embryos were allowed to develop to the tadpole stage and then examined by confocal or electron microscopy. DMM cells containing the truncated IF polypeptides displayed disorganized IF systems. While the alignment of Z-lines appeared unaffected, cells accumulating mutant IF polypeptides displayed abnormal organization at the intersomite junction. Myocyte termini are normally characterized by deep invaginations of the sarcolemma. In myocytes expressing mutated IF polypeptides, these membrane invaginations were reduced or completely absent. Furthermore, the attachment of myofibrils to the junctional membrane was often aberrant or completely disrupted. These results suggest that in active muscle IFs play an important role in the organization and/or stabilization of myofibril-membrane attachment sites.
An analysis of the response to gut induction in the C. elegans embryo
Authors: Goldstein Bob 
Published: April 2021

ABSTRACT
Establishment of the gut founder cell (E) in C. elegans involves an interaction between the P2 and the EMS cell at the four cell stage. Here I show that the fate of only one daughter of EMS, the E cell, is affected by this induction. In the absence of the P2-EMS interaction, both E and its sister cell, MS, produce pharyngeal muscle cells and body wall muscle cells, much as MS normally does.
By cell manipulations and inhibitor studies, I show first that EMS loses the competence to respond before it divides even once, but P2 presents an inducing signal for at least three cell cycles. Second, induction on one side of the EMS cell usually blocks the other side from responding to a second P2-derived signal. Third, microfilaments and microtubules may be required near the time of the interaction for subsequent gut differentiation. Lastly, cell manipulations in pie-1 mutant embryos, in which the P2 cell is transformed to an EMS-like fate and produces a gut cell lineage, revealed that gut fate is segregated to one of P2’s daughters cell-autonomously. The results contrast with previous results from similar experiments on the response to other inductions, and suggest that this induction may generate cell diversity by a different mechanism.
Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila
Authors: Frasch Manfred , Chen Xiaowei , Lufkin Thomas 
Published: April 2021

ABSTRACT
The HOM-C/Hox complexes are an evolutionary related family of genes that have been shown to direct regionspecific development of the animal body plan. We examined in transgenic mice the DNA regulatory elements that determine the temporal and spatially restricted expression of two of the earliest and most anteriorly expressed murine genes, Hoxa-1 and Hoxa-2, which are homologues of the labial and proboscipedia genes of Drosophila. In both mouse and Drosophila, these genes have been shown to play a critical role in head development. We identified three independent enhancers which direct distinct portions of the Hoxa-1 and Hoxa-2 expression domains during early murine embryogenesis. Two enhancers mediate hindbrainspecific expression, being active in either rhombomere 2, the most anterior rhombomere expressing Hoxa-2, or in rhombomere 4, a region where Hoxa-1 and Hoxa-2 have been shown to exert critical developmental roles. The third enhancer is essential for the most extensive expression domain of Hoxa-1 and contains a retinoic acid response element. Point mutations within the retinoic acid response element abolish expression in neuroepithelium caudal to rhombomere 4, supporting a natural role for endogenous retinoids in patterning of the hindbrain and spinal cord. Analysis of the murine Hoxa-2 rhombomere 2-specific enhancer in Drosophila embryos revealed a distinct expression domain within the arthropod head segments, which parallels the expression domain of the Hoxa-2 homologue proboscipedia. These results suggest an evolutionary conservation between HOM-C/Hox family members, which includes a conservation of certain DNA regulatory elements and possible regulatory cascades.