Limb development in mouse embryos: protection against teratogenic effects of 6-diazo-5 oxo-L-norleucine (DON) in vivo and in vitro

Development ◽  
1975 ◽  
Vol 33 (2) ◽  
pp. 355-370
Author(s):  
R. M. Greene ◽  
D. M. Kochhar
Keyword(s):  
Limb Development ◽  
Cleft Lip ◽  
Phenotypic Expression ◽  
Limb Bud ◽  
Concurrent Administration ◽  
Median Cleft ◽  
Mouse Limb ◽  
Limb Malformations

The glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), has been shown to inhibit biosynthesis of purines and glycosaminoglycans, presumably by blocking the glutaminedependent steps in the biosynthetic pathways. The teratogenic potential of DON on the developing mouse limb-bud in vivo and in vitro was studied in an attempt to discriminate whether DON is exerting its teratogenic effect by interfering with glycosaminoglycan orpurine metabolism. A single intramuscular injection of DON (0·5 mg/kg) to ICR/DUB mice on day 10 of gestation resulted in 76% resorption, while fetuses surviving to day 17 exhibited growth retardation, median cleft lip, and limb malformations. Concurrent administration of Lglutamine (250 mg/kg) provided no protection against resorption or malformations, while 5-aminoimidazolecarboxamide (AIC, 250 mg/kg) decreased the resorption rate to 34% without significantly altering the incidence of malformations. Injection of DON alone on day 11 resulted in 87% of fetuses exhibiting limb malformations, with only 2% resorption. Concurrent injection of AIC decreased the frequency of limb malformations to 32%. L-Glutamine, D-glucosamine, or inosinic acid were without any protective effect in vivo. DON (5 μg/ml medium) added in vitro to organ cultures of day 11 mouse limb-buds caused all limbs to evidence cartilage abnormalities. In this system, either L-glutamine or D-glucosamine (0·5 mg/ml medium) provided protection against DON effects while AIC (0·5 mg/ml medium) offered no protection in vitro. These data suggest that DON exerts its effects in vivo by interfering with purine metabolism while in vitro its teratogenic action may be interruption of glycosaminoglycan biosynthesis. This may reflect upon the relative importance of growth and differentiation to limb development in vivo and in vitro. These data infer that limb development in vitro relies more on the differentiative process (differentiation of cartilage) than on growth, whereas limb development in vivo is dependent, at this stage, to a greater extent on growth for normal phenotypic expression.

Vitamin A alters the internal viscosity of fragments of limb-bud mesenchyme

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 325-339
Author(s):  
T. E. Kwasigroch ◽  
D. M. Kochhar
Keyword(s):  
Retinoic Acid ◽  
Vitamin A ◽  
Mesenchymal Cells ◽  
Limb Bud ◽  
Mouse Limb ◽  
Vitamin A Acid ◽  
Internal Viscosity ◽  
Fusion Experiments

Two techniques were used to examine the effect of vitamin A compounds (vitamin A acid = retinoic acid and vitamin A acetate) upon the relative strengths of adhesion among mouse limb-bud mesenchymal cells. Treatment with retinoic acid in vivo and with vitamin A acetate in vitro reduced the rate at which the fragments of mesenchyme rounded-up when cultured on a non-adhesive substratum, but these compounds did not alter the behavior of tissues tested in fragment-fusion experiments. These conflicting results indicate that the two tests measure different activities of cells and suggest that treatment with vitamin A alters the property(ies) of cells which regulate the internal viscosity of tissues.

Changes in expression of the lysosomal membrane glycoprotein, LAMP-1 in interdigital regions during embryonic mouse limb development, in vivo and in vitro

2000 ◽  
Vol 201 (6) ◽  
pp. 483-490 ◽  
Author(s):  
Sharon Stewart ◽  
Scott Yi ◽  
Garo Kassabian ◽  
Mark Mayo ◽  
Anthony Sank ◽  
...  
Keyword(s):  
Limb Development ◽  
Lysosomal Membrane ◽  
Membrane Glycoprotein ◽  
Embryonic Mouse ◽  
Mouse Limb ◽  
Lysosomal Membrane Glycoprotein

Effects of cytosine arabinoside on in vivo and in vitro mouse limb development

In Vitro ◽  
1977 ◽  
Vol 13 (7) ◽  
pp. 434-442 ◽  
Author(s):  
Jeanne M. Manson ◽  
Michael L. Dourson ◽  
Carl C. Smith
Keyword(s):  
Cytosine Arabinoside ◽  
Limb Development ◽  
Mouse Limb

Maintenance of ZPA signaling in cultured mouse limb bud cells

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1421-1433 ◽  
Author(s):  
R. Anderson ◽  
M. Landry ◽  
K. Muneoka
Keyword(s):  
Culture Technique ◽  
Limb Bud ◽  
In Vivo Studies ◽  
Maintenance Activity ◽  
Vertebrate Limb ◽  
Mouse Limb ◽  
Zone Of Polarizing Activity

The positional signal localized to the posterior (zone of polarizing activity or ZPA) region of the vertebrate limb is transiently expressed during development and a decline in ZPA signaling is accelerated when posterior cells are dissociated and cultured in vitro. The evidence that cultured posterior cells display a precocious decline in ZPA signaling when compared to in vivo studies suggests that a factor present in the limb bud maintains or stabilizes ZPA signaling during limb outgrowth and that this maintenance factor is lost and/or exhausted in in vitro studies. We have developed a new culture technique, ‘microdissociation’, which preserves extracellular components that we have found to be necessary for ZPA signal maintenance. Our data suggest that the limb bud ectoderm produces a maintenance activity that becomes stored in the extracellular matrix where it acts on limb bud cells to stabilize the activity of the ZPA signal. Using our initial characterization of this maintenance activity, we have identified a growth factor, FGF-2 (bFGF), that can replace all of the ZPA signaling maintenance activity observed in microdissociate cultures. The existence of various members of the FGF family in the developing limb strongly argues a role for FGF in stabilizing ZPA signaling in vivo.

scholarly journals The ulnar-mammary syndrome gene, Tbx3, is a direct target of the retinoic acid signaling pathway, which regulates its expression during mouse limb development

2012 ◽  
Vol 23 (12) ◽  
pp. 2362-2372 ◽  
Author(s):  
Reyna Deeya Ballim ◽  
Cathy Mendelsohn ◽  
Virginia E. Papaioannou ◽  
Sharon Prince
Keyword(s):  
Retinoic Acid ◽  
Signaling Pathway ◽  
Limb Development ◽  
Postnatal Life ◽  
Functional Protein ◽  
Embryonic Limb ◽  
Mouse Limb ◽  
Direct Target

TBX3, a member of the T-box transcription factor gene family, is a transcriptional repressor that is required for the development of the heart, limbs, and mammary glands. Mutations in TBX3 that result in reduced functional protein lead to ulnar-mammary syndrome, a developmental disorder characterized by limb, mammary gland, tooth, and genital abnormalities. Increased levels of TBX3 have been shown to contribute to the oncogenic process, and TBX3 is overexpressed in several cancers, including breast cancer, liver cancer, and melanoma. Despite its important role in development and postnatal life, little is known about the signaling pathways that modulate TBX3 expression. Here we show, using in vitro and in vivo assays, that retinoic acid (RA) activates endogenous TBX3 expression, which is mediated by an RA–receptor complex directly binding and activating the TBX3 promoter, and we provide evidence that this regulation may be functionally relevant in mouse embryonic limb development. Our data identify TBX3 as a direct target of the RA signaling pathway and extend our understanding of the role and regulation of TBX3 in limb development.

scholarly journals Hst-1 (FGF-4) antisense oligonucleotides block murine limb development.

1995 ◽  
Vol 130 (4) ◽  
pp. 997-1003 ◽  
Author(s):  
T Ochiya ◽  
H Sakamoto ◽  
M Tsukamoto ◽  
T Sugimura ◽  
M Terada
Keyword(s):  
Organ Culture ◽  
Limb Development ◽  
Culture System ◽  
Inhibitory Effect ◽  
Defined Medium ◽  
Limb Bud ◽  
Embryonic Mouse ◽  
Organ Culture System ◽  
Mouse Limb

The initiation of limb development depends on the site specific proliferation of the mesenchyme by the signals from the apical ectodermal ridge (AER) in embryonic mouse. We have previously reported that the local expression of Hst-1/Fgf-4 transcripts in AER of the mouse limb bud is developmentally regulated, expressed at 11 and 12 days post coitus (p.c.) embryo. In an effort to further understand the role of Hst-1/FGF-4 in mouse limb development, an antisense oligodeoxynucleotides (ODNs) study was performed. We first established a novel organ culture system to study mouse limb development in vitro. This system allows mouse limb bud at 9.5-10-d p.c. embryo, when placed on a sheet of extracellular matrix in a defined medium, to differentiate into a limb at 12.5-d p.c. embryo within 4.5 d. Using this organ culture system, we have shown that exposure of 9.5-10-d p.c. embryonal limb bud explants to antisense ODNs of Hst-1/FGF-4 blocks limb development. In contrast, sense and scrambled ODNs have no inhibitory effect on limb outgrowth, suggesting that Hst-1/FGF-4 may work as a potent inducing factor for mouse limb development.

scholarly journals Experimental studies on the differentiation of embryonic tissues growing in vivo and in vitro .—II. The development of the isolated early embryonic eye of the fowl when cultivated in vitro

1926 ◽  
Vol 100 (703) ◽  
pp. 273-283 ◽  
Keyword(s):  
Medical Research ◽  
Research Council ◽  
Medical Research Council ◽  
Experimental Studies ◽  
Limb Bud ◽  
Progressive Development ◽  
Embryonic Tissues ◽  
Self Differentiation

In a previous communication (Strangeways and Fell, 1926) it was shown that if the undifferentiated limb-bud of the embryonic Fowl was cultivated in vitro , it underwent a considerable amount of progressive development. This capacity for independent development in vitro possessed by an isolated organ has been further investigated, and for these later experiments the writers have employed the early embryonic eye, a structure endowed with more complex potentialities than the limb-bud. As a result of these experiments it was found that the eyes of young Fowl embryos possess, in a remarkable degree, the faculty for self-differentiation in vitro and for “organotypic” growth as defined by Maximow (1925). The previous work on organotypic growth in vitro has already been briefly outlined in the writers’ earlier paper and need not be discussed here. The expenses connected with the experiments described in this communication were met by the Medical Research Council, to whom the writers desire to express their thanks.

FGF-4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 199-206 ◽  
Author(s):  
A. Vogel ◽  
C. Tickle
Keyword(s):  
Posterior Margin ◽  
Anterior Margin ◽  
Marked Decrease ◽  
Mesenchymal Cells ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Posterior Limb ◽  
Vertebrate Limb

The polarizing region is a major signalling tissue involved in patterning the tissues of the vertebrate limb. The polarizing region is located at the posterior margin of the limb bud and can be recognized by its ability to induce additional digits when grafted to the anterior margin of a chick limb bud. The signal from the polarizing region operates at the tip of the bud in the progress zone, a zone of undifferentiated mesenchymal cells, maintained by interactions with the apical ectodermal ridge. A number of observations have pointed to a link between the apical ectodermal ridge and signalling by the polarizing region. To test this possibility, we removed the posterior apical ectodermal ridge of chick wing buds and assayed posterior mesenchyme for polarizing activity. When the apical ectodermal ridge is removed, there is a marked decrease in polarizing activity of posterior cells. The posterior apical ectodermal ridge is known to express FGF-4 and we show that the decrease in polarizing activity of posterior cells of wing buds that normally follows ridge removal can be prevented by implanting a FGF-4-soaked bead. Furthermore, we show that both ectoderm and FGF-4 maintain polarizing activity of limb bud cells in culture.

Products, genetic linkage and limb patterning activity of a murine hedgehog gene

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3339-3353 ◽  
Author(s):  
D.T. Chang ◽  
A. Lopez ◽  
D.P. von Kessler ◽  
C. Chiang ◽  
B.K. Simandl ◽  
...  
Keyword(s):  
Limb Development ◽  
Genetic Linkage ◽  
Pcr Primers ◽  
Amino Acid Identity ◽  
Protein Product ◽  
Proteolytic Processing ◽  
Limb Bud ◽  
Gene Encoding ◽  
Mouse Limb ◽  
Drosophila Embryos

The hedgehog (hh) segmentation gene of Drosophila melanogaster encodes a secreted signaling protein that functions in the patterning of larval and adult structures. Using low stringency hybridization and degenerate PCR primers, we have isolated complete or partial hh-like sequences from a range of invertebrate species including other insects, leech and sea urchin. We have also isolated three mouse and two human DNA fragments encoding distinct hh-like sequences. Our studies have focused upon Hhg-1, a mouse gene encoding a protein with 46% amino acid identity to hh. The Hhg-1 gene, which corresponds to the previously described vhh-1 or sonic class, is expressed in the notochord, ventral neural tube, lung bud, hindgut and posterior margin of the limb bud in developing mouse embryos. By segregation analysis the Hhg-1 gene has been localized to a region in proximal chromosome 5, where two mutations affecting mouse limb development previously have been mapped. In Drosophila embryos, ubiquitous expression of the Hhg-1 gene yields effects upon gene expression and cuticle pattern similar to those observed for the Drosophila hh gene. We also find that cultured quail cells transfected with a Hhg-1 expression construct can induce digit duplications when grafted to anterior or mid-distal but not posterior borders within the developing chick limb; more proximal limb element duplications are induced exclusively by mid-distal grafts. Both in transgenic Drosophila embryos and in transfected quail cells, the Hhg-1 protein product is cleaved to yield two stable fragments from a single larger precursor. The significance of Hhg-1 genetic linkage, patterning activity and proteolytic processing in Drosophila and chick embryos is discussed.

scholarly journals SP1-Mediated Upregulation of Long Noncoding RNA ZFAS1 Involved in Non-syndromic Cleft Lip and Palate via Inactivating WNT/β-Catenin Signaling Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Shiyu Chen ◽  
Zhonglin Jia ◽  
Ming Cai ◽  
Mujie Ye ◽  
Dandan Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Noncoding Rna ◽  
Chondrogenic Differentiation ◽  
Cleft Lip ◽  
Cleft Lip And Palate ◽  
Tissue Sample ◽  
Human Umbilical Cord

Non-syndromic cleft lip and palate (NSCLP) is one of the most common congenital malformations with multifactorial etiology. Although long non-coding RNAs (lncRNAs) have been implicated in the development of lip and palate, their roles in NSCLP are not fully elucidated. This study aimed to investigate how dysregulated lncRNAs contribute to NSCLP. Using lncRNA sequencing, bioinformatics analysis, and clinical tissue sample detection, we identified that lncRNA ZFAS1 was significantly upregulated in NSCLP. The upregulation of ZFAS1 mediated by SP1 transcription factor (SP1) inhibited expression levels of Wnt family member 4 (WNT4) through the binding with CCCTC-binding factor (CTCF), subsequently inactivating the WNT/β-catenin signaling pathway, which has been reported to play a significant role on the development of lip and palate. Moreover, in vitro, the overexpression of ZFAS1 inhibited cell proliferation and migration in human oral keratinocytes and human umbilical cord mesenchymal stem cells (HUC-MSCs) and also repressed chondrogenic differentiation of HUC-MSCs. In vivo, ZFAS1 suppressed cell proliferation and numbers of chondrocyte in the zebrafish ethmoid plate. In summary, these results indicated that ZFAS1 may be involved in NSCLP by affecting cell proliferation, migration, and chondrogenic differentiation through inactivating the WNT/β-catenin signaling pathway.

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