Delta 1-activated notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis

Development ◽  
2000 ◽  
Vol 127 (23) ◽  
pp. 5213-5224 ◽  
Author(s):  
M. Delfini ◽  
E. Hirsinger ◽  
O. Pourquie ◽  
D. Duprez
Keyword(s):  
Limb Development ◽  
Notch Pathway ◽  
Muscle Differentiation ◽  
Notch Signalling ◽  
Limb Bud ◽  
Specific Gene ◽  
Expression Domain ◽  
Helix Loop Helix ◽  
The Right

The myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. In this paper, we show that Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signalling is known to prevent differentiation in different systems and species, we sought to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. We conclude that activation of Notch signalling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stage.

scholarly journals Wdpcp Regulates Proliferation and Differentiation in the Developing Limb via Hedgehog Signaling

2021 ◽  
Author(s):  
Mark Langhans ◽  
Rocky Tuan ◽  
Peter Alexander ◽  
Jingtao Gao ◽  
Bing Wang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Limb Development ◽  
Hedgehog Signaling ◽  
Proliferative Response ◽  
Limb Bud ◽  
In Vitro Assays ◽  
Loss Of Function ◽  
Growth Plates ◽  
Skeletal Phenotype

Abstract Background: Mice with a loss of function mutation in Wdpcp were described previously to display severe birth defects in the developing heart, neural tube, and limb buds. Further characterization of the skeletal phenotype of Wdpcp null mice was limited by perinatal lethality. Results: We utilized Prx1-Cre mice to generate limb bud mesenchyme specific deletion of Wdpcp. These mice recapitulated the appendicular skeletal phenotype of the Wdpcp null mice including polydactyl and limb bud signaling defects. Examination of later stages of limb development demonstrated decreased size of cartilage anlagen, delayed calcification, and abnormal growth plates. Utilizing in vitro assays, we demonstrated that loss of Wdpcp in skeletal progenitors lead to loss of hedgehog signaling responsiveness and associated proliferative response. In vitro chondrogenesis assays showed this loss of hedgehog and proliferative response was associated with decreased expression of early chondrogenic marker N-Cadherin. E14.5 forelimbs demonstrated delayed ossification and expression of osteoblast markers Runx2 and Sp7. P0 growth plates demonstrated loss of hedgehog signaling markers and expansion of the hypertrophic zones of the growth plate. In vitro osteogenesis assays demonstrated decreased osteogenic differentiation of Wdpcp null mesenchymal progenitors in response to hedgehog stimulation. Conclusions: These findings demonstrate how Wdpcp and associated regulation of the hedgehog signaling pathway plays an important role at multiple stages of skeletal development. Wdpcp is necessary for positive regulation of hedgehog signaling and associated proliferation is key to the initiation of chondrogenesis. At later stages, Wdpcp facilitates the robust hedgehog response necessary for chondrocyte hypertrophy and osteogenic differentiation.

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

Development ◽  
2001 ◽  
Vol 128 (1) ◽  
pp. 107-116 ◽  
Author(s):  
E. Hirsinger ◽  
P. Malapert ◽  
J. Dubrulle ◽  
M.C. Delfini ◽  
D. Duprez ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Muscle Differentiation ◽  
Detailed Examination ◽  
Notch Signalling ◽  
Myogenic Cells ◽  
Myogenic Markers ◽  
Notch Activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.

Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway

Development ◽  
1998 ◽  
Vol 125 (11) ◽  
pp. 2041-2051 ◽  
Author(s):  
D.B. Sparrow ◽  
W.C. Jen ◽  
S. Kotecha ◽  
N. Towers ◽  
C. Kintner ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Ectopic Expression ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Xenopus Embryo ◽  
Paraxial Mesoderm ◽  
Marker Genes ◽  
Transcription Activator ◽  
Presomitic Mesoderm ◽  
Helix Loop Helix

The presomitic mesoderm of vertebrates undergoes a process of segmentation in which cell-cell interactions mediated by the Notch family of receptors and their associated ligands are involved. The vertebrate homologues of Drosophila Δ are expressed in a dynamic, segmental pattern within the presomitic mesoderm, and alterations in the function of these genes leads to a perturbed pattern of somite segmentation. In this study we have characterised Thylacine 1 which encodes a basic helix-loop-helix class transcription activator. Expression of Thylacine is restricted to the presomitic mesoderm, localising to the anterior half of several somitomeres in register with domains of X-Delta-2 expression. Ectopic expression of Thylacine in embryos causes segmentation defects similar to those seen in embryos in which Notch signalling is altered, and these embryos also show severe disruption in the expression patterns of the marker genes X-Delta-2 and X-ESR5 within the presomitic mesoderm. Finally, the expression of Thylacine is altered in embryos when Notch signalling is perturbed. These observations suggest strongly that Thylacine 1 has a role in the segmentation pathway of the Xenopus embryo, by interacting with the Notch signalling pathway.

Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Multiple Myeloma Patients Is Associated with a Failure In the Deactivation of the NOTCH Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1894-1894
Author(s):  
Song Xu ◽  
Jinsong Hu ◽  
Dehui Xu ◽  
Isabelle Vande Broek ◽  
Xavier Leleu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Matrix Mineralization ◽  
Alp Activity ◽  
Hes1 Expression

Abstract Abstract 1894 Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The transcription factor LAG-1/CSL plays a Notch-independent role in controlling terminal differentiation, fate maintenance, and plasticity of serotonergic chemosensory neurons

PLoS Biology ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. e3001334
Author(s):  
Miren Maicas ◽  
Ángela Jimeno-Martín ◽  
Andrea Millán-Trejo ◽  
Mark J. Alkema ◽  
Nuria Flames
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Target Genes ◽  
Cell Signalling ◽  
Notch Pathway ◽  
Receptor Activation ◽  
Notch Signalling ◽  
Notch Receptor ◽  
Specific Gene ◽  
Effector Genes

During development, signal-regulated transcription factors (TFs) act as basal repressors and upon signalling through morphogens or cell-to-cell signalling shift to activators, mediating precise and transient responses. Conversely, at the final steps of neuron specification, terminal selector TFs directly initiate and maintain neuron-type specific gene expression through enduring functions as activators. C. elegans contains 3 types of serotonin synthesising neurons that share the expression of the serotonin biosynthesis pathway genes but not of other effector genes. Here, we find an unconventional role for LAG-1, the signal-regulated TF mediator of the Notch pathway, as terminal selector for the ADF serotonergic chemosensory neuron, but not for other serotonergic neuron types. Regulatory regions of ADF effector genes contain functional LAG-1 binding sites that mediate activation but not basal repression. lag-1 mutants show broad defects in ADF effector genes activation, and LAG-1 is required to maintain ADF cell fate and functions throughout life. Unexpectedly, contrary to reported basal repression state for LAG-1 prior to Notch receptor activation, gene expression activation in the ADF neuron by LAG-1 does not require Notch signalling, demonstrating a default activator state for LAG-1 independent of Notch. We hypothesise that the enduring activity of terminal selectors on target genes required uncoupling LAG-1 activating role from receiving the transient Notch signalling.

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.

The preliminary characterization of mitogens secreted by embryonic chick wing bud tissues in vitro

Development ◽  
1986 ◽  
Vol 93 (1) ◽  
pp. 257-265
Author(s):  
K. M. Bell
Keyword(s):  
Growth Factors ◽  
Limb Development ◽  
Rat Kidney ◽  
Active Regions ◽  
Limb Bud ◽  
Embryonic Chick ◽  
Bovine Endothelial Cells ◽  
Normal Rat ◽  
Wing Bud

Embryonic chick wing bud tissues secrete diffusible mitogens when cultured in vitro (Bell & McLachlan, 1985). These molecules may play an important role in limb development since media conditioned by morphogenetically active regions of the wing bud possess greater mitogenic activity than media conditioned by non-morphogenetic regions. These studies show that while the chick-derived growth factors were mitogenic for mouse-derived NIH 3T3,10T1/2 and NR6 cells and chick limb bud cells, they did not stimulate DNA synthesis in 3B11, PC13 END, normal rat kidney or bovine endothelial cells. Furthermore, the effects of the chick-derived mitogens were synergistically enhanced by insulin and PGF2α but remained unaffected by ECDGF, EGF, FGF and MSA. These findings indicate that embryonic chick limb bud cells synthesize and secrete growth factors which resemble in function other well-characterized growth factors and in particular PDGF.

scholarly journals Comparative and evolutionary analyses reveal conservation and divergence of the notch pathway in lophotrochozoa

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xin He ◽  
Fucun Wu ◽  
Linlin Zhang ◽  
Li Li ◽  
Guofan Zhang
Keyword(s):  
Molecular Basis ◽  
Notch Pathway ◽  
Sequence Divergence ◽  
Morphological Diversity ◽  
Notch Signalling ◽  
Specific Gene ◽  
Functional Diversification ◽  
Notch Signalling Pathway ◽  
Evolutionary Studies ◽  
Comprehensive Study

AbstractLophotrochozoan species exhibit wide morphological diversity; however, the molecular basis underlying this diversity remains unclear. Here, we explored the evolution of Notch pathway genes across 37 metazoan species via phylogenetic and molecular evolutionary studies with emphasis on the lophotrochozoans. We displayed the components of Notch pathway in metazoans and found that Delta and Hes/Hey-related genes, as well as their functional domains, are duplicated in lophotrochozoans. Comparative transcriptomics analyses allow us to pinpoint sequence divergence of multigene families in the Notch signalling pathway. We identified the duplication mechanism of a mollusc-specific gene, Delta2, and found it displayed complementary expression throughout development. Furthermore, we found the functional diversification not only in expanded genes in the Notch pathway (Delta and Hes/Hey-related genes), but also in evolutionary conservative genes (Notch, Presenilin, and Su(H)). Together, this comprehensive study demonstrates conservation and divergence within the Notch pathway, reveals evolutionary relationships among metazoans, and provides evidence for the occurrence of developmental diversity in lophotrochozoans, as well as a basis for future gene function studies.

The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2461-2470 ◽  
Author(s):  
J. Charite ◽  
D.G. McFadden ◽  
E.N. Olson
Keyword(s):  
Transcription Factor ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Target Genes ◽  
Ectopic Expression ◽  
Tissue Growth ◽  
Limb Bud ◽  
Bhlh Transcription Factor ◽  
Expression Domain ◽  
Zone Of Polarizing Activity

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

scholarly journals Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells

2018 ◽  
Vol 25 (2) ◽  
pp. 131-144 ◽  
Author(s):  
Y Capodanno ◽  
F O Buishand ◽  
L Y Pang ◽  
J Kirpensteijn ◽  
J A Mol ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Targeted Therapies ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Treatment Modalities ◽  
Stem Cell Markers ◽  
Gamma Secretase

Insulinomas (INS) are the most common neuroendocrine pancreatic tumours in humans and dogs. The long-term prognosis for malignant INS is still poor due to a low success rate of the current treatment modalities, particularly chemotherapy. A better understanding of the molecular processes underlying the development and progression of INS is required to develop novel targeted therapies. Cancer stem cells (CSCs) are thought to be critical for the engraftment and chemoresistance of many tumours, including INS. This study was aimed to characterise and target INS CSCs in order to develop novel targeted therapies. Highly invasive and tumourigenic human and canine INS CSC-like cells were successfully isolated. These cells expressed stem cell markers (OCT4,SOX9, SOX2, CD133 and CD34), exhibited greater resistance to 5-fluorouracil (5-FU) and demonstrated a more invasive and tumourigenic phenotypein vivocompared to bulk INS cells. Here, we demonstrated that Notch-signalling-related genes (NOTCH2andHES1)were overexpressed in INS CSC-like cells. Protein analysis showed an active NOTCH2-HES1 signalling in INS cell lines, especially in cells resistant to 5-FU. Inhibition of the Notch pathway, using a gamma secretase inhibitor (GSI), enhanced the sensitivity of INS CSC-like cells to 5-FU. When used in combination GSI and 5-FU, the clonogenicityin vitroand the tumourigenicityin vivoof INS CSC-like cells were significantly reduced. These findings suggested that the combined strategy of Notch signalling inhibition and 5-FU synergistically attenuated enriched INS CSC populations, providing a rationale for future therapeutic exploitation.

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