Regulation of Hedgehog Signaling in Chicken Embryonic Stem Cells Differentiation Into Male Germ Cells (<i>Gallus</i>)

Chen, Hao; Zuo, Qisheng; Wang, Yinjie; Ahmed, Mahmoud F.; Song, Jiuzhou; Zhang, Yani

doi:10.1002/jcb.25796

Cited by 11 publications

(15 citation statements)

References 24 publications

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“…Moreover, Wnt signaling pathway, a downstream signaling pathway of the Hedgehog signaling pathway plays a role in signal transduction in concert with other signal transduction pathways . It has been previously reported that Hedgehog signaling positively regulates the differentiation of chicken male germ cells . Thus, there is a crosstalk between Wnt signaling and Hedgehog signaling during the differentiation of male germ cells.…”

Section: Discussionmentioning

confidence: 99%

Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a

Wang

Zhang

et al. 2017

J of Cellular Biochemistry

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In this study, we investigated the mechanism of signaling pathway-mediated differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) in chicken. The Wnt signaling pathway was identified based on previous RNA Sequencing results and was proven a crucial signaling pathway that participates in the differentiation of ESCs into SSCs. In retinoic acid (RA) induction experiments in vitro, we found that Wnt signaling expression was inhibited by Wnt5a-shRNA, resulting in decreased expression of corresponding marker genes in SSCs, C-kit, Cvh, integrin α6 and integrin β1, but it was significantly promoted by RA treatment. Immunofluorescence assay showed that percentage of C-kit, Cvh, and integrin α6 and integrin β1-positive cells in RA treatment group and Wnt5a overexpression group was significantly higher than that in Wnt5a signaling interference group. Results of fluorescence-activated cell sorting analysis (FACS) also showed that proportion of germ-like cells was reduced by 14.3% (from 18.3% to 4.0%) at day 4 and 15.4% (from 18.6% to 3.2%) at day 12 after transfection, respectively. In experiments in vivo, shRNA-Wnt5a was stably expressed in fertilized chicken embryos and significantly reduced germ cell formation by 11.3% (from 21.7% to 10.4%) and 3.7% (6.4% from 10.1%). Results of quantitative PCR (qRT-PCR) and western blot assays showed that the expression of some specific germ cell marker genes, integrin α6 and integrin β1, was significantly suppressed following Wnt5a signaling interference in vivo. Taken together, our study suggests that Wnt signaling pathway could regulate positively the differentiation of chicken ESCs into SSCs through Wnt5a.

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Section: Discussionmentioning

confidence: 99%

Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a

Wang

Zhang

et al. 2017

J of Cellular Biochemistry

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“…Here, we found that C2EIP interacts with PTCH2 at the intracellular membrane and thus speculated that C2EIP may regulate PGC differentiation by influencing HH signaling pathway activation. Therefore, we constructed lentiviral shRNA-IHH 15 and shRNA-PTCH2 vectors to investigate the function of HH signaling in the process of PGC generation (Supplementary Figure 6a-c ). ESC were transfected with shRNA-IHH or shRNA-PTCH2 in parallel with in vitro RA induction experiments.…”

Section: Resultsmentioning

confidence: 99%

“…This hypothesis is supported by earlier studies showing that CRISPR/Cas9-mediated knockout of C2EIP in chick embryos significantly limited their development 13 and reduced PGC generation efficiency, and C2EIP overexpression promoted PGC generation. The GST pull-down assay showed that C2EIP interacted with PTCH2, an important membrane protein of the Hedgehog (HH) signaling pathway, which can upregulate germ cell differentiation and PGC generation 15 . However, whether C2EIP function is associated with HH signal activation remains unknown.…”

Section: Introductionmentioning

confidence: 99%

Interaction of the primordial germ cell-specific protein C2EIP with PTCH2 directs differentiation of embryonic stem cells via HH signaling activation

et al. 2018

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Although many marker genes for germ cell differentiation have been identified, genes that specifically regulate primordial germ cell (PGC) generation are more difficult to determine. In the current study, we confirmed that C2EIP is a PGC marker gene that regulates differentiation by influencing the expression of pluripotency-associated genes such as Oct4 and Sox2. Knockout of C2EIP during embryonic development reduced PGC generation efficiency 1.5-fold, whereas C2EIP overexpression nearly doubled the generation efficiency both in vitro and in vivo. C2EIP encodes a cytoplasmic protein that interacted with PTCH2 at the intracellular membrane, promoted PTCH2 ubiquitination, activated the Hedgehog (HH) signaling pathway via competitive inhibition of the GPCR-like protein SMO, and positively regulated PGC generation. Activation and expression of C2EIP are regulated by the transcription factor STAT1, histone acetylation, and promoter methylation. Our data suggest that C2EIP is a novel, specific indicator of PGC generation whose gene product regulates embryonic stem cell differentiation by activating the HH signaling pathway via PTCH2 modification.

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“…Exploring the potential mechanism of the regulation of FGSC proliferation or stemness is beneficial to understanding how normal egg and ovary function is maintained. As a canonical signaling pathway, the hedgehog (Hh) pathway plays a key role in the regulation of stem cell proliferation and embryonic development, as well as cellular communication [8][9][10]. Studies showed that mice were subjected to serious damage in folliculogenesis when a mutation was introduced in the Hh pathway [11].…”

Section: Introductionmentioning

confidence: 99%

Hedgehog pathway inhibition causes primary follicle atresia and decreases female germline stem cell proliferation capacity or stemness

et al. 2019

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Background: Follicle depletion is one of the causes of premature ovarian failure (POF) and primary ovarian insufficiency (POI). Hence, maintenance of a certain number of female germline stem cells (FGSCs) is optimal to produce oocytes and replenish the primordial follicle pool. The mechanism that regulates proliferation or stemness of FGSCs could contribute to restoring ovarian function, but it remains uncharacterized in postnatal mammalian ovaries. This study aims to investigate the mechanism by which inhibiting the activity of the hedgehog (Hh) signaling pathway regulates follicle development and FGSC proliferation. Methods and results: To understand the role of the Hh pathway in ovarian aging, we measured Hh signaling activity at different reproductive ages and the correlation between them in physiological and pathological mice. Furthermore, we evaluated the follicle number and development and the changes in FGSC proliferation or stemness after blocking the Hh pathway in vitro and in vivo. In addition, we aimed to explain one of the mechanisms for the FGSC phenotype changes induced by treatment with the Hh pathway-specific inhibitor GANT61 via oxidative stress and apoptosis. The results show that the activity of Hh signaling is decreased in the ovaries in physiological aging and POF models, which is consistent with the trend of expression levels of the germline stem cell markers Mvh and Oct4. In vitro, blocking the Hh pathway causes follicular developmental disorders and depletes ovarian germ cells and FGSCs after treating ovaries with GANT61. The proliferation or stemness of cultured primary FGSCs is reduced when Hh activity is blocked. Our results show that the antioxidative enzyme level and the ratio of Bcl-2/Bax decrease, the expression level of caspase 3 increases, the mitochondrial membrane potential is abnormal, and ROS accumulate in this system. Conclusions: We observed that the inhibition of the Hh signaling pathway with GANT61 could reduce primordial follicle number and decrease FGSC reproductive capacity or stemness through oxidative damage and apoptosis.

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Regulation of Hedgehog Signaling in Chicken Embryonic Stem Cells Differentiation Into Male Germ Cells (Gallus)

Cited by 11 publications

References 24 publications

Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a

Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a

Interaction of the primordial germ cell-specific protein C2EIP with PTCH2 directs differentiation of embryonic stem cells via HH signaling activation

Hedgehog pathway inhibition causes primary follicle atresia and decreases female germline stem cell proliferation capacity or stemness

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