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1.
Genome Biol ; 25(1): 58, 2024 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409044

RESUMO

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.


Assuntos
Degrons , Ácidos Indolacéticos , Humanos , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/metabolismo , Proteínas/metabolismo , Linhagem Celular , Proteólise
2.
Eur J Endocrinol ; 189(2): 271-280, 2023 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-37619992

RESUMO

OBJECTIVE: Congenital hypogonadotropic hypogonadism (CHH) is a rare, genetically heterogeneous reproductive disorder caused by gonadotropin-releasing hormone (GnRH) deficiency. Approximately half of CHH patients also have decreased or absent sense of smell, that is, Kallmann syndrome (KS). We describe a patient with White-Sutton syndrome (developmental delay and autism spectrum disorder) and KS due to a heterozygous de novo mutation in POGZ (c.2857C>T, p.(Gln953*)), a gene encoding pogo transposable element derived with zinc finger domain, which acts as a transcriptomic regulator of neuronal networks. DESIGN AND METHODS: We modeled the role of POGZ in CHH by generating 2 clonal human pluripotent stem cell lines with CRISPR/Cas9, carrying either the heterozygous patient mutation (H11 line) or a homozygous mutation (c.2803-2906del; p.E935Kfs*7 encoding a truncated POGZ protein; F6del line). RESULTS: During the differentiation to GnRH neurons, neural progenitors derived from F6del line displayed severe proliferation defect, delayed wound-healing capacity, downregulation of intermediate progenitor neuron genes TBR1 and TBR2, and immature neuron markers PAX6 and TUBB3 and gave rise to fewer neurons with shorter neurites and less neurite branch points compared to the WT and H11 lines (P < .005). Both lines, however, could be successfully differentiated to GnRH neurons. CONCLUSIONS: In conclusion, this is the first report on the overlap between White-Sutton syndrome and CHH. POGZ mutations do not hinder GnRH neuron formation but may cause CHH/KS by affecting the size and motility of the anterior neural progenitor pool and neurite outgrowth.


Assuntos
Transtorno do Espectro Autista , Síndrome de Kallmann , Humanos , Síndrome de Kallmann/genética , Neurônios , Hormônio Liberador de Gonadotropina , Mutação/genética
3.
Stem Cells ; 40(12): 1107-1121, 2022 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36153707

RESUMO

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons lay the foundation for human development and reproduction; however, the critical cell populations and the entangled mechanisms underlying the development of human GnRH neurons remain poorly understood. Here, by using our established human pluripotent stem cell-derived GnRH neuron model, we decoded the cellular heterogeneity and differentiation trajectories at the single-cell level. We found that a glutamatergic neuron population, which generated together with GnRH neurons, showed similar transcriptomic properties with olfactory sensory neuron and provided the migratory path for GnRH neurons. Through trajectory analysis, we identified a specific gene module activated along the GnRH neuron differentiation lineage, and we examined one of the transcription factors, DLX5, expression in human fetal GnRH neurons. Furthermore, we found that Wnt inhibition could increase DLX5 expression and improve the GnRH neuron differentiation efficiency through promoting neurogenesis and switching the differentiation fates of neural progenitors into glutamatergic neurons/GnRH neurons. Our research comprehensively reveals the dynamic cell population transition and gene regulatory network during GnRH neuron differentiation.


Assuntos
Hormônio Liberador de Gonadotropina , Células-Tronco Pluripotentes , Humanos , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Via de Sinalização Wnt/genética , Neurônios/metabolismo , Diferenciação Celular/genética , Células-Tronco Pluripotentes/metabolismo
4.
Dis Model Mech ; 15(8)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35833364

RESUMO

Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time- and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (P<0.05, Rs=0.49). However, cells treated with 100 ng/ml FGF8 for 2 days induced the expression of genes, such as FOXG1, ETV5 and SPRY2, and continued FGF8 treatment induced the dynamic expression of several other genes. Moreover, during exposure to FGF8, FGFR1 localized to the cell surface and its specific inhibition with the FGFR1 inhibitor PD166866 reduced expression of GNRH1 (P<0.05). In neurons, FGFR1 also localized to the nucleus. Our results suggest that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny. This article has an associated First Person interview with the first author of the paper.


Assuntos
Hormônio Liberador de Gonadotropina , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fator 8 de Crescimento de Fibroblasto/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Neurônios/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
5.
Dis Model Mech ; 13(3)2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-31996360

RESUMO

Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.


Assuntos
Genes Reporter , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Feto/citologia , Fator 8 de Crescimento de Fibroblasto/farmacologia , Humanos , Hipogonadismo/genética , Proteínas com Homeodomínio LIM/metabolismo , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
6.
Cell Mol Biol Lett ; 23: 5, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29456557

RESUMO

BACKGROUND: Perineuronal nets (PNNs), which are localized around neurons during development, are specialized forms of neural extracellular matrix with neuroprotective and plasticity-regulating roles. Hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R (TNR) and aggrecan (ACAN) are key elements of PNNs. In diseases characterized by neuritogenesis defects, the expression of these proteins is known to be downregulated, suggesting that PNNs may have a role in neural differentiation. METHODS: In this study, the mRNA and protein levels of HAPLN1, TNR and ACAN were determined and compared at specific time points of neural differentiation. We used PC12 cells as the in vitro model because they reflect this developmental process. RESULTS: On day 7, the HAPLN1 mRNA level showed a 2.9-fold increase compared to the non-differentiated state. However, the cellular HAPLN1 protein level showed a decrease, indicating that the protein may have roles in neural differentiation, and may be secreted during the early period of differentiation. By contrast, TNR mRNA and protein levels remained unchanged, and the amount of cellular ACAN protein showed a 3.7-fold increase at day 7. These results suggest that ACAN may be secreted after day 7, possibly due to its large amount of post-translational modifications. CONCLUSIONS: Our results provide preliminary data on the expression of PNN elements during neural differentiation. Further investigations will be performed on the role of these elements in neurological disease models.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Neurogênese , Neurônios/metabolismo , Agrecanas/genética , Agrecanas/metabolismo , Animais , Proteínas da Matriz Extracelular/genética , Neurogênese/genética , Neurônios/citologia , Células PC12 , Proteoglicanas/genética , Proteoglicanas/metabolismo , Ratos , Tenascina/genética , Tenascina/metabolismo
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