RESUMO
Microglia are the resident macrophages of the central nervous system (CNS). Gene expression profiling has identified Sall1, which encodes a transcriptional regulator, as a microglial signature gene. We found that Sall1 was expressed by microglia but not by other members of the mononuclear phagocyte system or by other CNS-resident cells. Using Sall1 for microglia-specific gene targeting, we found that the cytokine receptor CSF1R was involved in the maintenance of adult microglia and that the receptor for the cytokine TGF-ß suppressed activation of microglia. We then used the microglia-specific expression of Sall1 to inducibly inactivate the murine Sall1 locus in vivo, which resulted in the conversion of microglia from resting tissue macrophages into inflammatory phagocytes, leading to altered neurogenesis and disturbed tissue homeostasis. Collectively, our results show that transcriptional regulation by Sall1 maintains microglial identity and physiological properties in the CNS and allows microglia-specific manipulation in vivo.
Assuntos
Microglia/fisiologia , Fagócitos/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Inativação Gênica , Homeostase/genética , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/genética , Fatores de Transcrição/genética , Transcriptoma , Fator de Crescimento Transformador beta/metabolismoRESUMO
The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma , Fatores de Transcrição , Animais , Camundongos , Diferenciação Celular , Cromatina/metabolismo , Expressão Gênica , Mesoderma/metabolismo , Somitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.
Assuntos
Diferenciação Celular , Hemangioblastos , Células-Tronco Hematopoéticas , Fator de Células-Tronco , Trombopoetina , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Trombopoetina/metabolismo , Fator de Células-Tronco/metabolismo , Hemangioblastos/metabolismo , Hemangioblastos/citologia , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Transdução de Sinais , Hematopoese/fisiologia , Desenvolvimento Embrionário , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Fígado/embriologia , Fígado/metabolismo , Fígado/citologiaRESUMO
Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Glucose/metabolismo , Glicólise/genética , Botões de Extremidades/metabolismo , Camundongos KnockoutRESUMO
TET family enzymes successively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to eventual demethylation. 5hmC and TET enzymes occupy distinct chromatin regions, suggesting unknown mechanisms controlling the fate of 5hmC within diverse chromatin environments. Here, we report that SALL4A preferentially associates with 5hmC in vitro and occupies enhancers in mouse embryonic stem cells in a largely TET1-dependent manner. Although most 5hmC at SALL4A peaks undergoes further oxidation, this process is abrogated upon deletion of Sall4 gene, with a concomitant reduction of TET2 at these regions. Thus, SALL4A facilitates further oxidation of 5hmC at its binding sites, which requires its 5hmC-binding activity and TET2, supporting a collaborative action between SALL4A and TET proteins in regulating stepwise oxidation of 5mC at enhancers. Our study identifies SALL4A as a 5hmC binder, which facilitates 5hmC oxidation by stabilizing TET2 association, thereby fine-tuning expression profiles of developmental genes in mouse embryonic stem cells.
Assuntos
5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Metilação de DNA , Dioxigenases , Elementos Facilitadores Genéticos/fisiologia , Camundongos , Oxirredução , Proteínas Proto-Oncogênicas/metabolismo , Transcrição GênicaRESUMO
An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.
Assuntos
Heterocromatina/genética , Heterocromatina/metabolismo , Células-Tronco Embrionárias Murinas/fisiologia , Proteína Homeobox Nanog/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Regulação para Baixo , Deleção de Genes , Camundongos , Proteína Homeobox Nanog/genética , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Kinesin family member 26b (Kif26b) is essential for kidney development, and its deletion in mice leads to kidney agenesis. However, the roles of this gene in adult settings remain elusive. Thus, this study aims to investigate the role of Kif26b in the progression of renal fibrosis. A renal fibrosis model with adenine administration using Kif26b heterozygous mice and wild-type mice was established. Renal fibrosis and the underlying mechanism were investigated. The underlying pathways and functions of Kif26b were evaluated in an in vitro model using primary renal fibroblasts. Kif26b heterozygous mice were protected from renal fibrosis with adenine administration. Renal expressions of connective tissue growth factor (CTGF) and myofibroblast accumulation were reduced in Kif26b heterozygous mice. The expression of nonmuscle myosin heavy chain II (NMHCII), which binds to the C-terminus of Kif26b protein, was also suppressed in Kif26b heterozygous mice. The in vitro study revealed reduced expressions of CTGF, α-smooth muscle actin, and myosin heavy chain 9 (Myh9) via transfection with siRNAs targeting Kif26b in renal fibroblasts (RFB). RFBs, which were transfected by the expression vector of Kif26b, demonstrated higher expressions of these genes than non-transfected cells. Finally, Kif26b suppression and NMHCII blockage led to reduced abilities of migration and collagen gel contraction in renal fibroblasts. Taken together, Kif26b contributes to the progression of interstitial fibrosis via migration and myofibroblast differentiation through Myh9 in the renal fibrosis model. Blockage of this pathway at appropriate timing might be a therapeutic approach for renal fibrosis.
Assuntos
Rim , Cinesinas , Miofibroblastos , Animais , Camundongos , Actinas/genética , Actinas/metabolismo , Adenina/metabolismo , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Fibrose , Rim/metabolismo , Cinesinas/genética , Miofibroblastos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Diferenciação Celular , Movimento CelularRESUMO
Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/crescimento & desenvolvimento , Rim/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula , Regulação para Baixo , Rim/citologia , Rim/embriologia , Glomérulos Renais/citologia , Glomérulos Renais/embriologia , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/metabolismo , Transplante de Rim , Túbulos Renais/citologia , Túbulos Renais/embriologia , Túbulos Renais/crescimento & desenvolvimento , Túbulos Renais/metabolismo , Camundongos , Podócitos/citologia , Podócitos/metabolismo , RNA-Seq , Análise de Célula Única , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Microglia play many critical roles in neural development. Recent single-cell RNA-sequencing studies have found diversity of microglia both across different stages and within the same stage in the developing brain. However, how such diversity is controlled during development is poorly understood. In this study, we first found the expression of the macrophage mannose receptor CD206 in early-stage embryonic microglia on mouse brain sections. This expression showed a sharp decline between E12.5 and E13.5 across the central nervous system. We next tested the roles of the microglia-expressed zinc finger transcription factor SALL1 in this early transition of gene expression. By deleting Sall1 specifically in microglia, we found that many microglia continued to express CD206 when it is normally downregulated. In addition, the mutant microglia continued to show less ramified morphology in comparison with controls even into postnatal stages. Thus, SALL1 is required for early microglia to transition into a more mature status during development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Microglia , Neurogênese , Fatores de Transcrição , Dedos de Zinco , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Microglia/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Dedos de Zinco/fisiologiaRESUMO
Bi-potential neuromesodermal progenitors (NMPs) produce both neural and paraxial mesodermal progenitors in the trunk and tail during vertebrate body elongation. We show that Sall4, a pluripotency-related transcription factor gene, has multiple roles in regulating NMPs and their descendants in post-gastrulation mouse embryos. Sall4 deletion using TCre caused body/tail truncation, reminiscent of early depletion of NMPs, suggesting a role of Sall4 in NMP maintenance. This phenotype became significant at the time of the trunk-to-tail transition, suggesting that Sall4 maintenance of NMPs enables tail formation. Sall4 mutants exhibit expanded neural and reduced mesodermal tissues, indicating a role of Sall4 in NMP differentiation balance. Mechanistically, we show that Sall4 promotion of WNT/ß-catenin signaling contributes to NMP maintenance and differentiation balance. RNA-Seq and SALL4 ChIP-Seq analyses support the notion that Sall4 regulates both mesodermal and neural development. Furthermore, in the mesodermal compartment, genes regulating presomitic mesoderm differentiation are downregulated in Sall4 mutants. In the neural compartment, we show that differentiation of NMPs towards post-mitotic neuron is accelerated in Sall4 mutants. Our results collectively provide evidence supporting the role of Sall4 in regulating NMPs and their descendants.
Assuntos
Padronização Corporal/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/fisiologia , Mesoderma/citologia , Mesoderma/embriologia , Células-Tronco Neurais/citologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Mesoderma/metabolismo , Camundongos , Células-Tronco Neurais/fisiologia , Gravidez , Via de Sinalização Wnt/fisiologiaRESUMO
PURPOSE OF REVIEW: During embryogenesis, the kidney is mainly generated from three progenitor cells; nephron progenitors, ureteric bud progenitors and stromal progenitors. Mutual interactions of the all three progenitor populations are essential to form a functional kidney with the higher-order structure. Pluripotent stem cells have potential to differentiate into all cell types of the animal body, including the kidney. In this review, we will summarize recent advances in reconstructing kidney organoids from pluripotent stem cells. RECENT FINDINGS: In the past years, major advances were reported to induce nephron and ureteric bud progenitors from pluripotent stem cells in mice and humans, and to create kidney organoids of nephron and/or ureteric bud-derived collecting duct tissues in vitro. These kidney organoid technologies were applied to high-throughput genetic screenings and small chemical screenings to identify key factors for kidney development and disease. Furthermore, a novel method was established to induce stromal progenitors from pluripotent stem cells, leading to creation of kidney organoids with the higher-order structures completely derived from pluripotent stem cells. SUMMARY: These advances in kidney organoids from pluripotent stem cells should lay a foundation to establish a novel therapy for kidney disease, which ultimately eliminate the need of dialysis and kidney transplantation for patients with kidney disease in the future.
Assuntos
Nefropatias , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Humanos , Rim/metabolismo , Nefropatias/metabolismo , Camundongos , Organoides , Diálise RenalRESUMO
Newly synthesised glycoproteins enter the rough endoplasmic reticulum through a translocation pore. The translocon associated protein (TRAP) complex is located close to the pore. In a patient with a homozygous start codon variant in TRAPγ (SSR3), absence of TRAPγ causes disruption of the TRAP complex, impairs protein translocation into the endoplasmic reticulum and affects transport, for example, into the brush-border membrane. Furthermore, we observed an unbalanced non-occupancy of N-glycosylation sites. The major clinical features are intrauterine growth retardation, facial dysmorphism, congenital diarrhoea, failure to thrive, pulmonary disease and severe psychomotor disability.
Assuntos
Retículo Endoplasmático Rugoso/genética , Retardo do Crescimento Fetal/genética , Glicoproteínas/genética , Fosfatase Ácida Resistente a Tartarato/genética , Criança , Pré-Escolar , Diarreia/genética , Diarreia/patologia , Insuficiência de Crescimento/genética , Insuficiência de Crescimento/patologia , Feminino , Retardo do Crescimento Fetal/patologia , Glicoproteínas/biossíntese , Glicosilação , Humanos , Lactente , Recém-Nascido , Pneumopatias/genética , Pneumopatias/patologia , Masculino , Transtornos Psicomotores/genética , Transtornos Psicomotores/patologia , Fosfatase Ácida Resistente a Tartarato/deficiênciaRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease leading to renal failure, wherein multiple cysts form in renal tubules and collecting ducts derived from distinct precursors: the nephron progenitor and ureteric bud (UB), respectively. Recent progress in induced pluripotent stem cell (iPSC) biology has enabled cyst formation in nephron progenitor-derived human kidney organoids in which PKD1 or PKD2, the major causative genes for ADPKD, are deleted. However, cysts have not been generated in UB organoids, despite the prevalence of collecting duct cysts in patients with ADPKD. METHODS: CRISPR-Cas9 technology deleted PKD1 in human iPSCs and the cells induced to differentiate along pathways leading to formation of either nephron progenitor or UB organoids. Cyst formation was investigated in both types of kidney organoid derived from PKD1-deleted iPSCs and in UB organoids generated from iPSCs from a patient with ADPKD who had a missense mutation. RESULTS: Cysts formed in UB organoids with homozygous PKD1 mutations upon cAMP stimulation and, to a lesser extent, in heterozygous mutant organoids. Furthermore, UB organoids generated from iPSCs from a patient with ADPKD who had a heterozygous missense mutation developed cysts upon cAMP stimulation. CONCLUSIONS: Cysts form in PKD1 mutant UB organoids as well as in iPSCs derived from a patient with ADPKD. The organoids provide a robust model of the genesis of ADPKD.
Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Néfrons/patologia , Organoides/patologia , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Ureter/patologia , Técnicas de Cultura de Células , Humanos , Mutação de Sentido Incorreto/genética , Rim Policístico Autossômico Dominante/patologiaRESUMO
From March 27-29 2017, the RIKEN Center for Developmental Biology held a symposium entitled 'Towards Understanding Human Development, Heredity, and Evolution' in Kobe, Japan. Recent advances in technologies including stem cell culture, live imaging, single-cell approaches, next-generation sequencing and genome editing have led to an expansion in our knowledge of human development. Organized by Yoshiya Kawaguchi, Mitinori Saitou, Mototsugu Eiraku, Tomoya Kitajima, Fumio Matsuzaki, Takashi Tsuji and Edith Heard, the symposium covered a broad range of topics including human germline development, epigenetics, organogenesis and evolution. This Meeting Review provides a summary of this timely and exciting symposium, which has convinced us that we are moving into the era of science targeted on humans.
Assuntos
Desenvolvimento Humano , Animais , Evolução Biológica , Biologia do Desenvolvimento , Desenvolvimento Embrionário , Epigênese Genética , Hereditariedade , Humanos , OrganogêneseRESUMO
BACKGROUND: Previous research has elucidated the signals required to induce nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling the generation of kidney organoids. However, selectively controlling differentiation of NPCs to podocytes has been a challenge. METHODS: We investigated the effects of various growth factors in cultured mouse embryonic NPCs during three distinct steps of nephron patterning: from NPC to pretubular aggregate, from the latter to epithelial renal vesicle (RV), and from RV to podocyte. We then applied the findings to human PSC-derived NPCs to establish a method for selective induction of human podocytes. RESULTS: Mouse NPC differentiation experiments revealed that phase-specific manipulation of Wnt and Tgf-ß signaling is critical for podocyte differentiation. First, optimal timing and intensity of Wnt signaling were essential for mesenchymal-to-epithelial transition and podocyte differentiation. Then, inhibition of Tgf-ß signaling supported domination of the RV proximal domain. Inhibition of Tgf-ß signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resultant protocol enabled successful induction of human podocytes from PSCs with >90% purity. The induced podocytes exhibited global gene expression signatures comparable to those of adult human podocytes, had podocyte morphologic features (including foot process-like and slit diaphragm-like structures), and showed functional responsiveness to drug-induced injury. CONCLUSIONS: Elucidation of signals that induce podocytes during the nephron-patterning process enabled us to establish a highly efficient method for selective induction of human podocytes from PSCs. These PSC-derived podocytes show molecular, morphologic, and functional characteristics of podocytes, and offer a new resource for disease modeling and nephrotoxicity testing.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/transplante , Podócitos/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Camundongos , Camundongos Transgênicos , Néfrons/citologia , Organoides/metabolismo , Células-Tronco Pluripotentes/citologia , Transdução de SinaisRESUMO
The glomerular podocyte is one of the major targets of kidney research. Recent establishment of kidney organoids from pluripotent stem cells has enabled the detailed analysis of human podocytes in both development and disease. The podocytes in organoids express slit diaphragm-related genes and proteins and exhibit characteristic morphology, especially upon experimental transplantation. Organoid technology is now used to reproduce hereditary podocyte diseases, and selective podocyte induction methods have also been reported. Moreover, single-cell RNA-sequencing of human fetal and adult kidneys has revealed the detailed molecular features of this cell lineage, as well as serving as references for kidney organoids in which podocytes are still immature. Here, we discuss the recent progress and limitations of podocyte research from the viewpoint of developmental biology and kidney organoids.
Assuntos
Podócitos , Pesquisa com Células-Tronco , Animais , Humanos , Células-Tronco Pluripotentes Induzidas , OrganoidesRESUMO
Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Imunoprecipitação da Cromatina , Biologia Computacional , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Espectrometria de Massas , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Nucleossomos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
In mammals, the ovarian follicles are regulated at least in part by bone morphogenetic protein (BMP) family members. Dullard (also known as Ctdnep1) gene encodes a phosphatase that suppresses BMP signaling by inactivating or degrading BMP receptors. Here we report that the Col1a1-Cre-induced Dullard mutant mice displayed hemorrhagic ovarian cysts, with red blood cells accumulated in the follicles, resulting in infertility. Cells expressing Cre driven by Col1a1 2.3-kb promoter and their descendants were found in granulosa cells in the ovary and in Sertoli cells in the testis. DullardmRNA was localized to granulosa cells in the ovary. Genes involved in steroid hormone genesis including Cyp11a1, Hsd3b1 and Star were reduced, whereas expression of Smad6 and Smad7, BMP-inducible inhibitory Smads, was up-regulated in the Dullard mutant ovaries. Tamoxifen-inducible Dullard deletion in the whole body using Rosa26-CreER mice also resulted in hemorrhagic ovarian cysts in 2 weeks, which was rescued by administration of LDN-193189, a chemical inhibitor of BMP receptor kinase, suggesting that the hemorrhage in the Dullard-deficient ovarian follicles might be caused by increased BMP signaling. Thus, we conclude that Dullard is essential for ovarian homeostasis at least in part via suppression of BMP signaling.
Assuntos
Colágeno Tipo I/metabolismo , Hemorragia/patologia , Infertilidade Feminina/patologia , Cistos Ovarianos/patologia , Folículo Ovariano/patologia , Fosfoproteínas Fosfatases/deficiência , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hemorragia/metabolismo , Infertilidade Feminina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Cistos Ovarianos/metabolismo , Folículo Ovariano/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Proteínas Smad/metabolismo , Testículo/metabolismo , Testículo/patologiaRESUMO
It is difficult to restore kidney function following chronic kidney damage. Although dialysis is currently used to treat patients with chronic kidney disease, it does not cure the disease, while severely restricting the patient's daily and social activities. Kidney transplantation is an alternative and curative therapy, but donor numbers remain limited. However, the generation of kidney organoids from human induced pluripotent stem cells represents an important recent advance in regenerative medicine. Kidney organoids are expected to be used for disease modeling and drug discovery, and may eventually be applicable for transplantation. In this review, we describe the current status of kidney organoids and discuss the hurdles that need to be overcome to generate transplantable artificial kidneys.