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1.
Elife ; 102021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33587034

RESUMO

The canonical Wnt pathway transcriptional co-activator ß-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated ß-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of ß-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/ß-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/ß-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, ß-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising ß-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

3.
Am J Physiol Renal Physiol ; 319(3): F423-F435, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32657158

RESUMO

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.


Assuntos
Túbulos Renais Distais/enzimologia , Recombinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Recombinases/genética , Simportadores de Cloreto de Sódio/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Tamoxifeno/farmacologia
4.
Nat Metab ; 2(8): 732-743, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32694833

RESUMO

Acute kidney injury (AKI) is strongly associated with mortality, independently of its cause. The kidney contributes to up to 40% of systemic glucose production by gluconeogenesis during fasting and under stress conditions. Whether kidney gluconeogenesis is impaired during AKI and how this might influence systemic metabolism remain unknown. Here we show that glucose production and lactate clearance are impaired during human and experimental AKI by using renal arteriovenous catheterization in patients, lactate tolerance testing in mice and glucose isotope labelling in rats. Single-cell transcriptomics reveal that gluconeogenesis is impaired in proximal tubule cells during AKI. In a retrospective cohort of critically ill patients, we demonstrate that altered glucose metabolism during AKI is a major determinant of systemic glucose and lactate levels and is strongly associated with mortality. Thiamine supplementation increases lactate clearance without modifying renal function in mice with AKI, enhances glucose production by renal tubular cells ex vivo and is associated with reduced mortality and improvement of the metabolic pattern in a retrospective cohort of critically ill patients with AKI. This study highlights an unappreciated systemic role of renal glucose and lactate metabolism under stress conditions, delineates general mechanisms of AKI-associated mortality and introduces a potential intervention targeting metabolism for a highly prevalent clinical condition with limited therapeutic options.

5.
J Am Soc Nephrol ; 31(4): 701-715, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32034106

RESUMO

BACKGROUND: Gdf15 encodes a TGF-ß superfamily member that is rapidly activated in response to stress in multiple organ systems, including the kidney. However, there has been a lack of information about Gdf15 activity and effects in normal kidney and in AKI. METHODS: We used genome editing to generate a Gdf15 nuGFP-CE mouse line, removing Gdf15 at the targeted allele, and enabling direct visualization and genetic modification of Gdf15-expressing cells. We extensively mapped Gdf15 expression in the normal kidney and following bilateral ischemia-reperfusion injury, and quantified and compared renal responses to ischemia-reperfusion injury in the presence and absence of GDF15. In addition, we analyzed single nucleotide polymorphism association data for GDF15 for associations with patient kidney transplant outcomes. RESULTS: Gdf15 is normally expressed within aquaporin 1-positive cells of the S3 segment of the proximal tubule, aquaporin 1-negative cells of the thin descending limb of the loop of Henle, and principal cells of the collecting system. Gdf15 is rapidly upregulated within a few hours of bilateral ischemia-reperfusion injury at these sites and new sites of proximal tubule injury. Deficiency of Gdf15 exacerbated acute tubular injury and enhanced inflammatory responses. Analysis of clinical transplantation data linked low circulating levels of GDF15 to an increased incidence of biopsy-proven acute rejection. CONCLUSIONS: Gdf15 contributes to an early acting, renoprotective injury response, modifying immune cell actions. The data support further investigation in clinical model systems of the potential benefit from GDF15 administration in situations in which some level of tubular injury is inevitable, such as following a kidney transplant.


Assuntos
Lesão Renal Aguda/patologia , Fator 15 de Diferenciação de Crescimento/genética , Transplante de Rim , Polimorfismo Genético/genética , Traumatismo por Reperfusão/patologia , Lesão Renal Aguda/genética , Adulto , Animais , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Traumatismo por Reperfusão/genética
6.
Dev Dyn ; 249(6): 765-774, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32017326

RESUMO

BACKGROUND: Ureteric progenitor cells (UPCs) within the branch tips of the arborizing ureteric epithelium of the kidney's developing collecting system establish the shape and cellular organization of the collecting network, and drive the nephrogenic program through their interactions with nephron progenitor cells. In a previous study, expression screening identified a cohort of genes showing UPC-enriched expression including D17H6S56E-5, Hs3st3a1, Hs3st3b1, and Tmem59l. Each of these is also enriched in branch tips of assembling airways of the developing lungs. Here, we used Crispr-CAS9 directed gene editing to mutate each of these targets to address their potential role(s) in UPC programs. RESULTS: Single (D17H6S56E-5 and Tmem59l) and double (Hs3st3a1 and Hs3st3b1) mutants were viable, fertile, and displayed varying frequencies of ureter duplications and no overt lung phenotype. Ureter duplications arise spontaneously through multiple outgrowths of the ureteric bud at the onset of kidney development. Tmem59l mutants and Hs3st3a1/Hs3st3b1 compound mutants showed a weakly penetrant, but statistically significant increase in duplicated ureters compared to C57BL6/J and SW wild-type mouse strains. CONCLUSIONS: Tmem59l and Hs3st3a1/Hs3st3b1 activities contribute to the regulatory programs restricting ureteric outgrowth in the developing mouse kidney. However, the low penetrance of the observed phenotype precludes a detailed analysis of their specific actions.

7.
Dev Biol ; 458(2): 164-176, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31734175

RESUMO

The ureteric epithelial progenitor (UEP) population within the embryonic kidney generates the arborized epithelial network of the kidney's collecting system and plays a critical role in the expansion and induction of the surrounding nephron progenitor pool. Adamts18 shows UEP- restricted expression in the kidney and progenitor tip-restricted expression in several other organs undergoing branching epithelial growth. Adamts18 is encoded by 23 exons. Genetic removal of genomic sequence spanning exons 1 to 3 led to a specific loss of Adamts18 expression in UEPs, suggesting this region may encode a UEP-specific enhancer. Intron 2 (3 â€‹kb) was shown to have enhancer activity driving expression of the doxycycline inducible tet-on transcriptional regulator (rtTA) in an Adamts18en-rtTA transgenic mouse strain. Crossing Adamts18en-rtTA mice to a doxycycline dependent GFP reporter mouse enabled the live imaging of embryonic kidney explants. This facilitated the analysis of ureteric epithelial branching events at the cellular level. Ablation of UEPs at the initiation of ureteric bud outgrowth through the doxycycline-mediated induction of Diphtheria Toxin A (DTA) generated a range of phenotypes from complete kidneys agenesis, to duplex kidneys with double ureters. The latter outcome points to the potential of regulative processes to restore UEPs. In contrast, overexpression of YAP prior to ureteric bud outgrowth led to a complete failure of kidney development. Elevating YAP levels at later stages retarded branching growth. A similar phenotype was observed with the overexpression of MYC within the branch-tip localized UEP population. These experiments showcase the utility of the Adamts18en-rtTA transgenic model to the investigation of cellular and molecular events specific to branch tip progenitors within the mammalian kidney complementing existing CRE-dependent genetic tools. Further, the illustrative examples point to areas where new insight may be gained into the regulation of UEP programs.


Assuntos
Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Ureter/embriologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Feminino , Rim/metabolismo , Rim/patologia , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Morfogênese/genética , Néfrons/metabolismo , Organogênese/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ureter/metabolismo
8.
Dev Cell ; 51(3): 399-413.e7, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31689386

RESUMO

Chronic kidney disease affects 10% of the population with notable differences in ethnic and sex-related susceptibility to kidney injury and disease. Kidney dysfunction leads to significant morbidity and mortality and chronic disease in other organ systems. A mouse-organ-centered understanding underlies rapid progress in human disease modeling and cellular approaches to repair damaged systems. To enhance an understanding of the mammalian kidney, we combined anatomy-guided single-cell RNA sequencing of the adult male and female mouse kidney with in situ expression studies and cell lineage tracing. These studies reveal cell diversity and marked sex differences, distinct organization and cell composition of nephrons dependent on the time of nephron specification, and lineage convergence, in which contiguous functionally related cell types are specified from nephron and collecting system progenitor populations. A searchable database, Kidney Cell Explorer (https://cello.shinyapps.io/kidneycellexplorer/), enables gene-cell relationships to be viewed in the anatomical framework of the kidney.


Assuntos
Linhagem da Célula , Rim/citologia , Caracteres Sexuais , Análise de Célula Única , Animais , Células Epiteliais/citologia , Feminino , Rim/anatomia & histologia , Masculino , Camundongos , Néfrons/citologia , Fatores de Tempo
9.
iScience ; 20: 402-414, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31622881

RESUMO

Analysis of kidney disease-causing genes and pathology resulting from systemic diseases highlight the importance of the kidney's filtering system, the renal corpuscles. To elucidate the developmental processes that establish the renal corpuscle, we performed single-nucleus droplet-based sequencing of the human fetal kidney. This enabled the identification of nephron, interstitial, and vascular cell types that together generate the renal corpuscles. Trajectory analysis identified transient developmental gene expression, predicting precursors or mature podocytes express FBLN2, BMP4, or NTN4, in conjunction with recruitment, differentiation, and modeling of vascular and mesangial cell types into a functional filter. In vitro studies provide evidence that these factors exhibit angiogenic or mesangial recruiting and inductive properties consistent with a key organizing role for podocyte precursors in kidney development. Together these studies define a spatiotemporal developmental program for the primary filtration unit of the human kidney and provide novel insights into cell interactions regulating co-assembly of constituent cell types.

10.
Dev Cell ; 50(1): 102-116.e6, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31265809

RESUMO

The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Glomérulos Renais/citologia , Organoides/citologia , Podócitos/citologia , Análise de Célula Única/métodos , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomérulos Renais/metabolismo , Organoides/metabolismo , Podócitos/metabolismo
11.
Dev Biol ; 454(2): 156-169, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31242448

RESUMO

Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks.


Assuntos
Proteínas ADAMTS/metabolismo , Organogênese/fisiologia , Proteínas ADAMTS/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Rim/citologia , Rim/embriologia , Rim/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Masculino , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Knockout , Morfogênese , Néfrons/metabolismo , Técnicas de Cultura de Órgãos/métodos , Ureter/metabolismo
12.
Nat Commun ; 10(1): 1157, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30858375

RESUMO

The mechanisms initiating late immune responses to an allograft are poorly understood. Here we show, via transcriptome analysis of serial protocol biopsies from kidney transplants, that the initial responses to kidney injury correlate with a late B lymphocyte signature relating to renal dysfunction and fibrosis. With a potential link between dysfunctional repair and immunoreactivity, we investigate the immunological consequences of dysfunctional repair examining chronic disease in mouse kidneys 18 months after a bilateral ischemia/reperfusion injury event. In the absence of foreign antigens, a sustained immune response involving both innate and adaptive immune systems accompanies a transition to chronic kidney damage. At late stages, B lymphocytes exhibite an antigen-driven proliferation, selection and maturation into broadly-reacting antibody-secreting cells. These findings reveal a previously unappreciated role for dysfunctional tissue repair in local immunomodulation that may have particular relevance to transplant-associated immunobiology.


Assuntos
Lesão Renal Aguda/imunologia , Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim/efeitos adversos , Insuficiência Renal Crônica/imunologia , Lesão Renal Aguda/patologia , Lesão Renal Aguda/fisiopatologia , Imunidade Adaptativa , Adulto , Aloenxertos/citologia , Aloenxertos/imunologia , Aloenxertos/patologia , Aloenxertos/fisiopatologia , Animais , Biópsia , Modelos Animais de Doenças , Feminino , Fibrose , Perfilação da Expressão Gênica , Taxa de Filtração Glomerular , Rejeição de Enxerto/patologia , Rejeição de Enxerto/fisiopatologia , Humanos , Rim/citologia , Rim/imunologia , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Análise de Sequência de RNA , Transplante Homólogo/efeitos adversos
13.
Nat Commun ; 10(1): 239, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651543

RESUMO

Branching patterns and regulatory networks differ between branched organs. It has remained unclear whether a common regulatory mechanism exists and how organ-specific patterns can emerge. Of all previously proposed signalling-based mechanisms, only a ligand-receptor-based Turing mechanism based on FGF10 and SHH quantitatively recapitulates the lung branching patterns. We now show that a GDNF-dependent ligand-receptor-based Turing mechanism quantitatively recapitulates branching of cultured wildtype and mutant ureteric buds, and achieves similar branching patterns when directing domain outgrowth in silico. We further predict and confirm experimentally that the kidney-specific positive feedback between WNT11 and GDNF permits the dense packing of ureteric tips. We conclude that the ligand-receptor based Turing mechanism presents a common regulatory mechanism for lungs and kidneys, despite the differences in the molecular implementation. Given its flexibility and robustness, we expect that the ligand-receptor-based Turing mechanism constitutes a likely general mechanism to guide branching morphogenesis and other symmetry breaks during organogenesis.


Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Rim/crescimento & desenvolvimento , Modelos Biológicos , Organogênese , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Wnt/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Simulação por Computador , Embrião de Mamíferos , Retroalimentação Fisiológica , Feminino , Processamento de Imagem Assistida por Computador , Rim/diagnóstico por imagem , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Técnicas de Cultura de Órgãos , Transdução de Sinais/fisiologia , Imagem com Lapso de Tempo/métodos , Tomografia Óptica/métodos
15.
Elife ; 72018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30516471

RESUMO

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program.


Assuntos
Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Néfrons/metabolismo , Organogênese/genética , Células-Tronco/metabolismo , Proteínas Wnt/genética , Animais , Diferenciação Celular , Movimento Celular , Embrião de Mamíferos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Néfrons/citologia , Néfrons/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo
16.
JCI Insight ; 3(22)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30429361

RESUMO

BACKGROUND: The molecular understanding of the progression from acute to chronic organ injury is limited. Ischemia/reperfusion injury (IRI) triggered during kidney transplantation can contribute to progressive allograft dysfunction. METHODS: Protocol biopsies (n = 163) were obtained from 42 kidney allografts at 4 time points after transplantation. RNA sequencing-mediated (RNA-seq-mediated) transcriptional profiling and machine learning computational approaches were employed to analyze the molecular responses to IRI and to identify shared and divergent transcriptional trajectories associated with distinct clinical outcomes. The data were compared with the response to IRI in a mouse model of the acute to chronic kidney injury transition. RESULTS: In the first hours after reperfusion, all patients exhibited a similar transcriptional program under the control of immediate-early response genes. In the following months, we identified 2 main transcriptional trajectories leading to kidney recovery or to sustained injury with associated fibrosis and renal dysfunction. The molecular map generated by this computational approach highlighted early markers of kidney disease progression and delineated transcriptional programs associated with the transition to chronic injury. The characterization of a similar process in a mouse IRI model extended the relevance of our findings beyond transplantation. CONCLUSIONS: The integration of multiple transcriptomes from serial biopsies with advanced computational algorithms overcame the analytical hurdles related to variability between individuals and identified shared transcriptional elements of kidney disease progression in humans, which may prove as useful predictors of disease progression following kidney transplantation and kidney injury. This generally applicable approach opens the way for an unbiased analysis of human disease progression. FUNDING: The study was supported by the California Institute for Regenerative Medicine and by the Swiss National Science Foundation.


Assuntos
Nefropatias/genética , Transplante de Rim/efeitos adversos , Traumatismo por Reperfusão/genética , Transcrição Genética , Adulto , Idoso , Animais , Biópsia , Biologia Computacional , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Nefropatias/etiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Traumatismo por Reperfusão/etiologia , Transcriptoma
17.
Development ; 145(17)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30093555

RESUMO

The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6, which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size.


Assuntos
Ciclo Celular/fisiologia , Córtex Cerebral/embriologia , Quinase 6 Dependente de Ciclina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neuroglia/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Córtex Cerebral/citologia , Quinase 6 Dependente de Ciclina/genética , Feminino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Proteína Gli3 com Dedos de Zinco/genética
18.
Stem Cell Reports ; 11(2): 470-484, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30033089

RESUMO

Kidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células , Rim/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Biomarcadores , Diferenciação Celular , Fibrose , Técnicas de Inativação de Genes , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Rim/embriologia , Néfrons/citologia
19.
Dev Cell ; 45(5): 651-660.e4, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29870722

RESUMO

Mammalian nephrons arise from a limited nephron progenitor pool through a reiterative inductive process extending over days (mouse) or weeks (human) of kidney development. Here, we present evidence that human nephron patterning reflects a time-dependent process of recruitment of mesenchymal progenitors into an epithelial nephron precursor. Progressive recruitment predicted from high-resolution image analysis and three-dimensional reconstruction of human nephrogenesis was confirmed through direct visualization and cell fate analysis of mouse kidney organ cultures. Single-cell RNA sequencing of the human nephrogenic niche provided molecular insights into these early patterning processes and predicted developmental trajectories adopted by nephron progenitor cells in forming segment-specific domains of the human nephron. The temporal-recruitment model for nephron polarity and patterning suggested by direct analysis of human kidney development provides a framework for integrating signaling pathways driving mammalian nephrogenesis.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Néfrons/citologia , Organogênese/fisiologia , Animais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Néfrons/metabolismo , Transdução de Sinais , Análise de Célula Única , Fatores de Tempo
20.
PLoS Genet ; 14(4): e1007346, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29659575

RESUMO

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.


Assuntos
Condrócitos/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fatores de Transcrição SOX9/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Condrócitos/citologia , Condrogênese/genética , Condrogênese/fisiologia , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Feminino , Redes Reguladoras de Genes , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Modelos Biológicos , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Ativação Transcricional , Proteína GLI1 em Dedos de Zinco/genética
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